WO2015108203A1 - 抗slc6a6抗体を用いたがん治療用医薬組成物 - Google Patents
抗slc6a6抗体を用いたがん治療用医薬組成物 Download PDFInfo
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- A61K51/1072—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from the reproductive system, e.g. ovaria, uterus, testes or prostate
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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Definitions
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a monoclonal antibody that recognizes the extracellular domain of SLC6A6.
- an antibody conjugate in which a monoclonal antibody that recognizes the extracellular domain of SLC6A6 is conjugated with an anticancer agent having activity against hyperproliferative diseases such as cancer, and the conjugate
- the present invention relates to a pharmaceutical composition for treating cancer including colorectal cancer.
- colorectal cancer is a disease that ranks high in cancer mortality.
- Japan the number of patients with colorectal cancer has increased rapidly in recent years, and about 60,000 people suffer from colorectal cancer annually, and the number of deaths by organ is the third largest after gastric cancer and lung cancer.
- Colorectal cancer has a 5-year survival rate of about 90% or more when it stays only in the large intestine, and is known as a cancer whose early detection leads to a high cure rate. Nonetheless, colorectal cancer has a high number of deaths because of its high morbidity rate, 5-year survival rate is 70% when metastasis to lymph nodes occurs, and less than 25% when distant metastasis to the lungs and liver. And the mortality rate deteriorates rapidly as cancer progresses.
- surgical treatment and chemotherapy are common, but with the advent of molecular target drugs in recent years, new cancer-specific treatment methods have been sought.
- Avastin and Erbitux are known as antibody drugs approved in Japan as molecular target drugs for colorectal cancer. These are antibody drugs targeting growth factors such as vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF).
- VEGF vascular endothelial growth factor
- EGF epidermal growth factor
- Avastin has been approved for use in advanced and recurrent colorectal cancer that cannot be curatively excised. It binds to VEGF, suppresses angiogenesis by inhibiting binding to VEGF receptors, It works by the action mechanism of eliminating nutrition.
- Erbitux aims to stop the growth of cancer cells by binding to the EGF receptor (EGFR) and preventing the cell proliferation signal from EGF from working.
- EGFR EGF receptor
- ADCC Anti-Dependent Cellular Cytotoxicity
- NK cells natural killer cells
- macrophages Is also said to be working.
- Cancer cells have the characteristics of causing invasion and metastasis to surrounding tissues, which have no higher proliferation power and the number of cell divisions compared to normal cells. In recent years, not all cancer cells in cancer tissues have such properties, but some limited cells are considered to have such properties. That is, some of these cancer cells are common to stem cells such as embryonic stem cells and somatic stem cells, which have the ability to self-replicate to produce exactly the same cells as themselves and the multipotency that can differentiate into many types of cells. These characteristics are thought to be the basis for generating the majority of the surrounding cancer cells by differentiation while maintaining the same cells as themselves by self-replication in cancer tissues. ing. 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 1 GASTROENTERROLOGY, 138, 2151-2162, 2010. It only binds to cancer stem cells and is said to be ineffective as a therapeutic agent. Further, LGR5 interacts with R-spondin and has a mechanism of activating Wnt / ⁇ -catenin signal, suggesting the possibility of being used as a cancer stem cell marker (Patent Document 1: Special Table 2010). -532169).
- 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 agent that targets only cancer stem cells cannot kill the entire cancer cells.
- a therapeutic agent that targets 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 and also expressed in general cancer cells compared to normal tissues is This is an important issue.
- Molecular target drugs represented by antibodies are excellent substances in that they can kill cancer cells if they specifically recognize cancer. However, if 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 disorder, kidney disorder, and cerebrovascular disorder. Further, it is known that tissue staining strongly reacts with normal cardiomyocytes and causes severe heart damage (Non-patent Document 2: British Journal of Cancer, 94, 1016-1020, 2006). Furthermore, since 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- Patent 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 (for example, K-ras mutation) downstream of EGFR.
- a signal change for example, K-ras mutation
- Drug delivery systems that transport drugs using the property that molecular target drugs such as antibodies bind to the cell surface are also widely known. Particularly in recent years, as a method of effectively killing cancer cells after effectively transporting a drug to the surface of the cancer cells, a method of incorporating antibody molecules into the cells is taken. Antibody molecules are not normally taken up into cells because they cannot penetrate cell membranes. However, it is known that certain antibodies are taken into cells through a mechanism called internalization by binding to membrane protein molecules on the cell membrane.
- EGF receptor a molecule that binds to the receptor
- ligand a molecule that binds to the receptor
- clathrin a molecule that binds to the receptor
- a certain type of antibody binds to the EGF receptor, it is taken up into the cell in the same manner as when a ligand is bound. Utilizing this mechanism, a therapeutic method has been developed in which a drug is bound to an antibody and the antibody is efficiently transported into a cell.
- 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.
- transporters transport proteins
- Patent Document 2 shows that SLC6A6 is expressed in colorectal cancer and can be detected as a diagnostic agent by a monoclonal antibody that recognizes its extracellular domain.
- SLC6A6 (solute carrier family 6 (neurotransitter transporter, taurine), member 6) is a twelve-transmembrane membrane protein consisting of 620 amino acids, and Reference_SequenceS43NRefSequenceRIDNS: NP_003034.2 (SEQ ID NO: 1: nucleotide sequence (CDS: 296 to 2158), SEQ ID NO: 2: amino acid sequence).
- SLC6A6 is involved in the uptake of taurine into cells and co-transports taurine together with sodium ion and chloride ion.
- Patent Document 2 shows that the gene of SLC6A6 is expressed in all 5 cases of colon cancer tissue, while the transcription of the gene is not seen in 5 cases of normal tissue by in situ hybridization method. It is clear. Furthermore, it is described that the two clones (4B9b and 5H12d) of the obtained monoclonal antibodies have an epitope between the amino acid residues from the 145th to the 213rd of the SLC6A6 protein.
- Antibody drugs against cancer still have problems such as the occurrence of serious side effects and limited response to patients. Development was required.
- cancer surgery is not only difficult to treat metastases, but also involves a combination of invasion and complications.
- side effects are a problem in chemotherapy and radiotherapy.
- existing antibody drugs have cancers that do not show any response at all. Therefore, there has been a demand for the development of new drugs with cancer-specific molecular targets and fewer side effects.
- the present invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, specifically a pharmaceutical composition comprising an antibody conjugate in which one or more chemotherapeutic agents are combined with the antibody or antigen-binding fragment thereof, particularly cancer.
- the object is to provide a pharmaceutical composition for treatment.
- the present inventors include an epitope in the same region as the anti-SLC6A6 monoclonal antibody described in International Publication No. 2012/029990 (WO2012 / 029990), and increase the affinity. A new monoclonal antibody was obtained.
- SLC6A6 which is a membrane protein overexpressed in cancer tissue, is also useful as a cancer marker.
- the present inventor has found that SLC6A6 is expressed higher in cancer stem cells, which are the main cause of cancer recurrence and metastasis, than in normal cancer cells.
- the said antibody alone does not show the effect which kills a cancer cell, it discovered that the antibody couple
- a pharmaceutical composition for treating cancer comprising a monoclonal antibody or an antigen-binding fragment thereof that binds to a three-dimensional epitope in the extracellular region of SLC6A6.
- the monoclonal antibody is a conjugate of an anticancer agent, a toxin or a radioisotope.
- a cancer treatment comprising a monoclonal antibody that binds to a three-dimensional epitope in the extracellular region of SLC6A6 or an antigen-binding fragment thereof conjugated with an anticancer agent, a toxin or a radioisotope.
- the epitope includes at least one polypeptide selected from the following (a) to (c): (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted and / or inserted in the amino acid sequence shown in SEQ ID NO: 4, And a polypeptide functioning as an extracellular region of SLC6A6 (c) a polypeptide comprising an amino acid sequence having 70% or more homology to the amino acid sequence shown in SEQ ID NO: 4 and functioning as an extracellular region of SLC6A6.
- the monoclonal antibody is (A) a heavy chain variable region comprising the amino acid sequences of 50 to 54, 69 to 86 and 118 to 125 of SEQ ID NO: 24 as heavy chain CDR1, CDR2 and CDR3, respectively, and 44 to 54 of SEQ ID NO: 26, An antibody comprising a light chain variable region comprising the amino acid sequences of positions 70 to 76 and 109 to 116 as light chain CDR1, CDR2 and CDR3, respectively; (B) a heavy chain variable region comprising the amino acid sequences of 50 to 54, 69 to 86 and 118 to 126 of SEQ ID NO: 28 as heavy chain CDR1, CDR2 and CDR3, respectively, and 46 to 57 of SEQ ID NO: 30, An antibody comprising a light chain variable region comprising the amino acid sequences of 73 to 79 and 112 to 119 as light chain CDR1, CDR2 and CDR3, respectively; (C) a heavy chain variable region comprising the amino acid sequences of 50 to 54, 69 to 86 and
- the toxin is diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A, fungus, plant, antiviral protein, saporin, gelonin, momoridine, tricosanthin, barley toxin, Aleurites fordy protein, dian At least selected from the group consisting of a tin protein, a phytolacca americana protein, a momordica charantia inhibitor, a crucin, a crotin, a sapoaonaria officinalis inhibitor, a gelonin, a mitogen, a restrictocin, a phenomycin, a neomycin, and a trichothecene
- the present invention provides a monoclonal antibody that specifically binds to the extracellular region of SLC6A6.
- the present invention also provides an antibody conjugate comprising an anti-SLC6A6 monoclonal antibody or antigen-binding fragment bound to at least one anticancer agent, toxin or radioisotope, and a pharmaceutical composition comprising the antibody conjugate.
- the monoclonal antibody of the present invention specifically binds to and internalizes cancer cells expressing SLC6A6, the antibody conjugate and the pharmaceutical composition of the present invention are used for cancer treatment, particularly for colorectal cancer treatment. Useful as a product. Since the antibody may be humanized or chimerized, side effects when applied to humans can be reduced.
- SLC6A6 is highly expressed in cancer stem cells compared to normal tissues, and is also expressed in general cancer cells. Therefore, according to the present invention, not only cancer stem cells are targeted. New cancer therapeutics or treatments that target the entire cancer cell are also provided.
- “Isotype control” using Whole human IgG as a negative control
- “Cetuximab” using Whole human IgG as a negative control
- “430 human-mouse” directly labeled with a fluorescent dye
- the present invention is based on the knowledge that SLC6A6, which is a membrane protein, is a protein that is overexpressed in cancer tissues and is a useful marker for treatment. Furthermore, it is based on the knowledge that SLC6A6 is expressed more highly in cancer stem cells, which are the main cause of cancer recurrence and metastasis, than in normal cancer cells.
- an antibody having higher affinity than the conventional anti-SLC6A6 antibody is provided. And it has become clear that by using such an antibody of the present invention, an anticancer agent or the like can be effectively transported to a cancer cell and further incorporated into the cell.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a monoclonal antibody recognizing the extracellular region of a molecule called SLC6A6 (solute carrier family 6 (neurotransmitter transporter, taurine), member 6) or an antigen-binding fragment thereof. Since SLC6A6 is expressed in colon cancer, this antibody specifically binds to colon cancer cells.
- SLC6A6 synthetic carrier family 6 (neurotransmitter transporter, taurine), member 6) or an antigen-binding fragment thereof. Since SLC6A6 is expressed in colon cancer, this antibody specifically binds to colon cancer cells.
- the present invention uses an antibody conjugate containing an anti-cancer agent such as a chemotherapeutic agent, the antibody to which a toxin or a radioisotope is bound (hereinafter referred to as a conjugate), thereby allowing chemotherapy.
- an anti-cancer agent such as a chemotherapeutic agent, the antibody to which a toxin or a radioisotope is bound
- the present invention relates to a pharmaceutical composition containing an antibody conjugate whose action mechanism is that an agent or the like is taken into a cancer cell. Therefore, the pharmaceutical composition of the present invention is useful for the treatment of cancer, particularly colon cancer.
- Monoclonal Antibody of the Present Invention (1) Monoclonal Antibody of the Present Invention
- the monoclonal antibody used in the pharmaceutical composition of the present invention (hereinafter also referred to as “monoclonal antibody of the present invention”) can recognize native SLC6A6. “Native” means that the protein is in an intact three-dimensional structure in the environment of the living body.
- the monoclonal antibody of the present invention can recognize the extracellular region of SLC6A6. Specifically, the monoclonal antibody of the present invention binds to a three-dimensional epitope in the extracellular region of SLC6A6. In particular, as the extracellular region, the region of amino acid residues 143 to 216 (SEQ ID NO: 4) of SLC6A6 can be recognized.
- the monoclonal antibody of the present invention has SEQ ID NO: 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 polypeptide to be bound has a function as an extracellular region of SLC6A6.
- Substitution, deletion or insertion of one or several amino acids for example, 2 to 20, preferably 2 to 10, more preferably 2, 3, 4 or 5) of the amino acid sequence shown in FIG.
- the monoclonal antibody of the present invention is a polypeptide having a function as an extracellular region of SLC6A6, which is a polypeptide encoded by a polynucleotide consisting of the base sequence described in SEQ ID NO: 3, and described in SEQ ID NO: 3.
- hybridization can be performed according to a known method (for example, Molecular Cloning 2nd Ed (Cold Spring Harbor Lab.
- the extracellular region of SLC6A6 is responsible for binding with taurine and transporting taurine into the cell. Whether the mutant polypeptide has a function as an extracellular domain of SLC6A6 is determined by forcibly expressing the mutant polypeptide in animal cells and the like, and taking up taurine by the activation method (J. Membr. Biol, 76, 1). -15, 1983).
- the above-mentioned mutant polypeptide that can be bound by the monoclonal antibody of the present invention is a polypeptide having the amino acid sequence of antibody SEQ ID NO: 4. That is, it is included in a polypeptide having a function as an extracellular region of SLC6A6.
- the binding between the mutant polypeptide and the monoclonal antibody of the present invention can be confirmed by ELISA, immunoprecipitation, Western blotting or the like.
- the extracellular region of SLC6A6 is a cell surface site of a marker protein whose expression is increased in cancer cells. Whether or not the mutant polypeptide has a function as an extracellular domain of SLC6A6 is determined by immunostaining, ELISA, immunoprecipitation, Western blotting, FACS, etc. This can be confirmed by comparison.
- the monoclonal antibody of the present invention is a subclass IgG, and has higher affinity for SLC6A6 from immunohistochemical staining and / or analysis by flow cytometer. Is shown to have.
- the monoclonal antibody of the present invention has a property of being taken into cells via a mechanism called internalization.
- the monoclonal antibody of the present invention also recognizes a protein encoded by the slc6a6 mRNA variant. Since it can bind not only to the full-length SLC6A6 but also to a mutant lacking a part thereof, it can bind to cancer cells that express SLC6A6 extensively.
- an antigen-binding fragment that is a part of a monoclonal antibody for example, an antibody such as an antibody fragment, a low molecular weight antibody, a recombinant antibody, or an antibody modification product, by various genetic engineering and protein engineering techniques.
- a protein in which a molecule-like molecule or a monoclonal antibody is fused can be prepared. Specifically, for example, H chain, L chain, Fv, Fab, Fab ′, F (ab ′) 2, scFv, sdFv, sc (Fv) 2, (scFv) 2, DiAbody, chimeric antibody, humanized antibody And multispecific antibodies such as human antibodies, single chain antibodies, bispecific antibodies, and labeled antibodies. Any molecule that has the ability to bind to the extracellular region of SLC6A6 is included in the monoclonal antibody of the present invention.
- SLC6A6 recognized by the monoclonal antibody of the present invention is a molecular marker that is not expressed in normal cells, expressed in cancer cells, and is highly expressed in cancer stem cells compared to cancer cells in the cell population to be detected. . Therefore, the antibody binds to both cancer and cancer stem cells and binds more to cancer stem cells.
- cancer cell in the present invention refers to a cell population that has characteristics such as high proliferation ability and the number of cell divisions, as compared with normal cells, and invasion and metastasis to surrounding tissues. Say.
- the “cancer stem cell” in the present invention is a cell having the properties of a stem cell among cancer cells.
- Stem cells refer to cells that maintain their differentiation potential even after cell division.
- Cancer stem cells are stained with Hoechst fluorescent dye (Hoechst 33342), and when they are detected using UV laser (wavelength of about 350 nm) as excitation light using flow cytometry, they are concentrated in the Side Population (SP) fraction Recognized as SP fraction refers to the fraction that is not stained or weakly stained by discharging the pigment out of the cell via ABC transporter, etc., against the main population (MP) fraction that is stained with Hoechst fluorescent dye. (The American Journal of Pathology, 178, 4, 1805-1813, 2011).
- 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 monoclonal antibody of the present invention is preferably expressed in common to cancer cells of a plurality of cancer types and strongly binds to cancer stem cells, and can bind to a plurality of cancer types.
- cells such as heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, leukocyte, colon, stomach, bone marrow, colon and peripheral blood mononuclear cells
- it is a plurality of tissue-derived cancer types, more preferably colon cancer, breast cancer, or uterine cancer cancer cells.
- the monoclonal antibody of the present invention does not bind in normal cells.
- the monoclonal antibody of the present invention does not bind in normal cells.
- in 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.
- Preferred cell lines (hybridomas) producing the monoclonal antibodies of the invention are, for example: “Mouse-mouse hybridoma 204” (hereinafter referred to as “204”), “Mouse-mouse hybridoma 205” (hereinafter referred to as “205”), “Mouse-mouse hybrida 303” (hereinafter referred to as “303”), “Mouse-mouse hybridoma 419” (hereinafter referred to as “419”) “Mouse-mouse hybridoma 402” (hereinafter referred to as “402”), “Mouse-mouse hybridoma 422” (hereinafter referred to as “422”) and “mouse-mouse hybridoma 430” (hereinafter referred to as “430”).
- the present invention provides the hybridomas and the antibodies they produce.
- a homogeneous monoclonal antibody can be produced by culturing the hybrido
- the antibody produced by 205 includes a heavy chain variable region containing the 21st to 135th amino acid sequences of SEQ ID NO: 24 and a light chain variable region containing the 21st to 127th amino acid sequences of SEQ ID NO: 26. Further, the antibody produced by 205 comprises a heavy chain variable region comprising the amino acid sequences of the 50th to 54th, 69th to 86th and 118th to 125th amino acids of SEQ ID NO: 24 as heavy chain CDR1, CDR2 and CDR3, respectively, and SEQ ID NO: 26 A light chain variable region comprising the amino acid sequences of amino acids 44 to 54, 70 to 76, and 109 to 116 as light chain CDR1, CDR2 and CDR3, respectively.
- the antibody produced by 402 contains a heavy chain variable region comprising the 21st to 136th amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising the 23rd to 130th amino acid sequence of SEQ ID NO: 30.
- the antibody produced by 402 is a heavy chain variable region comprising the amino acid sequences of 50 to 54, 69 to 86 and 118 to 126 of SEQ ID NO: 28 as heavy chain CDR1, CDR2 and CDR3, respectively, and SEQ ID NO: 30
- a light chain variable region comprising the 46th to 57th, 73th to 79th and 112th to 119th amino acid sequences as light chain CDR1, CDR2 and CDR3, respectively.
- the antibody produced by 419 includes a heavy chain variable region comprising the 21st to 138th amino acid sequences of SEQ ID NO: 32 and a light chain variable region comprising the 25th to 131st amino acid sequences of SEQ ID NO: 34.
- the antibody produced by 419 has a heavy chain variable region comprising the amino acid sequences of the 50th to 54th, 69th to 86th and 118th to 128th amino acids of SEQ ID NO: 32 as heavy chain CDR1, CDR2 and CDR3, respectively, and SEQ ID NO: 34
- a light chain variable region comprising the amino acid sequences of Nos. 48 to 58, 74 to 80, and 113 to 120 as light chain CDR1, CDR2 and CDR3, respectively.
- the antibody produced by 303 includes a heavy chain variable region comprising the 21st to 141st amino acid sequences of SEQ ID NO: 36 and a light chain variable region comprising the 21st to 127th amino acid sequences of SEQ ID NO: 38.
- the antibody produced by 205 is a heavy chain variable region comprising the amino acid sequences of the 50th to 54th, 69th to 86th and 118th to 131st of SEQ ID NO: 36 as heavy chain CDR1, CDR2 and CDR3, and SEQ ID NO: 38.
- a light chain variable region comprising the amino acid sequences of amino acids 44 to 54, 70 to 76, and 109 to 116 as light chain CDR1, CDR2 and CDR3, respectively.
- the antibody produced by 422 includes a heavy chain variable region comprising the 21st to 139th amino acid sequences of SEQ ID NO: 40 and a light chain variable region comprising the 21st to 127th amino acid sequences of SEQ ID NO: 42.
- the antibody produced by 205 comprises a heavy chain variable region comprising the amino acid sequences of the 50th to 54th, 69th to 86th and 118th to 129th of SEQ ID NO: 40 as heavy chain CDR1, CDR2 and CDR3, and SEQ ID NO: 42.
- a light chain variable region comprising the amino acid sequences of amino acids 44 to 54, 70 to 76, and 109 to 116 as light chain CDR1, CDR2 and CDR3, respectively.
- the antibody produced by 430 includes a heavy chain variable region comprising the 21st to 136th amino acid sequences of SEQ ID NO: 44 and a light chain variable region comprising the 23rd to 130th amino acid sequences of SEQ ID NO: 46.
- the antibody produced by 205 comprises a heavy chain variable region comprising the amino acid sequences of 50 to 54, 69 to 86 and 118 to 126 of SEQ ID NO: 44 as heavy chains CDR1, CDR2 and CDR3, respectively, and SEQ ID NO: 46 A light chain variable region comprising the 46th to 57th, 73th to 79th and 112th to 119th amino acid sequences as light chain CDR1, CDR2 and CDR3, respectively.
- the monoclonal antibody of the present invention has a heavy chain of at least 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, an amino acid sequence represented by SEQ ID NO: 24, 28, 32, 36, 40 or 44. Amino sequence with 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity, light chain is represented by 26, 30, 34, 38, 42 or 46 At least 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%
- the antibody may be an amino sequence having identity.
- the monoclonal antibody of the present invention comprises The heavy chain variable region is the 21st to 135th amino acid sequence of SEQ ID NO: 24, the 21st to 136th amino acid sequence of SEQ ID NO: 28, the 21st to 138th amino acid sequence of SEQ ID NO: 32, and the 21st to 141st positions of SEQ ID NO: 36.
- the light chain variable region is the 21st to 127th amino acid sequence of SEQ ID NO: 26, the 23rd to 130th amino acid sequence of SEQ ID NO: 30, the 25th to 131st amino acid sequence of SEQ ID NO: 34, and the 21st to 127th sequence of SEQ ID NO: 38.
- amino acid sequence of SEQ ID NO: 42 the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence of SEQ ID NO: 46, and at least 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, It may be an antibody comprising amino sequences having 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity, respectively.
- the monoclonal antibody of the present invention comprises
- the heavy chain variable region is an amino acid sequence of 50-54, 69-86 and 118-125 of SEQ ID NO: 24, an amino acid sequence of 50-54, 69-86 and 118-126 of SEQ ID NO: 28, 50-54, 69-86 and 118-128 amino acid sequences of SEQ ID NO: 32, 50-54, 69-86 and 118-131 amino acid sequences of SEQ ID NO: 36, 50--50 of SEQ ID NO: 40 54th, 69th to 86th and 118th to 129th amino acid sequences or 50th to 54th, 69th to 86th and 118th to 126th amino acid sequences of SEQ ID NO: 44 and at least 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the amino acid sequence with it Re includes as
- amino acid sequences having a predetermined identity with the amino acid sequences of SEQ ID NOs: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46 are the sequences 21, 23, 25, 27, respectively. , 29, 31, 33, 35, 37, 39, 41, 43 or 45, with a predetermined identity, for example, at least 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, It may be encoded by a base sequence having 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity.
- the monoclonal antibody of the present invention having these characteristics is obtained by using an immunization method in which cells expressing a membrane protein are engrafted, as disclosed in WO2010 / 098471, unlike a conventional monoclonal antibody production method.
- a production method generally practiced by those skilled in the art. that is, (1) When preparing a membrane protein as an antigen, if a surfactant is used, the three-dimensional structure of the membrane protein is disrupted. On the other hand, if a surfactant is not used, the membrane protein aggregates between hydrophobic regions. (2) An immune response does not occur because the expression level on the cell surface is low or the extracellular region is small.
- the monoclonal antibody of the present invention has acquired characteristics superior to those of conventional antibodies as a result of the inventors of the present invention having devised the antibody production method (particularly the immunization method).
- the monoclonal antibody of the present invention has the following characteristics. Since the antibody of the present invention was produced based on the three-dimensional structure inherent to SLC6A6, it can recognize native SLC6A6. Therefore, the binding force is very high compared to a conventional antibody (HPA015028) that recognizes the same epitope, and it can sufficiently bind to SLC6A6 on the cell membrane, which was difficult with the conventional antibody. In addition, compared to the conventional monoclonal antibody (sc-166640) that recognizes the SLC6A6 membrane and intracellular regions, the recognition site of the antibody of the present invention is extracellular, so it can bind to living cells. It can be used as a carrier for therapeutic agents or anticancer agents.
- 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 monoclonal antibody in the present invention is a monoclonal antibody against SLC6A6 produced by the hybridoma 204, 205, 303, 419, 402, 422 or 430 (204, 205, 303, 419, 402, 422 or 430 antibody, respectively). Particularly preferred are 402 antibody or 430 antibody.
- the present invention is not limited to the monoclonal antibodies themselves produced by the hybridomas, but is included in the monoclonal antibodies against SLC6A6 of the present invention as long as the antibodies bind to epitopes recognized by the monoclonal antibodies produced by these hybridomas. .
- epitope refers to an epitope recognized by the monoclonal antibody produced by the hybridoma (amino acid residues from the 145th to the 213rd in the amino acid sequence of SLC6A6, as well as a part of these regions). May be).
- the monoclonal antibody of the present invention against SLC6A6 may be a chimerized antibody, a humanized antibody, or a fully human antibody.
- the humanized antibody is, for example, a mouse-human chimeric antibody
- an antibody gene is isolated from a mouse cell that produces an antibody against the SLC6A6 protein, and its heavy chain (H chain) constant region is converted into a human IgG H chain constant region gene. It can be prepared by introduction into recombinant, mouse myeloma cells.
- a human antibody can be prepared by immunizing a mouse in which the immune system is replaced with a human using the method disclosed in WO2010 / 098471 by SLC6A6 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.
- human-mouse chimerized antibody of a monoclonal antibody against SLC6A6 produced by the hybridoma 204, 205, 303, 419, 402, 422 or 430 is also included in the scope of the present invention.
- human-mouse chimeric antibody 205 includes a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 24 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 26, and human-mouse chimeric antibody 402 comprises
- human-mouse chimerized antibody 419 comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 28 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 30.
- the human-mouse chimerized antibody 303 comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 36 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 34.
- a human-mouse chimerized antibody 422 comprising a light chain comprising an amino acid sequence comprises the amino acid sequence represented by SEQ ID NO: 40
- the human-mouse chimerized antibody may be a human-chimerized antibody comprising a heavy chain variable region comprising a CDR derived from a mouse antibody and a light chain variable region comprising a CDR derived from a mouse antibody.
- the monoclonal antibody of the present invention (including an antigen-binding fragment thereof) is conjugated to at least one anticancer agent, toxin, or radioisotope. Further, the monoclonal antibody of the present invention conjugated with such an anticancer agent, toxin, or radioisotope is also included in the scope of the present invention.
- An antibody conjugate containing the monoclonal antibody of the present invention and at least one anticancer agent, toxin, or radioisotope (hereinafter also referred to as the antibody conjugate of the present invention) is also included in the scope of the present invention.
- the monoclonal antibody of the present invention used in the antibody conjugate of the present invention is preferably, for example, the 204, 205, 303, 419, 402, 422 or 430 antibody or the chimerized antibody thereof, the 402 antibody or the 430 antibody or the antibody thereof. Chimerized antibodies are more preferred.
- 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 radioisotope is, 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 a toxin, wherein the monoclonal antibody of the present invention is conjugated with an anticancer agent or a toxin.
- An antibody conjugate is provided.
- the anticancer agent is preferably a chemotherapeutic agent.
- toxins include Diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A, fungi (eg, ⁇ -salcin, restrictocin), plants (eg, abrin, lysine, Modesin, Viscomine, Pokeweed), antiviral protein, saporin, gelonin, momoridine, trichosanthin, barley toxin, Aleurites fordi protein, Giantin protein, Phytolacica protein (PAPI, PAPIII, and PAP-S), Momoldi Cha Lancia (Momordica charantia) inhibitor, curcin, Sapoaonaria officinalis (sapaonaria officinalis) inhibitors, gelonin, Maitojerin, restrictocin, fenofibrate mycin, neomycin, and the tricothecenes, and the like.
- fungi eg, ⁇ -salc
- chemotherapeutic agents are chemical compounds 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 (registered trademark), Genentech / OSI Pharm.), Docetaxel (TAXOTERE (registered trademark), Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS number 51- 21-8), gemcitabine (GEMZAR (registered trademark), Lilly), PD-0325901 (CAS number 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum (II), CAS number 15663-27-1 ), Carboplatin (CAS number 41575-94-4), paclitaxel (TAXOL (registered trademark), Bristol-MyersSquibb Oncology, Princeton, NJ), trastuzumab HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclic [4.3.0] nona-2,7,
- TEMODAR registered trademark
- TEMODAL registered trademark
- Schering Plow Tamoxifen ((Z) -2- [4- (1,2-diphenylbut-1- Enyl) phenoxy] -N, N-dimethyl-ethanamide, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1 / 2, HPPD and Rapamycin is included.
- chemotherapeutic agents include DM1 (N2′-Deacetyl-N2 ′-(3-mercapto-1-oxopropyl) -maytansine), DM4 (N2′-Deacetyl-N2 ′-(4-mercapto-4- (methyl-1-oxentyl) -maytansine), MMAE (Monomethyl auristatin E), oxaliplatin (ELOXATIN (registered trademark), Sanofi), bortezomib (VELCADE (registered trademark), Millennium PUM-Pl.
- DM1 N2′-Deacetyl-N2 ′-(3-mercapto-1-oxopropyl) -maytansine
- DM4 N2′-Deacetyl-N2 ′-(4-mercapto-4- (methyl-1-oxentyl) -maytansine
- MMAE Monomethyl auristatin E
- antibiotics such as enedyne antibiotics (eg calicheamicin, calicheamicin gamma 1I and calicheamicin omega I1, eg AgnewChew Intl. Ed. Engl.
- 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 C such as mito
- 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 the aromatase enzyme, which regulate estrogen production in the adrenal gland, eg (5) -Imidazoles, aminoglutethimide, MEGASE (registered trademark) (megestrol acetate), AROMASIN (registered trademark) (exemestane, Pfizer), formestine (formestanie), fadrozole, RIVISOR (registered trademark) (zolozole ( vorozole)), F
- SERM selective estrogen
- Inhibitors such as PKC- ⁇ , Raf and H-Ras, Oblimersen (GENAENSE®, Genta Inc.);
- ribozymes such as VEGF expression inhibitors (eg ANGIOZYME®) and HER2 expression inhibitors;
- Gene therapy vaccines such as ALLOVECTIN®, LEUVECTIN® and VAXID®; PROLEUKIN® rIL-2
- a topoisomerase 1 inhibitor such as LURTOTECAN®; an anti-angiogenic agent such as ABARELIX® rmRH;
- bevacizumab AVASTIN®, Genentech
- pharmaceutically acceptable salts of the above, Acids or derivatives are included.
- 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), Tositumab C and Toxumoma C (Bx) An antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth) is included.
- SLC6A6 monoclonal antibody and human monoclonal antibody or humanized monoclonal antibody having therapeutic ability as a chemotherapeutic agent combined therewith include alemtuzumab, apolizumab, acelizumab, atolizumab, bapineuzumab, bevacizumab, bibatuzumab mertansin, cantuzumab mertansin, certolizumab-Pegoru, Shidofushitsuzumabu, Shidotsuzumabu, daclizumab, eculizumab, efalizumab, epratuzumab, Erurizumabu, Ferubizumabu, fontolizumab, gemtuzumab ozogamicin, Inotsuzumabu ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizuma
- 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.
- a reactive moiety on an antibody such as an amino group of lysine, or a cysteine group (generated by reduction of a natural disulfide bond, or engineered using molecular biological methods to add an unnatural cysteine residue ).
- the antibody is first modified with a heterobifunctional linker reagent such as SPDB, SMCC and SIAB (US Patent No. 6,913,758 and US Patent Publication No. 20050169933) to provide a reactive group.
- a heterobifunctional linker reagent such as SPDB, SMCC and SIAB (US Patent No. 6,913,758 and US Patent Publication No. 20050169933)
- the reactive linker group incorporated into the antibody is then conjugated with a cytotoxic agent that contains a reactive moiety, such as a thiol group.
- a cytotoxic agent derivative containing a thiol reactive group eg haloacetamide or maleimide
- a thiol reactive group eg haloacetamide or maleimide
- N succinimide or maleimide which reacts with the amino group or carboxyl group of the antibody.
- the antibody is reacted with an oxidant to form an antibody with an aldehyde
- the antibody with an aldehyde is reacted with a PEG maleimide / hydrazide bifunctional linker to form a thiol reactive antibody
- the thiol reactive antibody is converted to a thiol reactive antibody.
- Reacting with an activating signal generating moiety to form an antibody-signal generating moiety conjugate Reacting an antibody with an oxidant to form an antibody with an aldehyde oxidizes the glycosylation region of the antibody (such as using periodate, bromine or iodine) and an antibody with an aldehyde Forming.
- the monoclonal antibody of the present invention can be bound (conjugate) directly or indirectly with an anticancer agent, toxin or 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.
- composition of the present invention a pharmaceutical composition comprising the monoclonal antibody of the present invention (including an antigen-binding fragment) is provided.
- the monoclonal antibody of the present invention is conjugated with an anticancer agent, a toxin or a radioisotope.
- a pharmaceutical composition comprising an antibody conjugate of the invention.
- the pharmaceutical composition of the present invention containing the monoclonal antibody (including antibody conjugate) of the present invention is used for cancer treatment.
- the pharmaceutical composition is preferably 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 anticancer agent, an SLC6A6 antibody or antibody conjugate conjugated with a toxin or a radioisotope, or a pharmaceutically acceptable anticancer agent or carrier as necessary.
- An appropriate combination with SLC6A6 antibody is included.
- 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 SLC6A6 expressed in cancer cells, binds to SLC6A6 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 SLC6A6 or for labeling cancer cells with radioisotopes.
- the chemotherapeutic agent conjugated to the antibody can also be taken up by cancer cells, so that the pharmaceutical composition of the present invention 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 may further contain at least one anticancer agent.
- an anticancer 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 which the pharmaceutical composition of the present invention is administered is not particularly limited as long as it expresses SLC6A6.
- the pharmaceutical composition of the present invention can 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 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 an anticancer 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 can contain various known shaping materials and additives according to various forms as long as the effects of the active ingredient are not impaired.
- 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 in the case of an antibody
- the above-mentioned active ingredient is 1 mg per kg body weight of the application subject (patient). It is preferably ⁇ 15 mg / day, more 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 comprising the monoclonal antibody of the present invention for treating cancer, for example, colorectal cancer. If the kit of this invention contains the monoclonal antibody of this invention conjugated with the anticancer agent, the toxin, or the radioisotope, the material which comprises it will not be specifically limited. In addition to the monoclonal antibody of the present invention conjugated with an anticancer agent, toxin or radioisotope, water, buffer solution, container, syringe, instruction manual and the like may be provided. The monoclonal 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 kit of the present invention may also contain at least one additional anticancer agent. By using the kit of the present invention, cancer can be effectively treated.
- 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 is conjugated with an anticancer 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 present invention includes the monoclonal antibody of the present invention used for the treatment of cancer, and the monoclonal antibody of the present invention is conjugated with an anticancer agent, a toxin or a radioisotope.
- the invention also includes a conjugate of the invention for use in the treatment of cancer.
- DLD-1, HCT116, Colo320 and WiDr were obtained from DS Pharma.
- Caco-2, COLO201, HCT15, HT-29, LOVO, SW480, SW620, 293T, and MDA-MB231 were obtained from the American Type Culture Collection (ATCC accession number HTB-26).
- MDA-MB231, SW620, DLD-1, Colo201, and Colo320 are RPMI1640 medium (Sigma) containing 10% (v / v) serum (Hyclone), and WiDr is an E-MEM medium (Sigma).
- HCT116 is McCoy's 5A medium (Sigma)
- HT-29, LoVo, SW480, 293T and the remaining cells are DMEM medium (Sigma) at 37 ° C., 5% CO 2 so as not to exceed 80% confluence. And subculture for 48 to 72 hours.
- cells not transfected with the gene and cells transfected with the gene were each planted in a 96-well plate so as to be 80% confluent, The cells were cultured for 16 hours under 5% CO 2 .
- Anti-c-myc antibody (Santa cruz, clone 9E10) was diluted to 1 ⁇ g / mL with TBS-T (25 mM Tris, 150 mM NaCl, 0.05% (v / v) Tween 20, pH 7.4), and primary antibody was added to the immobilized plate at 100 ⁇ L per well and allowed to react at room temperature for 1 hour. Each well was washed 3 times with 200 ⁇ L TBS-T.
- TBS-T 25 mM Tris, 150 mM NaCl, 0.05% (v / v) Tween 20, pH 7.4
- anti-mouse IgG polyclonal antibody-HRP-labeled (manufactured by BETHYL) was diluted 5000 times with TBS-T. 100 ⁇ L of the diluted antibody solution was added per well and reacted at room temperature for 30 minutes. Each well was washed 3 times with 200 ⁇ L TBS-T.
- Orthophenylenediamine (manufactured by Sigma) is diluted in 20 mM phosphate-citrate buffer (pH 5.0) to a final concentration of 0.5 mg / mL, and 35% of the volume of 1/2000 of this solution.
- W / w A mixed solution to which hydrogen peroxide solution (manufactured by WAKO) was added was added as a substrate solution to each well in an amount of 100 ⁇ L and allowed to react at room temperature for 10 minutes. The reaction was stopped by adding 25 ⁇ L of 3N sulfuric acid (manufactured by WAKO).
- Chloral hydrate (manufactured by Sigma) was dissolved in physiological saline to 3.5% (w / v) to prepare a 3.5% chloral hydrate physiological saline solution. Anesthesia was performed by administering 0.2 mL of a 3.5% chloral hydrate physiological saline solution into the abdominal cavity of a 6-8 week old nude mouse (BALB / cALcl-nu / nu strain (manufactured by CLEA Japan)). The cells suspended in Matrigel were transplanted into the 4th mammary gland of the mouse using a 24G injection needle at 1 ⁇ 10 6 cells per mammary gland so as not to protrude from the mammary gland. A single mouse was transplanted into the 4th mammary glands on both the left and right sides of the torso so as to have 2 transplants.
- SLC6A6 partial protein for screening From the full-length gene of SLC6A6 introduced into the pEF6 vector described in Example 1 (2), a region of amino acid residues 143 to 216 (SEQ ID NO: 4), which is an extracellular region, was obtained.
- the encoding DNA (SEQ ID NO: 3) was subcloned into the pET32 vector. PCR was performed using the following primers. Primer sequence
- the PCR reaction was performed at 94 ° C. for 2 minutes, followed by 30 cycles of 98 ° C., 10 seconds of denaturation, 58 ° C., 30 seconds of annealing, and 68 ° C., 30 seconds of elongation.
- the gene fragment was amplified.
- the obtained amplified fragment was incorporated into a pET32 vector (Novagen) using restriction enzymes (EcoRI and BamHI) arranged on the primer.
- BL21 (DE3) (Invitrogen) was transformed with this vector, and LB medium (1% (w / v) tryptone (Sigma), 0.5% (w / v) containing 1% (w / v) glucose) was obtained.
- Buffer A (1M guanidine hydrochloride (Sigma), 10 mM DTT (Sigma), 10 mM EDTA (Sigma)) and reacted at 37 ° C. for 1 hour.
- the reaction product was slowly added to 1 L of Buffer B (50 mM Tris, 150 mM NaCl, 5% glycerol, 0.4 mM oxidized glutathione (Sigma), pH 8.5) and stirred at 4 ° C. for 18 hours.
- Buffer C 50 mM potassium phosphate buffer, 150 mM NaCl, 200 mM imidazole, pH 8.0. Dialyzed against Buffer C not containing imidazole, the purified extracellular region partial protein of SLC6A6 was obtained.
- Skim milk (GIBCO) was added, and blocking was performed at room temperature for 30 minutes After washing each well 3 times with 200 ⁇ L of TBS-T, plasma collected from the tail vein of the mouse was multiplied by 1/2000 times with TBS-T. The plate was diluted to 100 ⁇ L per well to the ELISA plate and allowed to react for 1 hour at room temperature, and each well was washed 3 times with 200 ⁇ L TBS-T.
- anti-mouse IgG polyclonal antibody-HRP-labeled (manufactured by BETHYL) was diluted 5000 times with TBS-T. 100 ⁇ L of the diluted antibody solution was added per well and reacted at room temperature for 30 minutes. Each well was washed 3 times with 200 ⁇ L TBS-T.
- Orthophenylenediamine (manufactured by Sigma) is diluted in 20 mM carbonic acid-citrate buffer (pH 5.0) to a final concentration of 0.5 mg / mL, and 35% (w / 2000) of 1/2000 of this solution. w) 100 ⁇ L of the mixed solution to which hydrogen peroxide solution (manufactured by WAKO) was added as a substrate solution was allowed to react at room temperature for 10 minutes. The reaction was stopped by adding 25 ⁇ L of 3N sulfuric acid (manufactured by WAKO). Absorption at 492 nm was measured with a plate reader (SpectraMaxPure 384, manufactured by Molecular Devices) to analyze the antibody titer and used for selection of mice used for cell fusion.
- Cell fusion Lymphocytes derived from mouse spleen were electrically fused with mouse myeloma strain P3X63-Ag8 (ATCC accession number CRL-1580).
- P3X63-Ag8 ATCC accession number CRL-1580.
- EP Buffer 0.3 M Mannitol, 0.1 mM CaCl 2 , 0.1 mM MgCl 2
- the cell density was suspended at 0.25 ⁇ 10 8 cells / mL, and fusion was performed with an electrofusion apparatus LF201 (manufactured by Nepagene). The fusion conditions followed the manufacturer's recommended method.
- the fused cells were suspended in HAT medium (manufactured by Invitrogen) and spread on 30 96-well plates so that each well would be 100 ⁇ L. On the way, 200 ⁇ L of HAT medium was added, and when observed under a microscope after culturing for 11 to 16 days, 5 to 12 colonies were formed per well.
- HAT medium manufactured by Invitrogen
- the obtained hybridoma cells producing the monoclonal antibody are cultured to 10% 10 cm dish so as to be 90% confluent, and HT medium (manufactured by Invitrogen) and EX CELL Sp2 / 0 (manufactured by Nichirei Bioscience) are 1: 1. Culturing was carried out for 10 days in the medium mixed in the above. The culture supernatant was collected and purified using a Protein G column. A 0.5 mL Protein G column (GE Healthcare) was used for 100 mL of the culture supernatant.
- the hybridoma “4B9b” producing the antibody “4B9b” was dated July 21, 2010 as the accession number “FERM BP-11413” by Biomatrix Laboratories Co., Ltd. (105 Higashifui, Nagareyama, Chiba Prefecture 270-0101). Therefore, it is deposited internationally in accordance with the Budapest Treaty at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center ( ⁇ 305-866, 1-1-1 Higashi 1-1-1 Tsukuba City, Ibaraki Prefecture). In addition, the hybridoma “5H12d” producing “5H12d” was received from Biomatrix Laboratories Co., Ltd.
- the H chain expression vector and the L chain expression vector for expressing the mouse IgG antibody “4B9b” were obtained by the method described in PCT / JP2013 / 56884.
- the H chain expression vector and the L chain expression vector for expressing the “mouse-human chimeric 4B9b antibody” converted to human IgG were those obtained by the method described in PCT / JP2013 / 56884.
- HT-29 and LoVo were cultured to be 80% confluent, and seeded on a cover glass coated with Cellmatrix Type IA (Nitta Gelatin). After culturing for 2 days, the cells were fixed with 10% neutral buffered formalin (WAKO). The cover glass was treated with 0.3% (v / v) hydrogen peroxide solution for 20 minutes, washed with TBST three times, treated with TBST containing 5% (w / v) Skim milk, and then (7 ) Was added at 10 ⁇ g / mL and reacted at 4 ° C. for 16 hours.
- FIG. 1 shows the result of analyzing the fluorescence intensity for each antibody under the same conditions.
- a signal was observed in all clones (204, 205, 303, 419, 402, 422 and 430), in particular a clear signal was observed in clones 204, 205, 303, 419 and 402.
- the signals of clones 205, 303, and 419 were clearly high, indicating that the affinity was high. According to this example, a clone having a higher titer than the conventional SLC6A6 antibody was obtained.
- FACS analysis 293T which is a human embryonic kidney cell was cultured to become 90% confluent. The cells were washed twice with PBS ( ⁇ ), peeled off with a scraper, and collected in a 1.5 mL tube. Antibodies 204, 205 and 419 were added to the tube to a final concentration of 10 ⁇ g / mL, and reacted for 60 minutes. For comparison, 4B9b was added at the same concentration and allowed to react. After washing the cells twice with PBS ( ⁇ ) supplemented with 2% FBS (PBS + 2% FBS), AlexaFluor 488-labeled Goat-Anti-mouse IgG (Invitrogen) was diluted 1/1000 with PBS + 2% FBS and added. , Reacted for 30 minutes. After washing twice with PBS + 2% FBS, analysis was performed with FACS Verse (BD). The results are shown in FIG.
- antibodies 204, 205 and 419 reacted more strongly with 293T cells than 4B9b. Since 293T cells express SLC6A6, it was shown that the SLC6A6 antibody of the present invention recognizes the native structure of SLC6A6. It was also shown that the SLC6A6 antibody of the present invention can bind to native SLC6A6 with higher affinity than the conventional SLC6A6 antibody.
- FIG. 3 shows that antibody clones 204, 205, 303, 419, 402, 422, and 430 react clearly with human colon cancer cell HCT116.
- FIG. 4 shows that the antibody clones 205, 419, 402 and 430 antibodies clearly react to two types of cell lines.
- Example 3 When a monoclonal antibody is used as an ADC that is a conjugate with an anticancer agent, a toxin, a radioisotope, or the like, an antibody that recognizes the native structure of the protein more strongly is preferable. This is because cells exist in a living state in cancer tissue, and thus SLC6A6 expressed by cancer cells also has a native structure. That is, the FACS analysis of Example 3 would be a more appropriate method for selecting antibody clones suitable for ADC than the method shown in Example 1 (5) or (8).
- FIG. 5 shows the results (representative examples) of staining colon cancer tissue and normal tissue (normal tissue).
- ++ is indicated as being strongly positive
- ++ is indicated as being positive
- + is indicated as weak positivity that is distinguished from normal
- ⁇ is indicated when not being stained or normal tissue.
- the case where judgment was not made was described as +/-.
- Table 2 summarizes the origin and results of the samples used in Example 4.
- FIG. 7 shows that the fluorescence generated by binding of antibody clone 402 to HCT116 disappears when the extracellular region partial protein of SLC6A6 is added. This indicates that antibody clone 402 reacts with the extracellular region of SLC6A6. That is, it was shown that the antibody clone 402 reacts specifically with the extracellular region of SLC6A6.
- the target gene was amplified by performing PCR reaction using the obtained cDNA as a template and the following primers and using KOD PLUS (TOYOBO).
- PCR reaction (94 ° C., 2 minutes of denaturation was performed using primers of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, followed by 56 ° C., Fragments were obtained by 15 cycles of annealing, 68 ° C, 45 seconds elongation cycle (30 cycles).
- a PCR reaction (94 ° C., 2 minutes denaturation was performed using primers of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10, A fragment was obtained by 30 cycles of annealing at 56 ° C. for 15 seconds and elongation at 68 ° C. for 45 seconds.
- the purified PCR amplified fragment is mixed with 10 mM dNTP Mix (Invitrogen) and 2 ⁇ GoTaq Maxter Mix (Promega), reacted at 70 ° C. for 15 minutes, and then ice-cooled at 4 ° C. for 2 minutes to the 3 ′ end. dA was added. Thereafter, the H chain fragment and the L chain fragment were cloned by a so-called TA cloning method using pGEM-T-Easy Vector System (Promega).
- H chain variable region fragment of the 205 antibody was introduced into pEF6-hHchain-cloning
- the primers of SEQ ID NO: 16 and SEQ ID NO: 17 were used using the H chain variable region fragment cloned by the TA cloning method as a template.
- H chain variable region fragments were obtained by PCR reaction (94 ° C, 2 minutes of denaturation, followed by 30 cycles of 56 ° C, 15 seconds of annealing, 68 ° C, 45 seconds of elongation). .
- the amplified heavy chain variable region fragment was introduced into pEF6-hHchain-cloning using restriction enzymes (MluI / NheI) (hereinafter referred to as 205 chimeric heavy chain expression vector).
- the amplified human kappa chain constant region was introduced into the pEF1-myc / HisA vector (Invitrogen) using restriction enzymes (EcoRI / NotI). (This vector is called pEF1-hLchain-cloning.)
- the L chain signal sequence and variable region fragment cloned by TA cloning method were used as templates, and SEQ ID NO: 21 and SEQ ID NO: PCR was performed with 22 primers (94 ° C, 2 minutes of denaturation followed by 58 ° C, 15 seconds of annealing, 68 ° C, 30 seconds of 30 cycles). An L chain signal sequence and a variable region fragment were obtained.
- the amplified L chain signal sequence and variable region fragment were introduced into pEF1-hLchain-cloning using restriction enzymes (BamHI / BsiWI) (this is called 205 chimeric L chain expression vector).
- the DNA sequence of the H chain of the human-mouse chimerized 205 antibody is represented by SEQ ID NO: 23, and the amino acid sequence is represented by SEQ ID NO: 24. The same applies to the L chain.
- the DNA sequence is represented by SEQ ID NO: 25, and the amino acid sequence is represented by SEQ ID NO: 26.
- human-mouse chimeric antibodies were prepared for other antibodies.
- the DNA sequence of the H chain of the human-mouse chimerized 402 antibody is represented by SEQ ID NO: 27 and the amino acid sequence is represented by SEQ ID NO: 28. The same applies to the L chain.
- the DNA sequence is represented by SEQ ID NO: 29, and the amino acid sequence is represented by SEQ ID NO: 30.
- the DNA sequence of the H chain of the human-mouse chimerized 419 antibody is represented by SEQ ID NO: 31, and the amino acid sequence is represented by SEQ ID NO: 32. The same applies to the L chain.
- the DNA sequence is represented by SEQ ID NO: 33, and the amino acid sequence is represented by SEQ ID NO: 34.
- the DNA sequence of the H chain of the human-mouse chimeric antibody 303 is represented by SEQ ID NO: 35, and the amino acid sequence is represented by SEQ ID NO: 36.
- the DNA sequence is represented by SEQ ID NO: 37, and the amino acid sequence is represented by SEQ ID NO: 38.
- the DNA sequence of the H chain of the human-mouse chimerized 422 antibody is represented by SEQ ID NO: 39, and the amino acid sequence thereof is represented by SEQ ID NO: 40.
- the DNA sequence is represented by SEQ ID NO: 41, and the amino acid sequence is represented by SEQ ID NO: 42.
- the H-chain DNA sequence of the human-mouse chimerized 430 antibody is represented by SEQ ID NO: 43, and the amino acid sequence is represented by SEQ ID NO: 44.
- the DNA sequence is represented by SEQ ID NO: 45, and the amino acid sequence is represented by SEQ ID NO: 46.
- variable regions and the amino acid sequences of CDR1 to CDR3 of each human-mouse chimeric antibody obtained above is shown in Tables 3 and 4 below.
- the numbers described in the variable region and CDRs 1 to 3 indicate the corresponding amino acid number in the amino acid sequence represented by the SEQ ID NO.
- humanized IgG antibody was expressed by the following method. Expression of the antibody protein was performed using Expi293F Expression system (Life technologies). Using a 125 mL Erlenmeyer flask with a vent cap (Corning), 30 mL of a cell solution prepared to 2 ⁇ 10 6 cells / mL was subjected to swirl culture (125 rpm) at 37 ° C. and 8% CO 2 for 24 hours. Next, 81 ⁇ L of Expifectamine 293 reagent was mixed with 1.419 mL of Optimem (GIBCO), allowed to stand for 5 minutes, and then 15 ⁇ g each of the plasmids in which the genes encoding the antibody H chain and L chain genes were cloned.
- Expi293F Expression system Life technologies. Using a 125 mL Erlenmeyer flask with a vent cap (Corning), 30 mL of a cell solution prepared to 2 ⁇ 10 6 cells / mL was subjecte
- the 293F cells prepared above were resuspended in 25.5 mL of medium so that the concentration was 2.9 ⁇ 10 6 cells / ml, and 3 mL of the plasmid solution was added to start the culture. 20 hours after the start of the culture, 150 ⁇ L of Expifectine 293 transfection enhancer 1 and 1.5 mL of enhancer 2 were added and cultured for 3 days, and the culture supernatant was collected.
- Hydroxyapatite carrier was equilibrated with 10 mM phosphate buffer (10 mM Na 2 HPO 4 , 10 mM NaH 2 PO 4 (pH 6.7)), and 36 mL of 10 mM phosphate buffer with respect to 12 mL of the culture supernatant containing antibody protein. was added. To this diluted culture supernatant, an equilibrated hydroxyapatite carrier was added and stirred for 18 hours at 4 ° C. The carrier is packed in an Econopack column (Bio-Rad), washed with 10 volumes of 10 mM phosphate buffer, and then 50, 300, 500, 700, 800, 1000, 1300 mM in 10 mM phosphate buffer. Separation was carried out with a solution to which sodium chloride was added. More than 95% of the protein eluted between sodium chloride concentrations of 500 to 700 mM was antibody protein.
- the binding activity was measured in the same manner as in Example 1 (7) using 205 antibody as an example. At this time, the antibody concentration was 10 ⁇ g / mL.
- anti-mouse IgG polyclonal antibody-HRP label was used in the case of 205 antibody, and anti-human IgG polyclonal antibody-HRP label was used in the case of a chimeric antibody.
- SLC6A6 in HCT15 and HT-29 cells which are human colon cancer cells, was preserved even when the 205 antibody was a human-mouse chimerized antibody.
- SP cells Side population cells have attracted attention as a method for separating and identifying them.
- Stem cells have a high drug efflux ability and have a characteristic of releasing various substances to the outside of the cell, and a technique of analyzing the discharge of Hoechst 33342 as an index using the characteristics of this stem cell is widely used. In this technique, the cell fraction with weak color development in the Hoechst 33342 staining pattern is called SP cells. It is clear that such SP cells also exist for cancer cells, and it has been reported that SP cells are clearly higher in tumorigenicity than non-SP cells, and the fraction of cancer stem cells is concentrated. (Cancer Research 2005, 65, p. 6207-6219).
- Example 8 (1) After introduction of the gene by the method described in Example 8 (1) above, the cells were detached using DISSOCIATION BUFFER (Invitrogen) on the second day. Cells were stained with a solution prepared to be 10 ⁇ g / mL Hoechst 33342 in PBS ( ⁇ ) at 37 ° C. for 1 hour, and analyzed using a Violet laser (excitation wavelength: 407 nm) using FACS Canto II. In addition, in order to identify the SP fraction, an experiment was conducted in which Verapamil, an inhibitor of drug excretion transporter, was added at 30 ⁇ g / mL. The results are shown in FIG.
- FIG. 8 shows the result that the proportion of SP cells is increased when SLC6A6 is transiently expressed (“SLC6A6”) compared to the control vector (“Mock”). That is, it was shown that SLC6A6 is directly involved in an increase in the SP fraction in which the stem cell population is enriched.
- SLC6A6 expression of SLC6A6 was analyzed with respect to the SP fraction and non-SP fraction (major population (MP) fraction) of SW480 cells, which are human colon cancer cells.
- Cells were stained with Hoechst 33342 in the same manner as described in Example 8 (2), and then 1 ⁇ g / mL 430 antibody purified with Protein G and eluted with pH 3.0 eluate and cells were stained at 4 ° C. for 1 hour. After staining with anti-mouse IgG polyclonal antibody AlexaFluor 488 label as a secondary antibody, analysis was performed using FACS Canto II. The result is shown in FIG.
- FIG. 9 shows that when SP and MP cells are stained with SLC6A6, SP cells are stained more strongly with antibodies than MP cells. That is, it was shown that SP cells expressed more SLC6A6 than MP cells. When no antibody was added, no signal shift was observed in both SP cells and MP cells, indicating that 430 antibody specifically reacts with SLC6A6. Furthermore, since SP cells were not seen when verapamyl was added, it was confirmed that the fraction separated as SP cells was SP cells.
- SLC6A6 expression is directly involved in the formation of SP cells, a population enriched for cancer stem cells.
- SLC6A6 has been reported to be a transporter that transports taurine (FEBS Letter 1993, 318, p. 139-144). Therefore, it was analyzed whether the proportion of SP cells in the cell population changes when taurine is allowed to act on cells overexpressing SLC6A6.
- Colo320 cells which are human colon cancer cells, were overexpressed with SLC6A6, and 0.05 and 0.1, 0.5, 1 and 5 mM of taurine were added to the medium and cultured for 2 days. Then, SP cells were analyzed by the method described in Example 1 (3). The results are shown in FIG.
- FIG. 10 shows that the number of SP cells increases depending on the addition concentration of taurine in both Colo320-Mock and Colo320-SLC6A6-OEx that stably express SLC6A6. Show.
- the effect of taurine is particularly pronounced with Colo320-SLC6A6-OEx, the proportion of which is about 2.5 times (17.0% to 26%) when 5 mM taurine is added compared to when it is not added (0 mM). 0.7%). That is, when the expression of SLC6A6 is enhanced, taurine uptake increases, or SP cells increase as the concentration of taurine increases. Since SP cells are a fraction in which cancer stem cells are concentrated, the possibility that SLC6A6 is involved in stem cell transformation of cancer cells was shown.
- HT-29 or SW480 was cultured so as to be 90% confluent, and seeded on Glass Base Dish. The cells were cultured for 24 hours, washed with ice-cold PBS, and reacted with 10 ⁇ g / ml of 419, 402, or 430 antibody purified at pH 3.0 after expression for 1 hour at 4 ° C. The cells were washed with PBS, cultured at 4 ° C. or 37 ° C. for 20 minutes, and then fixed with 10% neutral buffered formalin for 10 minutes.
- the cells after the reaction were reacted with an anti-mouse IgG polyclonal antibody-AlexaFluor488 labeled 500-fold diluted at room temperature for 30 minutes, and then analyzed with a confocal laser microscope (Zeiss). The results are shown in FIG. 11A.
- FIG. 11A shows that the cell membrane is stained when the reaction is performed at 4 ° C., that is, under the condition that the cell activity is stopped, the intracellular transport mechanism does not function, and the internalization does not occur. This result indicates that the antibody is present on the cell membrane. On the other hand, when internalization was caused at 37 ° C., it was shown that the cell membrane was not stained. This result indicates that the antibody bound to the cell surface has migrated into the cell by internalization. Since the antibody taken up into the cells was collected in endosomes and lysosomes, a dark spot-like signal was observed in the cells.
- HCT116 cells which are human colon cancer cells, or SK-BR3, which is human breast cancer cells, are added to a glass bottom dish (MATSANAMI) at 1 ⁇ 10 4 cells / well, and the cells are added at 37 ° C., 5% CO 2. 2 and cultured for 16 hours. After removing the medium, a cooled medium was added on ice and allowed to stand on ice for 20 minutes.
- the primary antibody prepared in the medium so that the 402 human-mouse chimeric antibody purified with Protein G and eluted with an eluate of pH 3.0 was added to 20 ⁇ g / mL was added and reacted on ice for 30 minutes.
- the reaction was allowed to proceed on ice for 30 minutes with anti-human IgG polyclonal antibody-AlexaFluor488 label as a secondary antibody.
- the cells were cultured at 37 ° C. under 5% CO 2 for 0, 15, 30, 60, 120 minutes, washed twice with PBS ( ⁇ ), and then washed with a fluorescence microscope (BZ8100, Keyence). And analyzed. The result is shown in FIG.
- FIG. 11B shows that in HCT116 or SK-BR3, the antibody is taken up into cells over time. At 0 minutes, that is, immediately after staining, most of the antibody was bound on the cell membrane, but after 15 minutes, it was taken up into the cell, and after 60 minutes, most of the antibody was taken up into the cell. That is, it was confirmed that the 402 human-mouse chimeric antibody was taken up into cells after binding to SLC6A6 on the cell surface.
- 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. 12 shows that pretreatment with ConA inhibits the 402 human-mouse chimeric antibody internalization during the observed period.
- the left panel shows the change over time when ConA is not included, and the right panel shows the results when ConA is included.
- 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 402 human-mouse chimeric antibody bound to SLC6A6 on the cell membrane was taken up into the cells by clathrin-dependent endocytosis.
- Alexa Fluor 488-labeled, isotype control using Whole human IgG as negative control
- cetuximab cetuximab
- 402 human-mouse chimeric antibody chimera 402
- 430 human-mouse chimeric antibody chimera 430
- 4B9b human -Internalization was analyzed by flow cytometry using cells treated by the following two methods for mouse chimeric antibody (chimera 4B9b).
- the cell obtained by the first method was treated with Alexa.
- An antibody that binds to Fluor 488 and quenches fluorescence (Anti-Alexa Fluor 488, rabbit IgG antibody fraction: anti-Alexa Fluor 488 antibody) is allowed to react at a final concentration of 50 ⁇ g / mL at 4 ° C. for 1 hour to observe changes in fluorescence intensity. (FIG. 13B; surface quenching).
- FOG. 13B surface quenching
- FIG. 13A shows that the directly labeled 402 human-mouse chimeric antibody (c402) is taken up into cells even on ice.
- c402 human-mouse chimeric antibody c402
- FIG. 13A shows that the directly labeled 402 human-mouse chimeric antibody (c402) is taken up into cells even on ice.
- Alexa Fluor 488 fluorescence was found inside the cells.
- Alexa Fluor 488 fluorescence remained on the cell membrane surface. From this, it was confirmed that the internalization of the 402 human-mouse chimeric antibody proceeds even on ice (about 1 ° C.).
- FIG. 13B the internalization was analyzed by measuring by flow cytometry.
- the signal of isotype control was reduced to the signal of unstained cells by addition of anti-Alexa Fluor 488 antibody (surface quenching). This indicates that the fluorescence on the cell surface is quenched by the Anti-Alexa488 antibody.
- cetuximab registered trademark Arbitux
- cetuximab which is an anti-EGFR antibody and has been eagerly studied to bind to drugs and to be incorporated into cancer cells by internalization, reacting with anti-Alexa Fluor 488 antibody (surface quenching) ) Part of the fluorescence disappeared.
- the disappearance of fluorescence due to the addition of the anti-Alexa Fluor 488 antibody was not observed.
- the anti-SLC6A6 monoclonal antibody of the present invention is not simply bound to SLC6A6 on the cell surface, but is internalized in the cell, and more antibodies than cells in the case of cetuximab. It means that it is taken in.
- 402 and 430 human-mouse chimeric antibodies were taken up more intracellularly than the 4B9b human-mouse chimeric antibody.
- the antibody conjugate of the present invention has a greater cytotoxic activity. It is expected to show. Therefore, it is assumed that the antibody conjugate of the present invention and the composition containing the conjugate have a large effect as a pharmaceutical product (for example, a pharmaceutical product for cancer treatment).
- anti-ERBB2 antibody (Trastuzumab), which has been analyzed as an antibody to be internalized, has not been reported to be taken up into cells when reacted on ice. Therefore, the internalization of 402 human-mouse chimeric antibody is extremely It is thought that it is progressing efficiently. That is, it is considered that cancer cells can be killed even with a low concentration of antibody-drug conjugate due to the very good efficiency of incorporation.
- Saporin has ribosome-inactivating activity and suppresses cell growth only when it is taken into cells. These cells were cultured at 37 ° C. for 3 days, and then cell proliferation was measured using WST-8 (Roche).
- FIG. 14A shows the result.
- FIG. 14A black indicates cell growth when no saporin-labeled secondary antibody is contained, and gray indicates cell growth when saporin antibody is added to 10 nM.
- FIG. 14A shows that cell growth is suppressed by both HT-29 and SW480 when a saporin-labeled secondary antibody is added. In other words, it was shown that anti-mouse antibody conjugated with saporin, a ribosome-inhibiting toxin, was reacted with mouse monoclonal antibody, and these antibodies were taken up into cells, thereby suppressing the growth of cancer cells. .
- HCT116 cells which are human colon cancer cells, were seeded on 96-well plates at 2.5 ⁇ 10 3 cells / well and cultured at 37 ° C. under 5% CO 2 for 16 hours.
- a mixed solution prepared so as to be a mouse antibody having a final concentration of 0.1 or 0.3 ⁇ M and a 9 nM saporin-labeled anti-mouse IgG polyclonal antibody was added.
- Saporin has a ribosome inactivating activity and suppresses cell growth only when it is taken into cells. These cells were cultured at 37 ° C. under 5% CO 2 for 3 days, and then cell proliferation was measured using Cell Counting Kit-8 (Dojindo Laboratories). FIG. 14 shows the result.
- FIG. 14 shows that proliferation is suppressed in an antibody concentration-dependent manner only when mouse 402 and 430 antibodies are added to HCT116 cells. That is, it is shown that anti-mouse antibody conjugated with saporin, a ribosome-inhibiting toxin, reacts with mouse 402 and 430 antibodies, and these antibodies are taken into cells, thereby suppressing the growth of cancer cells. It was done. This result shows that the use of a conjugate obtained by binding a drug to the 402, 430 antibody has high cytotoxic activity, and it is expected to have a high antitumor effect when used as a pharmaceutical product.
- SEQ ID NO: 1 A nucleotide sequence encoding human SLC6A6.
- SEQ ID NO: 2 amino acid sequence of human SLC6A6.
- SEQ ID NO: 3 A nucleotide sequence encoding the amino acid sequence of amino acid residues 143 to 216 of human SLC6A6.
- SEQ ID NO: 4 amino acid sequence of amino acid residues 143 to 216 of human SLC6A6 SEQ ID NO: 5: Primer sequence, Forward SEQ ID NO: 6: Primer sequence, Reverse SEQ ID NO: 7: Primer sequence, Long SEQ ID NO: 8: Primer sequence, Short SEQ ID NO: 9: primer sequence, mIgG2a_CH1_reverse SEQ ID NO: 10: primer sequence, mIgkappa_R3 SEQ ID NO: 11: oligo DNA sequence, hHchain SEQ ID NO: 12: primer sequence, hCg1_F SEQ ID NO: 13: primer sequence, hCgI_R SEQ ID NO: 14: primer sequence, Hchain_signal_top SEQ ID NO: 15: primer sequence, Hchain_signal_bottom SEQ ID NO: 16: primer sequence, Forward SEQ ID NO: 17: primer sequence, Reverse SEQ ID NO: 18: oligo DNA sequence, hLchain SEQ ID NO: 19: primer sequence,
- the present invention provides a monoclonal antibody that specifically binds to the extracellular region of SLC6A6.
- the present invention also provides an antibody conjugate comprising an anti-SLC6A6 monoclonal antibody or antigen-binding fragment bound to at least one anticancer agent, toxin or radioisotope, and a pharmaceutical composition comprising the antibody conjugate.
- the monoclonal antibody of the present invention specifically binds to and internalizes cancer cells expressing SLC6A6, the antibody conjugate and the pharmaceutical composition of the present invention are used for cancer treatment, particularly for colorectal cancer treatment. Useful as a product. Since the antibody may be humanized or chimerized, side effects when applied to humans can be reduced.
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Abstract
Description
[1] SLC6A6の細胞外領域内の立体構造をとるエピトープに結合するモノクローナル抗体またはその抗原結合断片を含む、がん治療用医薬組成物。
[2] 前記モノクローナル抗体は、抗がん剤、トキシンまたはラジオアイソトープがコンジュゲートされているものである、[1]に記載の医薬組成物。
[3] SLC6A6の細胞外領域内の立体構造をとるエピトープに結合するモノクローナル抗体またはその抗原結合断片が、抗がん剤、トキシンまたはラジオアイソトープとコンジュゲートされた抗体コンジュゲートを含む、がん治療用医薬組成物。
[4] 前記エピトープが、以下の(a)~(c)から選択される少なくとも1つに示されるポリペプチドを含む、[1]~[3]のいずれか1つに記載の医薬組成物:
(a)配列番号4に示されるアミノ酸配列からなるポリペプチド
(b)配列番号4に示されるアミノ酸配列において1個もしくは数個のアミノ酸が置換、欠失および/または挿入されたアミノ酸配列からなり、かつ、SLC6A6の細胞外領域として機能するポリペプチド
(c)配列番号4に示されるアミノ酸配列に70%以上の相同性を有するアミノ酸配列からなり、かつ、SLC6A6の細胞外領域として機能するポリペプチド。
[5] モノクローナル抗体が、
(a)配列番号24の50~54番目、69~86番目および118~125番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号26の44~54番目、70~76番目および109~116番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体、
(b)配列番号28の50~54番目、69~86番目および118~126番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号30の46~57番目、73~79番目および112~119番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体、
(c)配列番号32の50~54番目、69~86番目および118~128番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号34の48~58番目、74~80番目および113~120番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体
(d)配列番号36の50~54番目、69~86番目および118~131番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号38の44~54番目、70~76番目および109~116番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体、
(e)配列番号40の50~54番目、69~86番目および118~129番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号42の44~54番目、70~76番目および109~116番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体、および
(f)配列番号44の50~54番目、69~86番目および118~126番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号46の46~57番目、73~79番目および112~119番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体、
からなる群から選択される少なくとも1つである、[1]~[4]のいずれか1つに記載の医薬組成物。
[6] 前記モノクローナル抗体が、[5]に記載のモノクローナル抗体又はその断片が認識するエピトープに結合するものである、[1]~[4]のいずれか1つに記載の医薬組成物。
[7] 前記モノクローナル抗体が、キメラ化抗体又はヒト化抗体である、[1]~[6]のいずれか1つに記載の医薬組成物。
[8] 上記トキシンが、ジフテリアトキシン、シュードモナスエンドトキシンおよびエキソトキシン、スタフィロコッカスエンテロトキシンA、真菌、植物、抗ウィルス性タンパク質、サポリン、ゲロニン、モモリジン、トリコサンチン、大麦トキシン、アレウリテス・フォーディタンパク質、ジアンチンタンパク質、フィトラッカ・アメリカーナタンパク質、モモルディカ・チャランチア阻害剤、クルシン、クロチン、サポアオナリア・オフィシナリス阻害剤、ゲロニン、マイトジェリン、レストリクトシン、フェノマイシン、ネオマイシン、およびトリコテセンからなる群から選択される少なくとも一つである、[1]~[7]のいずれか1つに記載の医薬組成物。
[9] 前記抗がん剤が、大腸がん、乳がん、または子宮がんを治療するための薬剤である、[1]~[7]のいずれか1つに記載の医薬組成物。
[10] 大腸がん、乳がん、または子宮がんを治療するための医薬組成物である、[1]~[9]のいずれか1つに記載の医薬組成物。
[11] 少なくとも1つの抗がん剤をさらに含む、[1]~[10]のいずれか1つに記載の医薬組成物。
(1)本発明のモノクローナル抗体
本発明の医薬組成物に使用されるモノクローナル抗体(以下「本発明のモノクローナル抗体」ともいう)は、ネイティブなSLC6A6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。
変異型ポリペプチドと本発明のモノクローナル抗体との結合は、ELISA、免疫沈降、ウエスタンブロッティングなどにより確認することができる。
「mouse−mouse hybridoma 204」(以下「204」という)、
「mouse−mouse hybridoma 205」(以下「205」という)、
「mouse−mouse hybridoma 303」(以下「303」という)、
「mouse−mouse hybridoma 419」(以下「419」という)
「mouse−mouse hybridoma 402」(以下「402」という)、
「mouse−mouse hybridoma 422」(以下「422」という)、および「mouse−mouse hybridoma 430」(以下「430」という)である。本発明は、当該ハイブリドーマおよびこれらが産生する抗体を提供する。当該ハイブリドーマを培養することにより、均質なモノクローナル抗体を製造することができる。
また、205の産生する抗体は、配列番号24の50~54番目、69~86番目および118~125番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号26の44~54番目、70~76番目および109~116番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む。
また、402の産生する抗体は、配列番号28の50~54番目、69~86番目および118~126番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号30の46~57番目、73~79番目および112~119番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む。
また、419の産生する抗体は、配列番号32の50~54番目、69~86番目および118~128番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号34の48~58番目、74~80番目および113~120番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む。
また、205の産生する抗体は、配列番号36の50~54番目、69~86番目および118~131番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号38の44~54番目、70~76番目および109~116番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む。
また、205の産生する抗体は、配列番号40の50~54番目、69~86番目および118~129番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号42の44~54番目、70~76番目および109~116番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む。
また、205の産生する抗体は、配列番号44の50~54番目、69~86番目および118~126番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号46の46~57番目、73~79番目および112~119番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む。
重鎖可変領域が、配列番号24の21~135番目のアミノ酸配列、配列番号28の21~136番目のアミノ酸配列、配列番号32の21~138番目のアミノ酸配列、配列番号36の21~141番目のアミノ酸配列、配列番号40の21~139番目のアミノ酸配列または配列番号44の21~136番目のアミノ酸配列と、少なくとも75、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98または99%の同一性を有するアミノ配列を含み、
軽鎖可変領域が、配列番号26の21~127番目のアミノ酸配列、配列番号30の23~130番目のアミノ酸配列、配列番号34の25~131番目のアミノ酸配列、配列番号38の21~127番目のアミノ酸配列、配列番号42の21~127番目のアミノ酸配列または配列番号46の23~130番目のアミノ酸配列と、少なくとも75、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98または99%の同一性を有するアミノ配列をそれぞれ含む抗体であってもよい。
重鎖可変領域が、配列番号24の50~54番目、69~86番目および118~125番目のアミノ酸配列、配列番号28の50~54番目、69~86番目および118~126番目のアミノ酸配列、配列番号32の50~54番目、69~86番目および118~128番目のアミノ酸配列、配列番号36の50~54番目、69~86番目および118~131番目のアミノ酸配列、配列番号40の50~54番目、69~86番目および118~129番目のアミノ酸配列または配列番号44の50~54番目、69~86番目および118~126番目のアミノ酸配列と少なくとも80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98または99%の同一性を有するアミノ配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含み、
軽鎖可変領域が、配列番号26の44~54番目、70~76番目および109~116番目のアミノ酸配列、配列番号30の46~57番目、73~79番目および112~119番目のアミノ酸配列、配列番号34の48~58番目、74~80番目および113~120番目のアミノ酸配列、配列番号38の44~54番目、70~76番目および109~116番目のアミノ酸配列、配列番号42の44~54番目、70~76番目および109~116番目のアミノ酸配列または配列番号46の46~57番目、73~79番目および112~119番目のアミノ酸配列と、少なくとも80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98または99%の同一性を有するアミノ配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む抗体であってもよい。
(1)抗原として膜タンパク質を調製する際に、界面活性剤を使用すると膜タンパク質の立体構造が崩れ、その一方、界面活性剤を使用しない場合は膜タンパク質が疎水性領域同士で凝集する、
(2)細胞表面の発現量が低い、または細胞外領域が小さいために免疫応答が起こらない、
などの理由による。
本発明のモノクローナル抗体は、本発明者らが抗体の製法(特に免疫方法)に工夫を施した結果、従来の抗体に対して優れた特徴を獲得したものである。
本発明の抗体は、SLC6A6が本来有する立体構造をもとに作製された故に、ネイティブなSLC6A6を認識することができる。そのため、同じエピトープを認識する従来の抗体(HPA015028)と比較して結合力が非常に高く、従来の抗体では困難であった細胞膜上のSLC6A6と十分に結合することができる。また、SLC6A6の膜内および細胞内の領域を認識する従来のモノクローナル抗体(sc−166640)に対して、本発明の抗体の認識部位は細胞外であるため、生きた細胞に結合することができ、治療薬あるいは抗がん剤のキャリアとして利用することが可能である。
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種類の抗がん剤、トキシン、またはラジオアイソトープにコンジュゲートされる、このような抗がん剤、トキシン、またはラジオアイソトープでコンジュゲートされた本発明のモノクローナル抗体も本発明の範囲に含まれる。また、本発明のモノクローナル抗体および少なくとも1種類の抗がん剤、トキシン、またはラジオアイソトープを含む抗体コンジュゲート(以下、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。本発明の抗体コンジュゲートに使用される本発明のモノクローナル抗体は、例えば、204、205、303、419、402、422もしくは430抗体またはそれらのキメラ化抗体が好ましく、402抗体もしくは430抗体またはそれらのキメラ化抗体がより好ましい。
本発明の抗体コンジュゲートにおいて、好ましい抗がん剤は、5−FU、ドキソルビシン(Dxosorubicin)、またはSN−38である。
本発明において、本発明のモノクローナル抗体(抗原結合断片を含む)を含む医薬組成物が提供される。ここで、前述のように、本発明のモノクローナル抗体は、抗がん剤、トキシンまたはラジオアイソトープとコンジュゲートされている。したがって、本発明の別の態様において、本発明の抗体コンジュゲートを含む、医薬組成物が提供される。
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。
(1)細胞
本願の実施例においては、DLD−1、HCT116、Colo320及びWiDrはDSファーマより入手した。Caco−2、COLO201、HCT15、HT−29、LOVO、SW480、SW620、293T、およびMDA−MB231はAmerican Type Culture Collection(ATCC受託番号 HTB−26)より入手した。
WO2012/029990公報実施例2に記載されたのと同じ方法で、pEF6ベクターにヒトSLC6A6遺伝子を組み込んだ発現用ベクターを作製した。このSLC6A6発現ベクターを、FUGENE6(ロシュ社製)を用いてMDA−MB231へ導入した。操作は当該キットに添付された説明書の記載に基づいて行った。ブラストサイジンS塩酸塩が10μg/mLとなるよう培地に添加し、3~5日毎に培地を交換して薬剤耐性を持つ細胞を選択した。得られた耐性株の中からSLC6A6を過剰発現している細胞を選び出すため、遺伝子導入を行っていない細胞、及び遺伝子導入した細胞それぞれを80%コンフルエントとなるよう96ウエルプレートに植え、37℃、5%CO2下で16時間培養した。
10cmシャーレに90%コンフルエントになるまで培養した細胞を、トリプシン(GIBCO社)を用いて回収し、PBS(−)(0.01M sodium−phosphate buffer、0.138M NaCl、0.0027M KCl、pH 7.4)で2回洗浄した。洗浄後の細胞を最終濃度が8.6×107細胞/mLとなるようグロースファクターリデューストマトリゲル(Becton Dickinson社製)に懸濁し、移植するまで氷上で保存した。
実施例1(2)に記載のpEF6ベクターに導入したSLC6A6全長遺伝子から、細胞外領域であるアミノ酸残基143~216の領域(配列番号4)をコードするDNA(配列番号3)をpET32ベクターにサブクローニングした。以下のプライマーを用いてPCRを行なった。
プライマー配列
上記(4)で得た組換えタンパク質10μg/mlを100μLずつ、MaxiSorp 96 wellプレート(ヌンク社)に入れ、室温で1時間プレートに吸着させた。吸着後、ウエルをTBS−T(TBS−T(25mM Tris、150mM NaCl、0.05%(v/v)Tween20、pH 7.4)で洗浄した後、5%となるようTBS−Tで希釈したスキムミルク(GIBCO社)を入れ、30分室温でブロッキングを行った。各ウエルを200μLのTBS−Tで3回洗浄した後、マウスの尾静脈より回収した血漿をTBS−Tで1/2000倍に希釈し、ELISAプレートへ1ウエルあたり100μL添加し、室温で1時間反応させた。各ウエルを200μLのTBS−Tで3回洗浄した。
マウスの脾臓由来のリンパ球をマウス骨髄腫株P3X63−Ag8(ATCC受託番号 CRL−1580)と電気的に融合させた。細胞融合に際しては、1×108個の脾臓細胞と0.25×108個の骨髄腫株とを混合し、EP Buffer(0.3M Mannitol、0.1mM CaCl2、0.1mM MgCl2)に細胞密度が0.25×108個/mLとなるよう懸濁し、電気融合装置LF201(ネッパジーン社製)で融合を行った。融合条件はメーカー推奨の手法に従った。
移植3~7ヶ月後にマウスから脾臓細胞を取り出し、上記(6)に記載の方法でハイブリドーマ細胞を作製した。SLC6A6を認識する抗体を選択するために、上記(5)記載の方法を用いてクローンの選択を行った。一次抗体としては、細胞の培養上清を用い、二次抗体としては、抗マウスIgG1ポリクローナル抗体−HRP標識、抗マウスIgG2aポリクローナル抗体−HRP標識、抗マウスIgG2bポリクローナル抗体−HRP標識(Bethyl社)をそれぞれ等量混合し、TBSTで100000倍希釈した溶液を用い、サブクラスIgGのクローンのみが検出されるようにして、目的の抗体を産生するハイブリドーマを選択した。
上記記載(7)で得られた7種類の抗体クローン「204」「205」、「303」、「419」、「402」、「422」及び「430」と、大腸がん細胞(HT−29及びLoVo)との反応性を解析した。また、比較対象として、WO2012/029990公報記載の抗SLC6A6抗体である「4B9b」及び「5H12d」を同様に解析した。以下では、ハイブリドーマ細胞が作る抗体をそれぞれのクローン番号で記載することがある。
ヒト胎児腎臓細胞である293T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、スクレーパーで剥がし、1.5mLチューブに回収した。204、205及び419抗体をそれぞれ最終濃度10μg/mLとなるようチューブに添加し、60分間反応させた。比較対象として、4B9bを同様の濃度で添加し、反応させた。
2%FBSを添加したPBS(−)(PBS+2%FBS)で細胞を2回洗浄後、AlexaFluor488標識されたGoat−Anti−mouse IgG(Invitrogen社)をPBS+2%FBSで1/1000希釈して添加し、30分間反応させた。PBS+2%FBSで2回洗浄後、FACS Verse(BD社)で解析を行った。結果を図2に示す。
実施例1(7)で得られたモノクローナル抗体に関して、ヒト大腸がん細胞であるHCT116に対して、抗体クローン204、205、303、419、402、422及び430抗体との反応性を実施例2と同様の方法で解析した。結果を図3に示す。
51症例の大腸がん組織と8症例の正常大腸粘膜をマウス抗体(クローン205)を用いて免疫組織染色を行った。パラフィン固定後、薄切したヒト大腸がん組織(US Biomax社)をキシレンで脱パラフィン処理し、エタノールで親水化した。得られた組織を0.3%(v/v)過酸化水素水で20分間処理した後、TBSTで3回洗浄し、1mg/mLのProteinase Kで37℃、15分間処理し、抗原賦活化を行った。ヒストファインSAB−POユニバーサルキット(ニチレイ社)を用い、添付のプロトコールに従って染色を行った。一次抗体の反応では、10μg/mLの205抗体を添加し、37℃、1時間反応させた。発色の際には、ImmPACT DAB Substrate(Vector社)で5分間発色させた。蒸留水で水洗後、Hematoxylin(WAKO)を用いて核を染色し、流水洗浄後、エタノール、キシレンの順に処理して、封入した。
上記、実施例4に記載した方法と同様に、マウス抗体(205クローン)を用いて乳がん及び子宮がんのがん組織(Cancer)、及び正常組織(Normal)を免疫組織染色した。その結果を図6に示す。乳がん及び子宮がんにおいても、本発明のSLC6A6抗体によって、がん部が明確に染色されることが示された。乳がんでは3症例中3症例で陽性(100%)であり、子宮がんでは3症例中2症例が強陽性(66%)であることが示された。
上記で得られたモノクローナル抗体の中で、抗体クローン402がSLC6A6の細胞外ドメインに結合しているか解析を行った。10μg/mLの抗体クローン402と実施例1(4)で作製したSLC6A6の細胞外領域部分タンパク質15μg/mLとを混合し、37℃で1時間反応させた。組み換えタンパク質を加えていないサンプルをコントロールとして同様に準備した。ガラス上で培養したHCT116細胞と4℃で1時間反応した後、実施例1(8)記載の方法で二次抗体と反応させ、検出を行った。結果を図7に示す。図7において「Phase」は、位相差顕微鏡を用いた可視光による画像である。
(1)遺伝子のクローニング
実施例1で得られたハイブリドーマ細胞を培養し、Qiagen社のRNeasy Miniキットを用いて、total RNAを抽出した。抽出したtotal RNA 2μgから、SuperScript III reverse transcriptase(Invitrogen)を用いて、逆転写(RT)反応を50℃で1時間行ってcDNAを合成し、85℃、5分間の加熱により、反応を停止させた。得られたcDNAを鋳型とし、以下のプライマーを用い、KOD PLUS(TOYOBO社)を用いたPCR反応を行なうことで、目的の遺伝子を増幅した。
まず、ヒトIgG1のH鎖定常領域を増幅するために、配列番号11で示されるオリゴDNAを化学合成し、配列番号12と配列番号13のプライマーでPCR反応(94℃、2分間のデナチュレーションを行ない、続いて、58℃、15秒間のアニーリング、68℃、60秒間のエロンゲーションのサイクルを30サイクル)を行ってヒトIgG1のH鎖定常領域断片を得た。
まず、ヒトkappa鎖定常領域を増幅するために、配列番号18で示されるオリゴDNAを化学合成し、配列番号19と配列番号20のプライマーでPCR反応(94℃、2分間のデナチュレーションを行ない、続いて、58℃、15秒間のアニーリング、68℃、30秒間のエロンゲーションのサイクルを30サイクル)を行ってヒトkappa鎖定常領域断片を得た。
6well plateに90%コンフルエントとなるようCHO細胞を培養し、(1)で作製したH鎖及びL鎖発現用ベクターをCHO細胞へFugene6(Roche社)を用いて導入し、タンパク質の一過性発現を行った。導入条件はメーカー推奨の方法で行った。24時間後にF12培地(GIBCO社)に交換し、で4日間培養を行った。培養上清を回収し、遠心分離により細胞を除き、上清を回収した。抗体の精製は実施例1(7)記載の方法と同様に行った。
次に、81μLのExpiFectamine 293 reagentを1.419mLのOptimem(GIBCO社)と混合し、5分間静置した後、抗体のH鎖、L鎖遺伝子それぞれをコードする遺伝子がクローニングされたプラスミドを各15μgとなるよう添加し、20分間室温で静置した。
上記で準備した293F細胞を2.9×106cells/mlとなるよう、25.5mLの培地に再懸濁し、プラスミド溶液3mLを添加して培養を開始した。培養開始後20時間後に150μLのExpiFectaine 293 transfection enhancer 1と1.5mLのenhancer 2を添加し、3日間培養し、培養上清を回収した。
Protein G sepharose担体を用いた抗体の精製は実施例1(7)記載の方法と同様に行うか、ハイドロキシアパタイト担体を用いた精製を行った。
ハイドロキシアパタイトを用いた精製法では、実施例7(2)記載の方法と同様の方法で培養した培養上清12mlに対し250mgのcheramic hydroxyapatite Type II、20μm(Bio−Rad社)を用いた。ハイドロキシアパタイト担体を10mMリン酸緩衝液(10mMNa2HPO4、10mM NaH2PO4(pH 6.7))で平衡化し、抗体タンパク質を含む培養上清の12mLに対し、36mLの10mMリン酸緩衝液を添加した。この希釈した培養上清に、平衡化したハイドロキシアパタイト担体を添加し、18時間4℃で撹拌した。担体をエコノパックカラム(Bio−Rad社)に充填し、10倍量の10mMリン酸緩衝液で洗浄した後、10mMリン酸緩衝液に50、300、500、700、800、1000、1300mMとなるよう塩化ナトリウムを添加した溶液で分離を実施した。500~700mMの塩化ナトリウム濃度の間で溶出してきたタンパク質の95%以上が抗体タンパク質であった。
実施例1(2)に記載のpEF6ベクターにSLC6A6遺伝子を組み込んだ発現用ベクターを、FUGENE6(ロシュ社製)を用いてHCT15及び、HT−29、およびSW480へ導入した。操作は当該キットに添付された説明書の記載に基づいて行った。
幹細胞は、様々な表面マーカーを用いて分離されているが、由来する組織により表面マーカーが異なることや、存在する割合が少ないことから、幹細胞の解析は非常に困難である。そこで、その分離・同定方法として、Side population(SP)細胞が注目されている。幹細胞は薬剤排出能が高く、様々な物質を細胞外に放出する特性を持っており、この幹細胞の特性を利用してHoechst33342の排出を指標に解析する技術が広く用いられている。この技術において、Hoechst33342の染色パターン中、発色が弱い細胞分画が、SP細胞と呼ばれる。がん細胞に関してもこのようなSP細胞が存在することが明らかになり、SP細胞は、非SP細胞に比べ明確に腫瘍形成能が高いことが報告され、がん幹細胞が濃縮される分画として知られるようになった(Cancer Research 2005,65,p.6207−6219)。
ヒト大腸がん細胞であるSW480細胞のSP分画及び非SP分画(Major population(MP)分画)に関して、SLC6A6の発現を解析した。実施例8(2)記載の方法と同様にHoechst33342で細胞を染色し、その後、ProteinGで精製しpH3.0の溶出液で溶出した1μg/mLの430抗体と細胞を4℃、1時間染色し、二次抗体として抗マウスIgGポリクローナル抗体AlexaFluor488標識で染色した後、FACS Canto IIを用いて解析を行った。その結果を図9に示す。
上記実施例9で示されているように、SLC6A6の発現は、がん幹細胞が濃縮される集団であるSP細胞の形成に直接関与している。一方で、SLC6A6はタウリンを輸送するトランスポーターであることが報告されている(FEBS Letter 1993,318,p.139−144)。そこで、SLC6A6を過剰発現させた細胞に対し、タウリンを作用させた場合、細胞集団におけるSP細胞の割合が変化するかを解析した。
(1)HT−29あるいはSW480を90%コンフルエントになるよう培養し、Glass Base Dishに播種した。細胞を24時間培養後、氷冷したPBSで洗浄し、発現後pH3.0で精製した419、402、または430抗体10μg/mlと1時間、4℃で反応させた。細胞をPBSで洗浄後、4℃もしくは37℃で20分間培養を行った後、10%中性緩衝ホルマリンで10分間固定した。反応後の細胞を500倍希釈した抗マウスIgGポリクローナル抗体−AlexaFluor488標識と室温で30分間反応させた後、共焦点レーザー顕微鏡(Zeiss社)で解析した。結果を図11Aに示す。
ヒト大腸がん細胞であるHCT116細胞あるいはヒト乳がん細胞であるSK−BR3をGlass Bottom Dish(MATSUNAMI社)上に、1×104個/wellとなるよう細胞を添加し、37℃、5%CO2下で16時間培養した。培地を除去後、氷上に冷却した培地を添加し、氷上で20分間静置した。培地を除去後、ProteinGで精製しpH3.0の溶出液で溶出した402ヒト−マウスキメラ抗体を20μg/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、二次抗体として抗ヒトIgGポリクローナル抗体−AlexaFluor488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分間培養を行ない、PBS(−)で2回洗浄た後に蛍光顕微鏡(BZ8100、キーエンス社)を用いて解析を行った。結果を図11Bに示す。
図11Bは、HCT116あるいはSK−BR3において、抗体が経時的に細胞に取り込まれることを示している。0分すなわち染色直後はほとんどの抗体が細胞膜上に結合しているが、15分以降では細胞内に取り込まれてゆき、60分以降ではほとんどの抗体が細胞内に取り込まれていた。すなわち、402ヒト−マウスキメラ抗体は、細胞表面上のSLC6A6と結合した後、細胞内に取り込まれることが確認された。
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11(2)記載の方法と同様にGlass Bottom Dish上で培養したHCT116細胞に0.5mg/mLとなるようConAを混合した培地を添加し、37℃、5%CO2下で60分間培養を行った。培地で2回洗浄後、実施例13(2)記載の方法と同様に一次抗体、二次抗体を用いて細胞を染色し、0、15、30、60、120分後に蛍光顕微鏡を用いて解析を行った。
402ヒト−マウスキメラ抗体をAlexa Fluor 488 Labeling Kit(Molecular Probe社)を用いて標識を行った。HCT116細胞をConA処理した場合としなかった場合それぞれを準備し、氷上で20分間静置した後、Alexa Fluor 488標識した402ヒト−マウスキメラ抗体を30μg/mLとなるよう培地で希釈した溶液と氷上で30分間反応させ、PBS(−)で2回洗浄した後に蛍光顕微鏡で解析を行った(図13A)。
細胞内に抗体が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。HT−29またはSW480細胞を10μg/mLのpH3.0で精製した419抗体と1時間、4℃で反応させた後、PBSで洗浄した。ここにサポリン標識された抗マウスIgGポリクローナル抗体(ADVANCED TARGETING SYSTEMS社)あるいは未標識の抗マウスIgGポリクローナル抗体を10nMとなるよう添加し、30分間、4℃で反応させた後、PBSで洗浄した。サポリン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。これらの細胞を37℃で3日間培養を行った後、細胞の増殖をWST−8(Roche社)を用いて測定した。図14Aにその結果を示す。
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。ヒト大腸がん細胞であるHCT116細胞を96wellプレートに、2.5×103個/wellとなるよう細胞を播き、37℃、5%CO2下で16時間培養した。HCT116細胞に、終濃度が0.1あるいは0.3μMのマウス抗体と9nMのsaporin標識された抗マウスIgGポリクローナル抗体(ADVANCED TARGETING SYSTEMS社)になるように調製した混合溶液を添加した。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。これらの細胞を37℃、5%CO2下で3日間培養を行った後、細胞の増殖をCell Counting Kit−8(同仁化学研究所)を用いて測定した。図14にその結果を示す。
配列番号2:ヒトSLC6A6のアミノ酸配列。
配列番号3:ヒトSLC6A6のアミノ酸残基143~216のアミノ酸配列をコードする塩基配列。
配列番号4:ヒトSLC6A6のアミノ酸残基143~216のアミノ配列。
配列番号5:プライマー配列、Forward
配列番号6:プライマー配列、Reverse
配列番号7:プライマー配列、Long
配列番号8:プライマー配列、Short
配列番号9:プライマー配列、mIgG2a_CH1_reverse
配列番号10:プライマー配列、mIgkappa_R3
配列番号11:オリゴDNA配列、hHchain
配列番号12:プライマー配列、hCg1_F
配列番号13:プライマー配列、hCgI_R
配列番号14:プライマー配列、Hchain_signal_top
配列番号15:プライマー配列、Hchain_signal_bottom
配列番号16:プライマー配列、Forward
配列番号17:プライマー配列、Reverse
配列番号18:オリゴDNA配列、hLchain
配列番号19:プライマー配列、hCk_F
配列番号20:プライマー配列、hCk_R
配列番号21:プライマー配列、Forward2
配列番号22:プライマー配列、Reverse2
配列番号23:ヒト‐マウスキメラ化205抗体の重鎖の塩基配列
配列番号24:ヒト‐マウスキメラ化205抗体の重鎖のアミノ酸配列
配列番号25:ヒト‐マウスキメラ化205抗体の軽鎖の塩基配列
配列番号26:ヒト‐マウスキメラ化205抗体の軽鎖のアミノ酸配列
配列番号27:ヒト‐マウスキメラ化402抗体の重鎖の塩基配列
配列番号28:ヒト‐マウスキメラ化402抗体の重鎖のアミノ酸配列
配列番号29:ヒト‐マウスキメラ化402抗体の軽鎖の塩基配列
配列番号30:ヒト‐マウスキメラ化402抗体の軽鎖のアミノ酸配列
配列番号31:ヒト‐マウスキメラ化419抗体の重鎖の塩基配列
配列番号32:ヒト‐マウスキメラ化419抗体の重鎖のアミノ酸配列
配列番号33:ヒト‐マウスキメラ化419抗体の軽鎖の塩基配列
配列番号34:ヒト‐マウスキメラ化419抗体の軽鎖のアミノ酸配列
配列番号35:ヒト‐マウスキメラ化303抗体の重鎖の塩基配列
配列番号36:ヒト‐マウスキメラ化303抗体の重鎖のアミノ酸配列
配列番号37:ヒト‐マウスキメラ化303抗体の軽鎖の塩基配列
配列番号38:ヒト‐マウスキメラ化303抗体の軽鎖のアミノ酸配列
配列番号39:ヒト‐マウスキメラ化422抗体の重鎖の塩基配列
配列番号40:ヒト‐マウスキメラ化422抗体の重鎖のアミノ酸配列
配列番号41:ヒト‐マウスキメラ化422抗体の軽鎖の塩基配列
配列番号42:ヒト‐マウスキメラ化422抗体の軽鎖のアミノ酸配列
配列番号43:ヒト‐マウスキメラ化430抗体の重鎖の塩基配列
配列番号44:ヒト‐マウスキメラ化430抗体の重鎖のアミノ酸配列
配列番号45:ヒト‐マウスキメラ化430抗体の軽鎖の塩基配列
配列番号46:ヒト‐マウスキメラ化430抗体の軽鎖のアミノ酸配列
Claims (11)
- SLC6A6の細胞外領域内の立体構造をとるエピトープに結合するモノクローナル抗体またはその抗原結合断片を含む、がん治療用医薬組成物。
- 前記モノクローナル抗体は、抗がん剤、トキシンまたはラジオアイソトープがコンジュゲートされているものである、請求項1に記載の医薬組成物。
- SLC6A6の細胞外領域内の立体構造をとるエピトープに結合するモノクローナル抗体またはその抗原結合断片が、抗がん剤、トキシンまたはラジオアイソトープとコンジュゲートされた抗体コンジュゲートを含む、がん治療用医薬組成物。
- 前記エピトープが、以下の(a)~(c)から選択される少なくとも1つに示されるポリペプチドを含む、請求項1~3のいずれか1項に記載の医薬組成物:
(a)配列番号4に示されるアミノ酸配列からなるポリペプチド
(b)配列番号4に示されるアミノ酸配列において1個もしくは数個のアミノ酸が置換、欠失および/または挿入されたアミノ酸配列からなり、かつ、SLC6A6の細胞外領域として機能するポリペプチド
(c)配列番号4に示されるアミノ酸配列に70%以上の相同性を有するアミノ酸配列からなり、かつ、SLC6A6の細胞外領域として機能するポリペプチド。 - モノクローナル抗体が、
(a)配列番号24の50~54番目、69~86番目および118~125番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号26の44~54番目、70~76番目および109~116番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体、
(b)配列番号28の50~54番目、69~86番目および118~126番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号30の46~57番目、73~79番目および112~119番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体、
(c)配列番号32の50~54番目、69~86番目および118~128番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号34の48~58番目、74~80番目および113~120番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体
(d)配列番号36の50~54番目、69~86番目および118~131番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号38の44~54番目、70~76番目および109~116番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体、
(e)配列番号40の50~54番目、69~86番目および118~129番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号42の44~54番目、70~76番目および109~116番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体、および
(f)配列番号44の50~54番目、69~86番目および118~126番目のアミノ酸配列をそれぞれ重鎖CDR1、CDR2およびCDR3として含む重鎖可変領域、および配列番号46の46~57番目、73~79番目および112~119番目のアミノ酸配列をそれぞれ軽鎖CDR1、CDR2およびCDR3として含む軽鎖可変領域を含む抗体、
からなる群から選択される少なくとも1つである、請求項1~4のいずれか1項に記載の医薬組成物。 - 前記モノクローナル抗体が、請求項5に記載のモノクローナル抗体又はその断片が認識するエピトープに結合するものである、請求項1~4のいずれか1項に記載の医薬組成物。
- 前記モノクローナル抗体が、キメラ化抗体又はヒト化抗体である、請求項1~6のいずれか1項に記載の医薬組成物。
- 上記トキシンが、ジフテリアトキシン、シュードモナスエンドトキシンおよびエキソトキシン、スタフィロコッカスエンテロトキシンA、真菌、植物、抗ウィルス性タンパク質、サポリン、ゲロニン、モモリジン、トリコサンチン、大麦トキシン、アレウリテス・フォーディタンパク質、ジアンチンタンパク質、フィトラッカ・アメリカーナタンパク質、モモルディカ・チャランチア阻害剤、クルシン、クロチン、サポアオナリア・オフィシナリス阻害剤、ゲロニン、マイトジェリン、レストリクトシン、フェノマイシン、ネオマイシン、およびトリコテセンからなる群から選択される少なくとも一つである、請求項1~7のいずれか1項に記載の医薬組成物。
- 前記抗がん剤が、大腸がん、乳がん、または子宮がんを治療するための薬剤である、請求項1~7のいずれか1項に記載の医薬組成物。
- 大腸がん、乳がん、または子宮がんを治療するための医薬組成物である、請求項1~9のいずれか1項に記載の医薬組成物。
- 少なくとも1つの抗がん剤をさらに含む、請求項1~10のいずれか1項に記載の医薬組成物。
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