WO2010016526A1 - 癌の治療及び予防用医薬組成物 - Google Patents
癌の治療及び予防用医薬組成物 Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—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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- C07K16/28—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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- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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- A61P35/00—Antineoplastic agents
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—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
- C07K16/3053—Skin, nerves, brain
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/734—Complement-dependent cytotoxicity [CDC]
Definitions
- the present invention relates to a novel pharmaceutical use of an antibody against CAPRIN-1 or a fragment thereof as a therapeutic and / or prophylactic agent for cancer.
- Cancer is a disease that occupies the top cause of all deaths, and the current treatment is a combination of radiation therapy and chemotherapy, mainly surgery. Despite the recent development of new surgical methods and the discovery of new anti-cancer drugs, the therapeutic results of cancer have not improved much except for some cancers. In recent years, advances in molecular biology and cancer immunology have identified antibodies that react specifically with cancer, cancer antigens that are recognized by cytotoxic T cells, genes that encode cancer antigens, There is an increasing expectation for a specific cancer therapy targeting cancer antigens (Non-patent Document 1).
- Non-Patent Documents 4 to 4 9
- clinical studies of cell therapy using immune cells that specifically react with cancer antigens and cancer-specific immunotherapy such as vaccines containing cancer antigens are being conducted targeting a part of them.
- Cytoplasma-and promotion-associated protein 1 (CAPRIN-1) is expressed when quiescent normal cells are activated or undergo cell division, and also forms RNA and intracellular stress granules within the cell to transport mRNA It is an intracellular protein known to be involved in the control of translation.
- CAPRIN-1 Cytoplasma-and promotion-associated protein 1
- examples include GPI-anchored membrane protein 1 and Membrane component surface marker protein 1 (M11S1), and this protein is a cell membrane protein. There is a name as if it was known. These aliases originally originated from a report (Non-patent Document 10) that the gene sequence of CAPRIN-1 has a GPI-binding region and is expressed in colon cancer cells.
- the CAPRIN-1 gene sequence is incorrect, and the CAPRIN-1 gene sequence currently registered in GenBank et al. Lacks one base, resulting in a frameshift resulting in a deletion of 80 amino acids from the C-terminus.
- the resulting artifact (74 amino acids) is the GPI-binding portion in the previous report, and further there is a gene sequence error on the 5 ′ side, and 53 amino acids are deleted from the N-terminus (non-patent literature). 11). Further, it has been reported that the protein encoded by the gene sequence of CAPRIN-1 currently registered in GenBank et al. Is not a cell membrane protein (Non-patent Document 11).
- Patent Documents 2 and 3 include the name M11S1, and CAPRIN-1 is one of the cell membrane proteins and is used as a target for antibody drugs. It is described that it can be used for treatment (there is no mention of treatment using an antibody against this protein in the Examples).
- Non-Patent Document 11 from the time of filing of Patent Document 2 to the present, it has become common knowledge that CAPRIN-1 is not expressed on the cell surface, and CAPRIN-1 is a cell membrane protein. It is clear that the contents of Patent Documents 2 and 3 based only on false information that is not to be understood as technical common knowledge for those skilled in the art.
- An object of the present invention is to identify a cancer antigen protein that is specifically expressed on the surface of a cancer cell, and to provide a use of an antibody targeting it as a therapeutic and / or prophylactic agent for cancer.
- CAPRIN-1 having an amino acid sequence represented by an even sequence number among SEQ ID NOs: 2 to 30 based on the obtained gene and its human, bovine, horse, mouse, chicken homologous gene Antibodies against CAPRIN-1 were generated.
- CAPRIN-1 is specifically expressed in breast cancer, brain tumor, leukemia, lymphoma, lung cancer, esophageal cancer, colon cancer, gastric cancer and kidney cancer cells, and part of the CAPRIN-1 protein is a cell of these cancer cells. It was found that it was specifically expressed on the surface. And it discovered that the antibody with respect to the part expressed on the cell surface of each cancer cell of these CAPRIN-1 impaired the cancer cell which expresses CAPRIN-1, and came to complete this invention.
- the present invention has the following features.
- the present invention relates to an amino acid sequence represented by an even sequence number among SEQ ID NOs: 2 to 30, or 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more with the amino acid sequence.
- a CAPRIN-1 protein having an amino acid sequence having the same sequence identity, or a fragment thereof containing 7 or more consecutive amino acids, and an antibody or fragment thereof having immunological reactivity as an active ingredient,
- a pharmaceutical composition for the treatment and / or prevention of cancer is provided.
- the cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, esophageal cancer, colon cancer, stomach cancer or kidney cancer.
- the antibody is a monoclonal antibody or a polyclonal antibody.
- the antibody is a human antibody, a humanized antibody, a chimeric antibody, a single chain antibody, or a bispecific antibody.
- the antibody has the amino acid sequence represented by SEQ ID NO: 37 or 136, or 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more with the amino acid sequence.
- the antibody is any one of the following antibodies (a) to (k), an antibody having immunological reactivity with the CAPRIN-1 protein, or the antibody as an active ingredient: (A) a heavy chain variable region comprising SEQ ID NOs: 40, 41 and 42 and a light chain comprising SEQ ID NOs: 44, 45 and 46, which is a pharmaceutical composition for the treatment and / or prevention of cancer, characterized in that An antibody comprising a chain variable region.
- An antibody comprising a heavy chain variable region comprising SEQ ID NOs: 40, 41 and 42 and a light chain variable region comprising SEQ ID NOs: 65, 66 and 67.
- An antibody comprising a heavy chain variable region comprising SEQ ID NOs: 70, 71 and 72 and a light chain variable region comprising SEQ ID NOs: 74, 75 and 76.
- An antibody comprising a heavy chain variable region comprising SEQ ID NOs: 90, 91 and 92 and a light chain variable region comprising SEQ ID NOs: 94, 95 and 96.
- An antibody comprising a heavy chain variable region comprising SEQ ID NOs: 100, 101 and 102 and a light chain variable region comprising SEQ ID NOs: 104, 105 and 106.
- the antibody against CAPRIN-1 used in the present invention damages cancer cells. Therefore, antibodies against CAPRIN-1 are useful for the treatment and prevention of cancer.
- FIG. 2 is a graph showing cytotoxicity against a breast cancer cell line MDA-MB-157 expressing CAPRIN-1 by monoclonal antibodies (# 1 to # 11) against CAPRIN-1 that react with the cell surface of cancer cells.
- the antitumor activity of the antibody against the polypeptide having an even sequence number among SEQ ID NOs: 2 to 30 used in the present invention is, as described later, by examining the suppression of tumor growth in a cancer-bearing animal in vivo, or It can be evaluated by examining whether tumor cells expressing the polypeptide in vitro exhibit cytotoxic activity via immune cells or complement.
- nucleotide sequences of the polynucleotides encoding the proteins consisting of the amino acid sequences of even numbers among SEQ ID NOs: 2 to 30 are respectively SEQ ID NOs: 1 to It is shown by the odd number sequence number (namely, sequence number 1,3,5..27,29) among 29.
- amino acid sequences shown in SEQ ID NOs: 6, 8, 10, 12, and 14 in the sequence listing disclosed by the present invention are derived from cancer-bearing dogs by the SEREX method using a dog testis tissue-derived cDNA library and the serum of a dog with breast cancer.
- the amino acid sequences shown in SEQ ID NOs: 2 and 4 are human homologues (homologs), the amino acid sequence shown in SEQ ID NO: 16 is As the bovine homologous factor, the amino acid sequence shown in SEQ ID NO: 18 is the equine homologous factor, the amino acid sequence shown in SEQ ID NO: 20-28 is the mouse homologous factor, the amino acid sequence shown in SEQ ID NO: 30 is the It is an amino acid sequence of CAPRIN-1 isolated as a chicken homologous factor (see Example 1 described later). CAPRIN-1 is known to be expressed when quiescent normal cells are activated or undergo cell division.
- CAPRIN-1 was known not to be expressed on the cell surface, this study revealed that a part of the CAPRIN-1 protein is expressed on the cell surface of various cancer cells.
- an antibody that binds to a portion of the CAPRIN-1 protein that is expressed on the cell surface of a cancer cell is preferably used.
- a polypeptide comprising a sequence of 7 or more amino acids in the region of number (aa) 50-98 or amino acid residue number (aa) 233-305, specifically, for example, SEQ ID NO: 37 or SEQ ID NO: 136 (among the amino acid sequences represented by SEQ ID NO: 136, the region of the amino acid sequence represented by SEQ ID NO: 137 or SEQ ID NO: 138 is preferred), or 80% or more of the amino acid sequence, preferably Is an amino acid sequence having a sequence identity of 85% or more, more preferably 90% or more, and still more preferably 95% or more
- Antibodies of the invention bind to these peptides, and includes all antibodies that exhibit anti-tumor activity.
- the antibody against CAPRIN-1 used in the present invention may be any kind of antibody as long as it can exhibit antitumor activity, such as a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a multispecific antibody, a human antibody, Humanized antibodies, chimeric antibodies, single chain antibodies (scFV), antibody fragments such as Fab and F (ab ′) 2 are included. These antibodies and fragments thereof can also be prepared by methods known to those skilled in the art.
- an antibody capable of specifically binding to the CAPRIN-1 protein is desirable, and a monoclonal antibody is preferable, but a polyclonal antibody may be used as long as a homogeneous antibody can be stably produced. .
- a test subject is a human, it is desirable that it is a human antibody or a humanized antibody in order to avoid or suppress rejection.
- CAPRIN-1 protein specifically binds to CAPRIN-1 protein and does not substantially bind to other proteins.
- the antitumor activity of the antibody that can be used in the present invention is determined by examining the suppression of tumor growth in cancer-bearing animals in vivo, or against tumor cells that express the polypeptide in vitro. Thus, it can be evaluated by examining whether or not it exhibits cytotoxic activity via immune cells or complement.
- the subject to be treated and / or prevented for cancer in the present invention is a mammal such as a human, a pet animal, livestock, a sport animal, etc., and a preferred subject is a human.
- the protein or fragment thereof used as a sensitizing antigen for obtaining an antibody against CAPRIN-1 used in the present invention is used for the animal species from which it is derived, such as humans, dogs, cows, horses, mice, rats, and chickens. Not limited. However, it is preferable to select in consideration of compatibility with the parent cell used for cell fusion. In general, a protein derived from a mammal is preferable, and a protein derived from a human is particularly preferable. For example, when CAPRIN-1 is human CAPRIN-1, human CAPRIN-1 protein, a partial peptide thereof, cells expressing human CAPRIN-1 and the like can be used.
- GenBank GenBank
- FASTA Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.
- the base sequence or amino acid sequence of these ORFs or mature portions is 70% to 100%, preferably 80% to 100%, more preferably 90% to 100%, even more preferably 95% to 100%, such as 97% to 100%, 98% to 100%, 99% to 100% or 99.100%.
- a target is a nucleic acid or protein consisting of a sequence having 5% to 100% sequence identity.
- “% sequence identity” refers to the amino acid (or base) of two sequences when they are aligned (aligned) for maximum similarity with or without gaps. The percentage (%) of the same amino acid (or base) relative to the total number.
- the CAPRIN-1 protein fragment has a length less than the total length of the protein from the amino acid length of the epitope (antigenic determinant), which is the smallest unit recognized by the antibody.
- An epitope refers to a polypeptide fragment that has antigenicity or immunogenicity in a mammal, preferably a human, and its minimum unit consists of about 7 to 12 amino acids, such as 8 to 11 amino acids.
- Specific examples include the amino acid sequence represented by SEQ ID NO: 37, SEQ ID NO: 137, or SEQ ID NO: 138, or 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% with the amino acid sequence. Examples include amino acid sequences having the above sequence identity.
- polypeptides including human CAPRIN-1 protein and partial peptides thereof are synthesized according to chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method), for example.
- Fmoc method fluorenylmethyloxycarbonyl method
- tBoc method t-butyloxycarbonyl method
- the polynucleotide encoding the above-mentioned polypeptide can be easily prepared by a known genetic engineering technique or a conventional method using a commercially available nucleic acid synthesizer.
- DNA containing the nucleotide sequence of SEQ ID NO: 1 is subjected to PCR using a pair of primers designed to amplify the nucleotide sequence described in SEQ ID NO: 1, using human chromosomal DNA or cDNA library as a template.
- PCR reaction conditions can be appropriately set. For example, using a thermostable DNA polymerase (eg, Taq polymerase) and a Mg 2+ -containing PCR buffer, the reaction temperature is 94 ° C.
- the reaction process consisting of 1 minute (annealing) and 2 minutes (elongation) at 72 ° C. is defined as one cycle.
- the reaction may be performed at 72 ° C. for 7 minutes after 30 cycles, but is not limited thereto.
- the PCR method, conditions, etc. are described in, for example, Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, A compendium of Methods from Current Protocols in Molecular Biology (1995), J ing.
- probes and primers are prepared based on the nucleotide sequence and amino acid sequence information shown in SEQ ID NOs: 1 to 30 in the sequence listing in this specification, and a human or other cDNA library is screened using the probes and primers. By doing so, the desired DNA can be isolated.
- the cDNA library is preferably prepared from cells, organs or tissues expressing a protein having an even sequence number among SEQ ID NOs: 2 to 30. Examples of such cells and tissues are cells or tissues derived from cancer or tumors such as testis, leukemia, breast cancer, lymphoma, brain tumor, lung cancer, colon cancer and the like.
- the host cell may be any cell that can express the polypeptide.
- prokaryotic cells include Escherichia coli
- examples of eukaryotic cells include monkey kidney cells COS1, Chinese hamster ovary cells.
- examples include, but are not limited to, mammalian cells such as CHO, human embryonic kidney cell line HEK293, mouse embryonic skin cell line NIH3T3, yeast cells such as budding yeast and fission yeast, silkworm cells, and Xenopus egg cells.
- the expression vector When a prokaryotic cell is used as a host cell, the expression vector includes an origin, promoter, ribosome binding site, multicloning site, terminator, drug resistance gene, auxotrophic complementary gene, etc. that can be replicated in the prokaryotic cell. Is used. Examples of the expression vector for E. coli include pUC system, pBluescript II, pET expression system, pGEX expression system and the like.
- a prokaryotic host cell is transformed with the vector, and the resulting transformant is cultured, the polypeptide encoded by the DNA is prokaryotic. It can be expressed in a host cell. In this case, the polypeptide can also be expressed as a fusion protein with another protein.
- an expression vector for a eukaryotic cell having a promoter, a splicing region, a poly (A) addition site and the like is used as an expression vector.
- expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pYES2, and the like.
- pIND / V5-His pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, etc.
- a His tag eg (His) 6 to (His) 10
- FLAG tag eg (His) 10
- myc tag eg (His) 6 to (His) 10
- the polypeptide can be expressed as a fusion protein to which various tags such as HA tag and GFP are added.
- a well-known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, virus infection, lipofection, binding to a cell membrane-permeable peptide, etc. can be used. .
- An antibody is usually a heteromultimeric glycoprotein comprising at least two heavy chains and two light chains. Apart from IgM, it is an approximately 150 kDa heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to the heavy chain by one disulfide covalent bond, but the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has intrachain disulfide bonds. Each heavy chain has at one end a variable domain (VH region) followed by several constant regions. Each light chain has a variable domain (VL region) and one constant region at the opposite end.
- VH region variable domain
- VL region variable domain
- the constant region of the light chain is aligned with the first constant region of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
- the variable domain of an antibody confers binding specificity on the antibody by exhibiting specific variability, in which a specific region is called a complementarity determining region (CDR).
- CDR complementarity determining region
- the relatively conserved portion of the variable region is called the framework region (FR).
- the complete heavy and light chain variable domains each contain 4 FRs linked by 3 CDRs.
- the three CDRs are called CDRH1, CDRH2, CDRH3 from the N-terminal in the heavy chain, and similarly called CDRL1, CDRL2, CDRL3 in the light chain.
- CDRH3 is most important for the binding specificity of the antibody to the antigen.
- the CDRs of each chain are held together in a state closer to the FR region, and contribute to the formation of an antigen binding site of the antibody together with the CDR from the other chain.
- the constant region does not contribute directly to the binding of the antibody to the antigen, but is involved in various effector functions such as antibody-dependent cellular cytotoxicity (ADCC), phagocytosis through binding to Fc ⁇ receptors,
- Figure 6 shows half-life / clearance rate through neonatal Fc receptor (FcRn), complement dependent cytotoxicity (CDC) through the C1q component of the complement cascade.
- the anti-CAPRIN-1 antibody in the present invention means an antibody having immunological reactivity with the full length of a CAPRIN-1 protein or a fragment thereof.
- immunological reactivity means the property of binding between an antibody and a CAPRIN-1 antigen in vivo, and damages the tumor through such binding (eg, death, suppression or regression). Function. That is, the antibody used in the present invention can bind to the CAPRIN-1 protein and can damage tumors such as leukemia, lymphoma, breast cancer, brain tumor, lung cancer, esophageal cancer, stomach cancer, kidney cancer, colon cancer and the like. For example, it doesn't matter.
- antibodies include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, antibody fragments (eg, Fab and F (ab ′) 2 ), and the like.
- the antibody may also be any class of immunoglobulin molecules, such as IgG, IgE, IgM, IgA, IgD and IgY, or any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and the like.
- the antibody may be further modified by acetylation, formylation, amidation, phosphorylation, or PEGylation.
- the antibody is a monoclonal antibody
- a breast cancer cell line SK-BR-3 expressing CAPRIN-1 is administered to the mouse for immunization, the spleen is extracted from the mouse, the cells are separated, A cell and a mouse myeloma cell are fused, and a clone producing an antibody having a cancer cell growth inhibitory action is selected from the obtained fused cells (hybridoma).
- It can be prepared by isolating a monoclonal antibody-producing hybridoma having cancer cell growth inhibitory action, culturing the hybridoma, and purifying the antibody from the culture supernatant by a general affinity purification method.
- a hybridoma producing a monoclonal antibody can also be produced, for example, as follows.
- an animal is immunized with a sensitizing antigen according to a known method.
- a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously.
- the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, or the like, and mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified.
- PBS Phosphate-Buffered Saline
- physiological saline or the like
- an ordinary adjuvant for example, Freund's complete adjuvant, if necessary, and emulsified.
- the mammal is dosed several times every 4-21 days.
- an appropriate carrier can be used during immunization with the sensitizing antigen.
- immune cells are collected from the mammal and subjected to cell fusion. Can be mentioned.
- Mammalian myeloma cells are used as the other parent cell to be fused with the immune cells.
- This myeloma cell is known in various known cell lines such as P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-511) (Margulies.
- the cell fusion between the immune cell and myeloma cell is basically performed by a known method, for example, the method of Kohler and Milstein et al. (Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46. ) And the like.
- the cell fusion is performed, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter.
- a cell fusion promoter for example, polyethylene glycol (PEG), Sendai virus (HVJ), or the like is used as the fusion promoter, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency as desired.
- the usage ratio of immune cells and myeloma cells can be arbitrarily set.
- the number of immune cells is preferably 1 to 10 times that of myeloma cells.
- the culture solution used for the cell fusion for example, RPMI1640 culture solution suitable for growth of the myeloma cell line, MEM culture solution, and other normal culture solutions used for this kind of cell culture can be used.
- Serum replacement fluid such as fetal calf serum (FCS) can be used in combination.
- a predetermined amount of the immune cells and myeloma cells are mixed well in the culture medium, and a PEG solution (for example, an average molecular weight of about 1000 to 6000) preliminarily heated to about 37 ° C. is usually 30 to 60% (
- the desired hybridoma is formed by adding at a concentration of w / v) and mixing.
- cell fusion agents and the like that are undesirable for the growth of the hybridoma are removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.
- the hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing with the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fusion cells) to die. Subsequently, the usual limiting dilution method is performed, and the hybridoma producing the target antibody is screened and single-cloned.
- a normal selective culture solution for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing with the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fusion cells) to die. Subsequently, the usual limiting dilution method is performed, and the hybridoma producing the target antibody is screened
- human lymphocytes such as human lymphocytes infected with EB virus are sensitized in vitro with proteins, protein-expressing cells or lysates thereof. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, for example, U266 (Registration No. TIB196) to obtain a hybridoma that produces a human antibody having a desired activity (for example, cell growth inhibitory activity).
- a desired activity for example, cell growth inhibitory activity
- the hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution and can be stored for a long time in liquid nitrogen.
- a desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen and immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. And can be prepared by screening monoclonal antibody-producing cells (hybridomas) by a normal screening method.
- a polyclonal antibody can be obtained, for example, as follows.
- This is prepared by, for example, purification using ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with CAPRIN-1 protein or synthetic peptide, or the like.
- a rabbit polyclonal antibody against a partial peptide (expressed by SEQ ID NO: 37) of a region expressed on the cell surface of a cancer cell in the amino acid sequence of the CAPRIN-1 protein was prepared, and the antitumor effect was exhibited. It has been confirmed.
- human antibody-producing mice for example, KM mice (Kirin Pharma / Medarex) and Xeno mice (Amgen) are known (for example, International Publication Nos. WO02 / 43478, WO02 / 092812, etc.).
- fully human polyclonal antibodies can be obtained from blood.
- spleen cells can be removed from the immunized mouse and a human monoclonal antibody can be prepared by a fusion method with myeloma cells.
- the antigen can be prepared according to, for example, a method using animal cells (Japanese Patent Publication No. 2007-530068), a method using baculovirus (eg, International Publication No. WO 98/46777).
- immunization may be performed by binding to an immunogenic macromolecule such as albumin.
- a recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing it into a host, and producing it using a gene recombination technique (for example, Carl, AK Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL, ANTIBODIES, Published in the United KingdomMIMILLAN PUBLISHERS 19).
- a gene recombination technique for example, Carl, AK Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL, ANTIBODIES, Published in the United KingdomMIMILLAN PUBLISHERS 19.
- V region an antibody variable region
- DNA encoding the V region of the target antibody is obtained, it is ligated with DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector.
- DNA encoding the V region of the antibody may be incorporated into an expression vector containing DNA of the antibody C region. It is incorporated into an expression vector so as to be expressed under the control of an expression control region such as an enhancer or promoter.
- host cells can be transformed with this expression vector to express the antibody.
- the anti-CAPRIN-1 antibody of the present invention is preferably a monoclonal antibody. However, it may be a polyclonal antibody, a genetically modified antibody (such as a chimeric antibody or a humanized antibody) and the like.
- Monoclonal antibodies include human monoclonal antibodies, non-human animal monoclonal antibodies (eg, mouse monoclonal antibody, rat monoclonal antibody, rabbit monoclonal antibody, chicken monoclonal antibody, etc.). Monoclonal antibodies can be produced by culturing hybridomas obtained by fusion of spleen cells and myeloma cells from non-human mammals (eg, mice, human antibody-producing mice, etc.) immunized with CAPRIN-1 protein. In the examples described later, a mouse monoclonal antibody was produced, and the antitumor effect was confirmed.
- non-human animal monoclonal antibodies eg, mouse monoclonal antibody, rat monoclonal antibody, rabbit monoclonal antibody, chicken monoclonal antibody, etc.
- Monoclonal antibodies can be produced by culturing hybridomas obtained by fusion of spleen cells and myeloma cells from non-human mammals (eg, mice, human antibody-producing mice, etc.) imm
- VH heavy chain variable
- CDR1 represented by the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: 60, SEQ ID NO: 65, SEQ ID NO: 74, SEQ ID NO: 84, SEQ ID NO: 94, SEQ ID NO: 104, SEQ ID NO: 114, or SEQ ID NO: 124.
- CDR2 and SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 57, SEQ ID NO: 62, SEQ ID NO: 67, SEQ ID NO: 76, SEQ ID NO: 8 , SEQ ID NO: 96, SEQ ID NO: 106 include CDR3 represented by the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 126.
- a chimeric antibody is an antibody prepared by combining sequences derived from different animals, such as a mouse antibody heavy chain, light chain variable region and human antibody heavy chain, light chain constant region antibody, etc. .
- a chimeric antibody can be prepared using a known method. For example, a DNA encoding an antibody V region and a DNA encoding a human antibody C region are ligated, incorporated into an expression vector, and introduced into a host. It is obtained by producing.
- Polyclonal antibodies include antibodies obtained by immunizing human antibody-producing animals (eg, mice) with CAPRIN-1 protein.
- a humanized antibody is a modified antibody also called a reshaped human antibody.
- Humanized antibodies are constructed by transplanting CDRs of antibodies from immunized animals into the complementarity determining regions of human antibodies. The general gene recombination technique is also known.
- oligos were prepared so that the DNA sequence designed to link the CDR of the mouse antibody and the framework region (FR) of the human antibody had an overlapping portion at the end. It is synthesized from nucleotides by PCR. The obtained DNA is obtained by ligating with the DNA encoding the human antibody constant region, then incorporating it into an expression vector, introducing it into a host and producing it (European Patent Application Publication No. EP239400, International Publication No. WO96). No. 02576). As the FR of a human antibody linked through CDR, a complementarity determining region that forms a favorable antigen binding site is selected.
- the amino acid of the framework region in the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen binding site (Sato K. et al., Cancer Research). 1993, 53: 851-856). Moreover, you may substitute by the framework area
- a region in which the complementarity determining region forms a favorable antigen binding site is selected. If necessary, the amino acid of the framework region in the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen binding site (Sato K. et al., Cancer). Research 1993, 53: 851-856).
- variable region e.g, FR
- constant region amino acids in the variable region or constant region may be substituted with other amino acids.
- the amino acid substitution is, for example, less than 15, less than 10, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 2, preferably 1 to 5, more preferably 1 or 2 amino acids
- the substituted antibody should be functionally equivalent to the unsubstituted antibody.
- the substitution is preferably a conservative amino acid substitution, which is a substitution between amino acids with similar properties such as charge, side chain, polarity, aromaticity and the like.
- Amino acids with similar properties include, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar It can be classified into sex amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched chain amino acids (threonine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine).
- basic amino acids arginine, lysine, histidine
- acidic amino acids aspartic acid, glutamic acid
- uncharged polar amino acids glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine
- modified antibody examples include antibodies bound to various molecules such as polyethylene glycol (PEG).
- PEG polyethylene glycol
- the substance to be bound is not limited. In order to obtain such a modified antibody, it can be obtained by chemically modifying the obtained antibody. These methods are already established in this field.
- “functionally equivalent” means that the target antibody has the same biological or biochemical activity as the antibody of the present invention, specifically, a function of damaging a tumor, and is applied to humans. Sometimes refers to not essentially causing rejection. Examples of such activity include cell growth inhibitory activity or binding activity.
- an antibody that recognizes the epitope of the CAPRIN-1 protein recognized by the anti-CAPRIN-1 antibody can be obtained by methods known to those skilled in the art.
- the epitope of the CAPRIN-1 protein recognized by the anti-CAPRIN-1 antibody is determined by an ordinary method (eg, epitope mapping), and an antibody is produced using a polypeptide having the amino acid sequence contained in the epitope as an immunogen.
- This method can be obtained by a method, a method of determining an epitope of an antibody prepared by a usual method, and selecting an antibody having the same epitope as the anti-CAPRIN-1 antibody.
- epitope refers to a polypeptide fragment having antigenicity or immunogenicity in a mammal, preferably a human, and its minimum unit consists of about 7 to 12 amino acids, preferably 8 to 11 amino acids.
- the affinity constant Ka (k on / k off ) of the antibody of the present invention is preferably at least 10 7 M ⁇ 1 , at least 10 8 M ⁇ 1 , at least 5 ⁇ 10 8 M ⁇ 1 , at least 10 9 M ⁇ 1 , At least 5 ⁇ 10 9 M ⁇ 1 , at least 10 10 M ⁇ 1 , at least 5 ⁇ 10 10 M ⁇ 1 , at least 10 11 M ⁇ 1 , at least 5 ⁇ 10 11 M ⁇ 1 , at least 10 12 M ⁇ 1 , or alternatively At least 10 13 M ⁇ 1 .
- the antibody of the present invention can be conjugated with an antitumor agent.
- the bond between the antibody and the antitumor agent is a group reactive with an amino group, carboxyl group, hydroxy group, thiol group, etc. (for example, succinate imidyl group, formyl group, 2-pyridyldithio group, maleimidyl group, alkoxycarbonyl group). , A hydroxy group, etc.).
- antitumor agents include the following antitumor agents known in the literature, such as paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan, piperosulfan, benzodopa (benzodopa) ), Carbocone, methredopa, uredopa, uretopa, altreamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolothramine , Camptothecin, bryostatin, calistatin ( allystatin), cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleuterbin, panclastatin, sarcodictin, spongestatin, chlorambucil, chloronaphazine, cholophosphamide, estram Ifosfamide, mechlore
- the antibodies of the present invention include radioactive substances such as 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 SM, 212 Bi, 32 P, 175 Lu, 176 Lu, which are known in the literature. It is also possible to combine isotopes. It is desirable that the radioisotope is effective for tumor treatment and diagnosis.
- the antibody of the present invention is an antibody immunologically reactive with CAPRIN-1 or an antibody that specifically recognizes CAPRIN-1.
- the antibody should be an antibody having a structure such that little or no rejection is avoided in the subject animal to which it is administered.
- examples of such antibodies include human antibodies, humanized antibodies, chimeric antibodies (eg, human-mouse chimeric antibodies), single chain antibodies, bispecific antibodies and the like when the target animal is human.
- the heavy and light chain variable regions are derived from human antibodies, or the heavy and light chain variable regions are derived from non-human animal antibody complementarity determining regions (CDR1, CDR2 and CDR3).
- a framework region derived from a human antibody, or the variable regions of the heavy and light chains are derived from a non-human animal antibody, and the constant regions of the heavy and light chains are human antibodies. It is a recombinant antibody that is derived from. Preferred antibodies are the previous two antibodies.
- DNA encoding a monoclonal antibody against human CAPRIN-1 eg, human monoclonal antibody, mouse monoclonal antibody, rat monoclonal antibody, rabbit monoclonal antibody, chicken monoclonal antibody, etc.
- DNAs encoding the light chain variable region and heavy chain variable region of the antibody were prepared by RT-PCR method, etc., and Kabat EU numbering system (Kabat et al., Sequences of Proteins of Immunological Institute, 5th Ed. of Health, Bethesda, Md. (1991)) to determine the sequence or sequences of the CDR1, CDR2, CDR3 of the variable region of the light and heavy chains based on.
- DNA encoding each of these variable regions or DNA encoding each CDR can be obtained using a gene recombination technique (Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press (1989)) or a DNA synthesizer. Make it.
- the human monoclonal antibody-producing hybridoma is prepared by immunizing a human antibody-producing animal (eg, mouse) with human CAPRIN-1, and then fusing spleen cells excised from the immunized animal with myeloma cells. Can do.
- DNA encoding the variable region and constant region of the light chain or heavy chain derived from a human antibody is prepared as necessary using a gene recombination technique or a DNA synthesizer.
- the CDR coding sequence in the DNA encoding the variable region of the light chain or heavy chain derived from the human antibody is derived from a non-human animal (for example, mouse, rat, chicken, etc.) corresponding thereto.
- a humanized antibody is encoded by preparing a DNA in which the CDR coding sequence of the antibody is replaced, and ligating the resulting DNA with a DNA encoding a constant region of a light chain or heavy chain derived from a human antibody, respectively. DNA can be produced.
- DNA encoding the light chain or heavy chain variable region of an antibody derived from an animal other than a human is used.
- a DNA encoding a chimeric antibody can be prepared by linking with a DNA encoding a region.
- this antibody is an antibody in which a heavy chain variable region and a light chain variable region are linearly linked via a linker, DNA encoding the heavy chain variable region, DNA encoding the linker And a DNA encoding a light chain variable region can be combined to produce a DNA encoding a single chain antibody.
- each of the heavy chain variable region and the light chain variable region is derived from a human antibody, or only the CDR is replaced by the CDR of an antibody derived from a non-human animal (eg, mouse, rat, chicken, etc.). Derived from human antibodies.
- the linker consists of 12 to 19 amino acids, and examples thereof include 15 amino acids (G 4 S) 3 (G. B. Kim et al., Protein Engineering Design and Selection 2007, 20 (9): 425-432). .
- this antibody is an antibody that can specifically bind to two different epitopes, such as DNA encoding heavy chain variable region A, light chain variable region B.
- DNA, DNA encoding heavy chain variable region B, and DNA encoding light chain variable region A are joined in this order (however, DNA encoding light chain variable region B and DNA encoding heavy chain variable region B) Are linked via a DNA encoding a linker as described above), whereby a DNA encoding a bispecific antibody can be prepared.
- each of the heavy chain variable region and the light chain variable region is derived from a human antibody, or only the CDR is replaced by the CDR of an antibody derived from a non-human animal (eg, mouse, rat, chicken, etc.). Derived from human antibodies.
- Recombinant DNA produced as described above is incorporated into one or more appropriate vectors, introduced into host cells (eg, mammalian cells, yeast cells, insect cells, etc.), and (co) expressed
- host cells eg, mammalian cells, yeast cells, insect cells, etc.
- a recombinant antibody can be prepared (PJ Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY, P. Shepherd and C. Dean. SNT. W. Gododing., Monoclonal Antibodies: principals and practices., 1993 ACADEMI PRESS).
- Examples of the antibody of the present invention produced by the above method include the following antibodies (a) to (k).
- An antibody composed of a light chain variable region
- an antibody comprising a heavy chain variable region comprising SEQ ID NOs: 70, 71 and 72 and a light chain variable region comprising SEQ ID NOs: 74, 75 and 76 (preferably a heavy chain variable region of SEQ ID NO: 73 and SEQ ID NO: 77
- An antibody composed of a light chain variable region An antibody composed of a light chain variable region.
- An antibody composed of a light chain variable region
- an antibody comprising a heavy chain variable region comprising SEQ ID NOs: 100, 101 and 102 and a light chain variable region comprising SEQ ID NOs: 104, 105 and 106 (preferably a heavy chain variable region of SEQ ID NO: 103 and SEQ ID NO: 107
- Antibody composed of light chain variable region (J) an antibody comprising a heavy chain variable region comprising SEQ ID NOS: 110, 111 and 112 and a light chain variable region comprising SEQ ID NOS: 114, 115 and 116 (preferably a heavy chain variable region of SEQ ID NO: 113 and SEQ ID NO: 117
- An antibody composed of a light chain variable region An antibody composed of a light chain variable region).
- SEQ ID NO: 40, 41 and 42, SEQ ID NO: 70, 71 and 72, SEQ ID NO: 80, 81 and 82, SEQ ID NO: 90, 91 and 92, SEQ ID NO: 100, 101 and 102, SEQ ID NO: 110, 111 and 112 , SEQ ID NOs: 120, 121 and 122 are the CDR1, CDR2 and CDR3 of the mouse antibody heavy chain variable region, respectively, and SEQ ID NOs: 44, 45 and 46, SEQ ID NOs: 50, 51 and 52, SEQ ID NO: 55, 56 and 57, SEQ ID NO: 60, 61 and 62, SEQ ID NO: 65, 66 and 67, SEQ ID NO: 74, 75 and 76, SEQ ID NO: 84, 85 and 86, SEQ ID NO: 94, 95 and 96, SEQ ID NO: 104, 105 and 106, SEQ ID NOs 114, 115 and 116, SEQ ID NOs 124 and 12 And the amino
- the humanized antibody, chimeric antibody, single chain antibody or bispecific antibody of the present invention is, for example, the following antibody (exemplified by antibody (a)).
- variable region of the heavy chain comprises the amino acid sequence of SEQ ID NOs: 40, 41 and 42 and the amino acid sequence of the framework region derived from a human antibody
- variable region of the light chain comprises the amino acids of SEQ ID NOs: 44, 45 and 46
- An antibody comprising the sequence and the amino acid sequence of the framework region derived from a human antibody preferably an antibody comprising the amino acid sequence of SEQ ID NO: 43 in the heavy chain variable region and the amino acid sequence of SEQ ID NO: 47 in the light chain variable region).
- variable region of the heavy chain comprises the amino acid sequence of SEQ ID NOs: 40, 41 and 42 and the amino acid sequence of the framework region derived from a human antibody
- constant region of the heavy chain comprises the amino acid sequence derived from a human antibody
- variable region of the light chain comprises the amino acid sequence of SEQ ID NOs: 44, 45 and 46 and the amino acid sequence of the framework region derived from a human antibody
- constant region of the light chain comprises the amino acid sequence derived from a human antibody.
- variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 43
- constant region of the heavy chain comprises an amino acid sequence derived from a human antibody
- variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 47
- constant region of the light chain comprises an amino acid sequence derived from a human antibody
- human IgG1 heavy chain constant region has registration number J00228, human IgG2 Registration number J00230 for the heavy chain constant region, registration number X03604 for the human IgG3 heavy chain constant region, registration number K01316 for the human IgG4 heavy chain constant region, registration numbers V00557, X64135, X64133, etc. for the human light chain kappa constant region
- sequences such as registration numbers X64132 and X64134 can be referred to.
- the antibody preferably has a cytotoxic activity, and can thereby exert an antitumor effect.
- a hybridoma capable of producing another human antibody or non-human animal antibody (eg, mouse antibody) against human CAPRIN-1 is prepared, and the monoclonal antibody produced by the hybridoma is recovered, and immunological binding to human CAPRIN-1 and It is determined whether or not the antibody is the target antibody using the cytotoxic activity as an index. After identifying the target monoclonal antibody-producing hybridoma, DNAs encoding the variable regions of the heavy and light chains of the target antibody were prepared from the hybridoma and sequenced as described above. Use for production.
- the antibody of the present invention has the specificity of specifically recognizing CAPRIN-1, in particular the sequence of the framework region and / or the sequence of the constant region of each antibody of (i) to (iv) above There may be 1 or several (preferably 1 or 2) amino acid substitutions, deletions or additions. Here, several means 2 to 5, preferably 2 or 3.
- the present invention further provides DNA encoding the antibody of the present invention, DNA encoding the heavy chain or light chain of the antibody, or DNA encoding the variable region of the heavy chain or light chain of the antibody.
- DNA is, for example, in the case of antibody (a), a DNA encoding a heavy chain variable region comprising a base sequence encoding the amino acid sequences of SEQ ID NOs: 40, 41 and 42, and amino acid sequences of SEQ ID NOs: 44, 45 and 46 DNA encoding a light chain variable region comprising a nucleotide sequence encoding
- complementarity determining regions (CDRs) encoded by the DNA of these sequences are regions that determine the specificity of the antibody
- sequences that encode other regions of the antibody May be a sequence derived from another antibody.
- other antibodies include antibodies derived from organisms other than humans, but those derived from humans are preferable from the viewpoint of reducing side effects. That is, in the above DNA, the regions encoding the heavy chain and light chain framework regions and the constant regions preferably include a base sequence encoding a corresponding amino acid sequence derived from a human antibody.
- DNA encoding the antibody of the present invention is, for example, in the case of the antibody (a), a DNA encoding a heavy chain variable region comprising a base sequence encoding the amino acid sequence of SEQ ID NO: 43, a light chain variable region And the like.
- the DNA coding region includes DNA containing the base sequence encoding the amino acid sequence of SEQ ID NO: 47
- an example of the base sequence encoding the amino acid sequence of SEQ ID NO: 43 is the base sequence of SEQ ID NO: 48.
- An example of the base sequence encoding the amino acid sequence of SEQ ID NO: 47 is the base sequence of SEQ ID NO: 49.
- the region encoding each heavy chain and light chain constant region includes a base sequence encoding a corresponding amino acid sequence derived from a human antibody.
- the DNA of the present invention can be obtained, for example, by the above method or the following method.
- total RNA is prepared from a hybridoma related to the antibody of the present invention using a commercially available RNA extraction kit, and cDNA is synthesized by reverse transcriptase using a random primer or the like.
- the cDNA encoding the antibody is amplified by a PCR method using oligonucleotides having conserved sequences as primers.
- the sequence encoding the constant region can be obtained by amplifying a known sequence by the PCR method.
- the base sequence of DNA can be determined by a conventional method by incorporating it into a sequencing plasmid or phage.
- the anti-tumor effect of the anti-CAPRIN-1 antibody used in the present invention on CAPRIN-1-expressing cancer cells is considered to occur by the following mechanism.
- ADCC Effector cell antibody-dependent cytotoxicity
- CDC complement-dependent cytotoxicity
- the activity of the anti-CAPRIN-1 antibody used in the present invention is evaluated by the above-mentioned ADCC activity or CDC activity against cancer cells expressing CAPRIN-1 in vitro, as specifically shown in the Examples below. It can be evaluated by measuring.
- the anti-CAPRIN-1 antibody used in the present invention binds to the CAPRIN-1 protein on cancer cells and exhibits an antitumor action due to the above activity, and thus is considered useful for the treatment or prevention of cancer. That is, the present invention provides a pharmaceutical composition for treating and / or preventing cancer comprising an anti-CAPRIN-1 antibody as an active ingredient.
- the anti-CAPRIN-1 antibody is used for the purpose of administering it to the human body (antibody treatment), it is preferable to use a human antibody or a humanized antibody in order to reduce immunogenicity.
- the binding constant (affinity constant) Ka (k on / k off ) is preferably at least 10 7 M ⁇ 1 , at least 10 8 M ⁇ 1 , at least 5 ⁇ 10 8 M as the high binding affinity.
- ⁇ Binding to antigen-expressing cells The ability of an antibody to bind to CAPRIN-1 can be identified using binding assays such as those described in the Examples using, for example, ELISA, Western blotting, immunofluorescence and flow cytometry analysis.
- Antibodies that recognize CAPRIN-1 are inoculated with tissue obtained from patients during surgery or cell lines expressing CAPRIN-1 naturally or after transfection by immunohistochemistry in a manner well known to those skilled in the art
- tissue obtained from animals bearing selected xenografts using paraformaldehyde or acetone-fixed frozen sections or paraformaldehyde-fixed tissue sections Can do.
- an antibody reactive to CAPRIN-1 can be stained by various methods. For example, it can be visualized by reacting horseradish peroxidase-conjugated goat anti-mouse antibody or goat anti-rabbit antibody.
- the target of the pharmaceutical composition for the treatment and / or prevention of cancer of the present invention is not particularly limited as long as it is a cancer (cell) expressing the CAPRIN-1 gene.
- tumor and cancer refer to malignant neoplasms and are used interchangeably.
- the target cancer in the present invention is a cancer expressing a gene encoding a polypeptide containing an even numbered amino acid sequence of SEQ ID NOs: 2 to 30 or a partial sequence consisting of 7 or more consecutive amino acids.
- Yes preferably breast cancer, brain tumor, leukemia, lung cancer, lymphoma, mastocytoma, esophageal cancer and colon cancer.
- These specific cancers include, for example, breast cancer, complex breast cancer, malignant mixed breast tumor, intraductal papillary carcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, neuroepithelial tissue Tumor glioma, ventricular ependymoma, neuronal tumor, fetal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal type Lymphoma, gastrointestinal lymphoma, small to medium cell lymphoma, cecal cancer, ascending colon cancer, descending colon cancer, transverse colon cancer, sigmoid colon cancer, rectal cancer are included, but are not limited thereto.
- the target animals are mammals, for example, mammals including primates, pet animals, domestic animals, sport animals and the like, and humans, dogs and cats are particularly preferable.
- the antibody used in the present invention when used as a pharmaceutical composition, it can be formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension.
- a pharmacologically acceptable carrier or medium specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative
- a pharmaceutical preparation by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.
- a sterile composition for injection can be formulated in accordance with normal pharmaceutical practice using a vehicle such as distilled water for injection.
- Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride.
- Suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-60 may be used in combination.
- oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent.
- oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent.
- buffer for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant.
- the prepared injection solution is usually filled into a suitable ampoule.
- Administration is oral or parenteral, preferably parenteral administration. Specific examples include injection, nasal administration, pulmonary administration, and transdermal administration. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.
- the administration method can be appropriately selected depending on the age, weight, sex, symptoms, etc. of the patient.
- the dosage of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg body weight. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values.
- the dose and administration method vary depending on the weight, age, sex, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.
- the present invention further provides the following polypeptides and DNAs related to the antibodies (a) to (k).
- a polypeptide comprising the amino acid sequences of SEQ ID NO: 43, SEQ ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 93, SEQ ID NO: 103, SEQ ID NO: 113 and SEQ ID NO: 123, and DNA encoding the polypeptide.
- DNA comprising the nucleotide sequences of SEQ ID NO: 48, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 98, SEQ ID NO: 108, SEQ ID NO: 118, and SEQ ID NO: 128.
- V SEQ ID NO: 40, 41 and 42, SEQ ID NO: 70, 71 and 72, SEQ ID NO: 80, 81 and 82, SEQ ID NO: 90, 91 and 92, SEQ ID NO: 100, 101 and 102, SEQ ID NO: 110, 111 and 112
- a heavy chain CDR polypeptide selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 120, 121 and 122, and DNA encoding the polypeptide.
- SEQ ID NO: 44, 45 and 46, SEQ ID NO: 50, 51 and 52, SEQ ID NO: 55, 56 and 57, SEQ ID NO: 60, 61 and 62, SEQ ID NO: 65, 66 and 67, SEQ ID NO: 74, 75 and 76 Selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 84, 85 and 86, SEQ ID NOs: 94, 95 and 96, SEQ ID NOs: 104, 105 and 106, SEQ ID NOs: 114, 115 and 116, SEQ ID NOs: 124, 125 and 126 A light chain CDR polypeptide, and a DNA encoding the polypeptide.
- polypeptides and DNA can be prepared using gene recombination techniques as described above.
- RNA was extracted from the testis tissue of a healthy dog by the acid guanidinium-phenol-chloroform method (Acid guanidinium-Phenol-Chloroform method) PolyA RNA was purified using Oligotex-dT30 mRNA purification Kit (Takara Shuzo) according to the protocol attached to the kit.
- a dog testis cDNA phage library was synthesized using the obtained mRNA (5 ⁇ g).
- the cDNA phage library was prepared using cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, ZAP-cDNA GigapackIII Gold Clonig Kit (manufactured by STRATAGENE) according to the protocol attached to the kit.
- the size of the prepared cDNA phage library was 7.73 ⁇ 10 5 pfu / ml.
- the membrane was recovered, immersed in TBS (10 mM Tris-HCl, 150 mM NaCl pH 7.5) containing 0.5% nonfat dry milk, and shaken at 4 ° C. overnight to suppress nonspecific reaction.
- TBS 10 mM Tris-HCl, 150 mM NaCl pH 7.5
- This filter was reacted with serum of a patient dog diluted 500 times at room temperature for 2 to 3 hours.
- the serum pretreatment method is as follows. Specifically, ⁇ ZAP Express phage into which no foreign gene was inserted was infected with host E. coli (XL1-Blue MRF ′), and then cultured overnight at 37 ° C. on NZY plate medium. Next, a buffer of 0.2 M NaHCO 3 pH 8.3 containing 0.5 M NaCl was added to the plate and allowed to stand at 4 ° C. for 15 hours, and then the supernatant was recovered as an E. coli / phage extract. Next, the recovered E.
- coli / phage extract was passed through an NHS-column (GE Healthcare Bio-Science) to immobilize the protein derived from E. coli / phage.
- Serum dog serum was passed through and reacted with this protein-immobilized column, and antibodies adsorbed to E. coli and phage were removed from the serum.
- the serum fraction passed through the column was diluted 500 times with TBS containing 0.5% nonfat dry milk, and this was used as an immunoscreening material.
- phagemid host Escherichia coli prepared to have an absorbance OD 600 of 1.0 and 10 ⁇ l of the purified phage solution were mixed and reacted at 37 ° C. for 15 minutes, and 50 ⁇ l was treated with ampicillin (final concentration 50 ⁇ g / ml).
- SOLR phagemid host Escherichia coli
- the purified plasmid was analyzed for the full-length insert sequence by the primer walking method using the T3 primer shown in SEQ ID NO: 31 and the T7 primer shown in SEQ ID NO: 32.
- the gene sequences described in SEQ ID NOs: 5, 7, 9, 11, and 13 were obtained by this sequence analysis.
- the homology search program BLAST search http://www.ncbi.nlm.nih.gov/BLAST/
- BLAST search http://www.ncbi.nlm.nih.gov/BLAST/
- sequence identity of this gene with the gene encoding the human homologous factor was 94% for the nucleotide sequence and 98% for the amino acid sequence in the region translated into protein.
- the base sequences of human homologous factors are shown in SEQ ID NOs: 1 and 3, and amino acid sequences are shown in SEQ ID NOs: 2 and 4.
- sequence identity of the obtained canine gene with the gene encoding the bovine homologous factor was 94% for the nucleotide sequence and 97% for the amino acid sequence in the region translated into protein.
- the base sequence of the bovine homologous factor is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16.
- sequence identity between the gene encoding the human homologous factor and the gene encoding the bovine homologous factor was 94% in the base sequence and 93-97% in the amino acid sequence in the region translated into the protein. Further, the sequence identity of the obtained canine gene with the gene encoding the equine homologous factor was 93% nucleotide sequence and 97% amino acid sequence in the region translated into protein.
- the base sequence of the equine homologous factor is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18.
- sequence identity between the gene encoding the human homologous factor and the gene encoding the equine homologous factor was 93% nucleotide sequence and 96% amino acid sequence in the region translated into protein.
- sequence identity of the obtained canine gene with the gene encoding the mouse homologous factor was 87 to 89% of the base sequence and 95 to 97% of the amino acid sequence in the region translated into the protein.
- the base sequence of the mouse homologous factor is shown in SEQ ID NO: 19, 21, 23, 25, 27, and the amino acid sequence is shown in SEQ ID NO: 20, 22, 24, 26, 28.
- sequence identity between the gene encoding the human homologous factor and the gene encoding the mouse homologous factor was 89 to 91% of the base sequence and 95 to 96% of the amino acid sequence in the region translated into the protein.
- sequence identity with the gene which codes the chicken homologous factor of the acquired canine gene was the base sequence 82% and the amino acid sequence 87% in the area
- the nucleotide sequence of the chicken homologous factor is shown in SEQ ID NO: 29, and the amino acid sequence is shown in SEQ ID NO: 30.
- the sequence identity between the gene encoding the human homologous factor and the gene encoding the chicken homologous factor was 81 to 82% of the base sequence and 86% of the amino acid sequence in the region translated into the protein.
- the above gene-specific primers are 206 to 632 in the nucleotide sequence of SEQ ID NO: 5 (canine CAPRIN-1 gene) and 698 to 1124 in the nucleotide sequence of SEQ ID NO: 1 (human CAPRIN-1 gene). It was intended to amplify the base region.
- GAPDH specific primers (described in SEQ ID NOs: 35 and 36) were also used. As a result, as shown in FIG. 1, strong expression was observed in testis in healthy dog tissues, while expression was observed in canine breast cancer and adenocarcinoma tissues.
- the expression of the human homologous factor of the obtained gene was also confirmed, it was found only in the testis that the expression was confirmed in normal tissues as in the canine CAPRIN-1 gene, but in cancer cells, breast cancer, brain tumor, leukemia Expression was detected in many types of cancer cell lines such as lung cancer and esophageal cancer cell lines, and in particular, expression was confirmed in many breast cancer cell lines. From this result, it was confirmed that CAPRIN-1 was not expressed in normal tissues other than testis, but was expressed in many cancer cells, particularly in breast cancer cell lines.
- reference number 1 on the vertical axis indicates the expression pattern of the gene identified above, and reference number 2 indicates the expression pattern of the GAPDH gene as a comparative control.
- a CAPRIN-1-derived peptide (Arg-Asn-Leu-Glu-Lys-Lys-Lys-Gly-Lys-Leu) -Asp-Asp-Tyr-Gln (SEQ ID NO: 37)) was synthesized. 1 mg of this peptide was mixed with an equal volume of incomplete Freund's adjuvant (IFA) solution as an antigen, and this was administered subcutaneously to rabbits 4 times every 2 weeks. Thereafter, blood was collected to obtain an antiserum containing a polyclonal antibody.
- IFA incomplete Freund's adjuvant
- this antiserum was purified using a protein G carrier (manufactured by GE Healthcare Bioscience) to obtain a polyclonal antibody against the CAPRIN-1-derived peptide. Further, a serum obtained by purifying rabbit serum not administered with an antigen in the same manner as described above using a protein G carrier was used as a control antibody.
- a protein G carrier manufactured by GE Healthcare Bioscience
- the same operation as described above was performed using the control antibody prepared in the above (5) instead of the polyclonal antibody against the CAPRIN-1-derived peptide as a control.
- the cells to which the anti-human CAPRIN-1 antibody was added all had a fluorescence intensity of 30% or higher compared to the control.
- MDA-MB-157 is 184%
- T47D is 221%
- MRK-nu-1 is 115%
- MDA-MB-231V is 82%
- BT20 is 32%
- SK-BR-3 is 279%.
- MDA-MB-231T showed 80% enhancement of fluorescence intensity. This confirmed that the CAPRIN-1 protein was expressed on the cell membrane surface of the human cancer cell line.
- the enhancement rate of the fluorescence intensity is represented by the increase rate of the average fluorescence intensity (MFI value) in each cell, and was calculated by the following calculation formula.
- Average fluorescence intensity increase rate ((MFI value of cells reacted with anti-human CAPRIN-1 antibody) ⁇ (control MFI value)) ⁇ (control MFI value) ⁇ 100 (7) Immunohistochemical staining (7) -1 Expression of CAPRIN-1 in mice and normal dog tissues Mice (Balb / c, females) and dogs (beagle dogs, females) under ether anesthesia and ketamine / isoflurane anesthesia After exsanguination and laparotomy, each organ (stomach, liver, eyeball, thymus, muscle, bone marrow, uterus, small intestine, esophagus, heart, kidney, salivary gland, large intestine, mammary gland, brain, lung, skin, adrenal gland, ovary, pancreas, spleen , Bladder) were transferred to 10 cm dishes each containing PBS.
- Each organ was cut in PBS and fixed at reflux overnight with 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde (PFA). The reflux solution is discarded, and the tissue surface of each organ is rinsed with PBS.
- a PBS solution containing 10% sucrose is placed in a 50 ml centrifuge tube, and each tissue is placed therein and shaken at 4 ° C. for 2 hours using a rotor. That ’s it.
- the solution was replaced with a PBS solution containing 20% sucrose, allowed to stand at 4 ° C. until the tissue subsided, then replaced with a PBS solution containing 30% sucrose, and allowed to stand at 4 ° C. until the tissue subsided.
- the tissue was removed and the necessary part was cut out with a scalpel.
- an OCT compound manufactured by Tissue Tek
- Cryomold is placed on dry ice and rapidly frozen, then sliced into 10-20 ⁇ m using a cryostat (LEICA) and air-dried with a hair dryer for 30 minutes together with the slide glass, and the sliced tissue is placed.
- a slide glass was prepared.
- an operation of replacing PBS-T every 5 minutes in a staining bottle filled with PBS-T (physiological saline containing 0.05% Tween 20) was performed three times.
- the mouse tissue was MOM mouse Ig blocking reagent (VECTASTAIN) as the blocking solution, and the dog tissue was PBS containing 10% FBS.
- VECTASTAIN MOM mouse Ig blocking reagent
- Each -T solution was placed and allowed to stand at room temperature for 1 hour on a moist chamber.
- a monoclonal antibody (monoclonal antibody # 6) against CAPRIN-1 having a heavy chain variable region of SEQ ID NO: 73 and a light chain variable region of SEQ ID NO: 77 that reacts with the surface of cancer cells prepared in Example 4 is used.
- the solution prepared to 10 ⁇ g / ml with a blocking solution was placed and allowed to stand overnight at 4 ° C.
- a MOM biotin-labeled anti-IgG antibody manufactured by VECTASTAIN
- a blocking solution was placed and allowed to stand at room temperature for 1 hour in a moist chamber.
- avidin-biotin ABC reagent manufactured by VECTASTAIN was placed and allowed to stand at room temperature for 5 minutes in a moist chamber.
- a DAB coloring solution (DAB 10 mg + 30% H 2 O 2 10 ⁇ l / 0.05 M Tris-HCl (pH 7.6) 50 ml) was placed on it and placed in a moist chamber at room temperature for 30 minutes. Let stand for a minute. After rinsing with distilled water, a hematoxylin reagent (manufactured by DAKO) was placed and allowed to stand at room temperature for 1 minute, and then rinsed with distilled water. Each of the 70%, 80%, 90%, 95%, and 100% ethanol solutions was sequentially placed for 1 minute, and then allowed to stand overnight in xylene.
- DAB coloring solution (DAB 10 mg + 30% H 2 O 2 10 ⁇ l / 0.05 M Tris-HCl (pH 7.6) 50 ml) was placed on it and placed in a moist chamber at room temperature for 30 minutes. Let stand for a minute. After rinsing with distilled water, a hematoxylin reagent (manufacture
- CAPRIN-1 was slightly expressed in cells in the salivary gland, kidney, colon, and stomach tissues, but was not observed on the cell surface, and in tissues derived from other organs. No expression was observed. This result was the same when using a monoclonal antibody (monoclonal antibody # 9) against CAPRIN-1 having the heavy chain variable region of SEQ ID NO: 103 and the light chain variable region of SEQ ID NO: 107.
- Example 2 Anti-tumor effect of an antibody against CAPRIN-1 on cancer cells (ADCC activity) Next, it was examined whether antibodies against CAPRIN-1 can damage tumor cells expressing CAPRIN-1. Evaluation was carried out using a polyclonal antibody against the human CAPRIN-1-derived peptide prepared in Example 1. Caprin-1 2 kinds expression have been identified in human breast cancer cell lines, were collected T47D and MDA-MB-157 to centrifuge tubes 106 50ml ml respectively, incubated for 2 hours at 37 ° C. was added chromium 51 100 ⁇ Ci did. Thereafter, the plate was washed 3 times with RPMI 1640 medium containing 10% fetal bovine serum, and 10 3 pieces were added per well of a 96-well V-bottom plate.
- the cytotoxic activity was determined by mixing 10 3 leukemia cell lines incorporating the antibody against CAPRIN-1 used in the present invention, mouse lymphocytes and chromium 51 and culturing for 4 hours, It is the result of measuring the amount of chromium 51 released into the culture medium after culturing and showing the cytotoxic activity against each leukemia cell line calculated by the following calculation formula * .
- Cytotoxic activity (%) chromium 51 release from T47D and MDA-MB-157 upon addition of antibody against CAPRIN-1 and mouse lymphocytes ⁇ chromium 51 release from target cells with 1N hydrochloric acid added Amount x100.
- Example 3 Production of Novel Human Cancer Antigen Protein
- a recombinant protein of human homologous gene was produced by the following method. For PCR, 1 ⁇ l of a cDNA whose expression by RT-PCR method was confirmed from various tissue and cell cDNAs prepared in Example 1 and two kinds of primers containing SacI and XhoI restriction enzyme cleavage sequences (described in SEQ ID NOs: 38 and 39).
- the purified DNA fragment was ligated to a cloning vector PCR-Blunt (manufactured by Invitrogen). After transforming this into E. coli, the plasmid was recovered, and it was confirmed by sequencing that the amplified gene fragment matched the target sequence.
- a plasmid corresponding to the target sequence was treated with SacI and XhoI restriction enzymes, purified with QIAquick Gel Extraction Kit, and then inserted into an expression vector pET30a (manufactured by Novagen) for Escherichia coli treated with SacI and XhoI restriction enzymes. . By using this vector, a His-tagged recombinant protein can be produced. This plasmid was transformed into E.
- the cells were suspended in phosphate buffered physiological saline and sonicated on ice.
- the E. coli sonication solution was centrifuged at 6000 rpm for 20 minutes, and the resulting supernatant was used as a soluble fraction and the precipitate was used as an insoluble fraction.
- the soluble fraction was added to a nickel chelate column (carrier: Chelating Sepharose (trademark) Fast Flow (GE HealthCare), column volume 5 ml, equilibration buffer 50 mM hydrochloric acid buffer (pH 8.0)) prepared according to a conventional method.
- the unadsorbed fraction was washed with 10 mM column volume of 50 mM hydrochloric acid buffer (pH 8.0) and 20 mM imidazole-containing 20 mM phosphate buffer (pH 8.0), and immediately, 100 mM imidazole-containing 20 mM.
- Six beds were eluted with phosphate buffer (pH 8.0).
- the unadsorbed fraction was washed with 20 mM phosphate buffer solution (pH 7.0) 10 times the column volume and 20 mM phosphate buffer solution (pH 7.0) containing 200 mM sodium chloride, and immediately, 400 mM chloride was added. Five bed elution was performed with sodium-containing 20 mM phosphate buffer (pH 7.0) to obtain a purified fraction of each protein having the amino acid sequence shown in SEQ ID NO: 2.
- Example 4 Production of Monoclonal Antibody to CAPRIN-1 100 ⁇ g of the antigen protein (human CAPRIN-1) shown in SEQ ID NO: 2 prepared in Example 3 was mixed with an equal amount of MPL + TDM adjuvant (manufactured by Sigma), Antigen solution per mouse was used. After the antigen solution was administered intraperitoneally to 6-week-old Balb / cc mice (Japan SLC), immunization was completed by further 3 and 24 administrations per week. Each spleen removed 3 days after the last immunization was crushed between two sterilized glass slides, washed with PBS (-) (manufactured by Nissui), centrifuged at 1500 rpm for 10 minutes, and the supernatant was obtained.
- PBS -
- the removal operation was repeated 3 times to obtain spleen cells.
- the obtained spleen cells and mouse myeloma cells SP2 / 0 (purchased from ATCC) were mixed at a ratio of 10: 1, and 200 ⁇ l of RPMI 1640 medium containing 10% FBS heated to 37 ° C. and PEG 1500 (Boehringer) PEG solution prepared by mixing 800 ⁇ l was added and allowed to stand for 5 minutes for cell fusion.
- the cells were suspended in 150 ml of RPMI 1640 medium (HAT selective medium) containing 15% FBS to which 2% equivalent of Gibco's HAT solution was added, and a 96-well plate (Nunk) 100 ⁇ l per well of (made) was seeded on 15 plates.
- RPMI 1640 medium HAT selective medium
- Nunk 96-well plate
- Hybridomas were selected using as an index the binding affinity of the antibody produced by the prepared hybridomas to the CAPRIN-1 protein.
- 100 ⁇ l of 1 ⁇ g / ml of the CAPRIN-1 protein solution prepared in Example 3 was added per well of a 96-well plate, and allowed to stand at 4 ° C. for 18 hours. Each well was washed 3 times with PBS-T, and then added with 400 ⁇ l of 0.5% Bovine Serum Albumin (BSA) solution (manufactured by Sigma) per well and allowed to stand at room temperature for 3 hours.
- BSA Bovine Serum Albumin
- the selected hybridomas were added to the plate at a ratio of 0.5 per well of the 96-well plate and cultured. One week later, hybridomas forming a single colony in the well were observed. The cells in these wells were further cultured, and hybridomas were selected using the binding affinity of the antibody produced by the cloned hybridomas to the CAPRIN-1 protein as an index. 100 ⁇ l of 1 ⁇ g / ml of the CAPRIN-1 protein solution prepared in Example 3 was added per well of a 96-well plate, and allowed to stand at 4 ° C. for 18 hours. After washing each well 3 times with PBS-T, 400 ⁇ l of 0.5% BSA solution was added per well and allowed to stand at room temperature for 3 hours.
- Example 5 Characterization of selected antibodies (1) Cloning of variable region gene of anti-CAPRIN-1 monoclonal antibody mRNA was extracted from each hybridoma strain producing each of the 11 monoclonal antibodies selected in Example 4, and mouse The RT-PCR method using primers specific to the FR1-derived sequence and the mouse FR4-derived sequence was used to determine the heavy chain variable (VH) region and light chain variable (VL) region genes of all anti-CAPRIN-1 monoclonal antibodies. I got it. The genes were cloned into pCR2.1 vector (Invitrogen) for sequencing.
- RT-PCR MRNA was prepared from 10 6 hybridoma strains using mRNA micro purification kit (manufactured by GE Healthcare), and the resulting mRNA was reverse transcribed using SuperScript II 1st strand synthesis kit (manufactured by Invitrogen). cDNA was synthesized. These operations were performed according to the protocol attached to each kit.
- antibody genes were amplified by PCR.
- a primer specific to the mouse heavy chain FR1 sequence SEQ ID NO: 130
- a primer specific to the mouse heavy chain FR4 sequence SEQ ID NO: 131
- a primer specific for the mouse light chain FR1 sequence SEQ ID NO: 132
- a primer specific for the mouse light chain FR4 SEQ ID NO: 133
- a cDNA sample was added to 5 ⁇ l of 10 ⁇ EX Taq Buffer, 4 ⁇ l of dNTP Mixture (2.5 mM), 2 ⁇ l of each primer (1.0 ⁇ M) and 0.25 ⁇ l of Ex Taq (5 U / ⁇ l), and the total amount was made 50 ⁇ l with sterilized water. After treatment at 94 ° C. for 2 minutes, denaturation at 94 ° C. for 1 minute, annealing at 58 ° C. for 30 seconds, and extension reaction at 72 ° C. for 1 minute were performed under 30 cycles.
- the amino acid sequence of the heavy chain variable region of the obtained monoclonal antibody is shown in SEQ ID NO: 43, SEQ ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 93, SEQ ID NO: 103, SEQ ID NO: 113, and SEQ ID NO: 123 as the amino acid of the light chain variable region.
- the sequences are shown in SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 63, SEQ ID NO: 68, SEQ ID NO: 77, SEQ ID NO: 87, SEQ ID NO: 97, SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 127.
- monoclonal antibody # 1 is composed of a heavy chain variable region of SEQ ID NO: 43 and a light chain variable region of SEQ ID NO: 47
- # 2 is a heavy chain variable region of SEQ ID NO: 43 and light chain variable of SEQ ID NO: 53.
- # 3 consists of a heavy chain variable region of SEQ ID NO: 43 and a light chain variable region of SEQ ID NO: 58
- # 4 consists of a heavy chain variable region of SEQ ID NO: 43 and a light chain variable of SEQ ID NO: 63
- # 5 consists of a heavy chain variable region of SEQ ID NO: 43 and a light chain variable region of SEQ ID NO: 68
- # 6 consists of a heavy chain variable region of SEQ ID NO: 73 and a light chain variable region of SEQ ID NO: 77
- # 7 consists of a heavy chain variable region of SEQ ID NO: 83 and a light chain variable region of SEQ ID NO: 87
- # 8 consists of a heavy chain variable region of SEQ ID NO: 93 and a light chain variable of SEQ ID NO: 97 # 9 is the heavy chain variable region of SEQ ID NO: 103 and SEQ ID NO: 103 Consisting of a light chain variable region of SEQ ID NO: 113 and a
- IN-1 protein was examined whether the expression. 10 6 cells of each cell line were centrifuged in a 1.5 ml microcentrifuge tube. To this was added a hybridoma supernatant (100 ⁇ l) containing monoclonal antibodies against CAPRIN-1 of # 1 to # 11, which reacts with the cancer cell surface prepared in Example 4, and allowed to stand on ice for 1 hour. After washing with PBS, the suspension was suspended in FITC-labeled goat anti-mouse IgG antibody (manufactured by Invitrogen) diluted 500-fold with PBS containing 0.1% FBS, and allowed to stand on ice for 1 hour. After washing with PBS, the fluorescence intensity was measured with a FACS caliber from Becton Dickinson.
- the same operation as described above was performed using the control antibody prepared in (5) above instead of the hybridoma supernatant containing monoclonal antibodies against CAPRIN-1 of # 1 to # 11, and used as a control.
- the cells to which the monoclonal antibodies # 1 to # 11 were added all had a fluorescence intensity of 30% or more compared to the control.
- MDA-MB-157 was 211%
- T47D was 145%
- MRK-nu-1 was 123%
- MDA-MB-231V was 251%
- BT20 168% and MDA-MB-231T showed 94% enhancement of fluorescence intensity.
- the enhancement rate of the fluorescence intensity is represented by the increase rate of the average fluorescence intensity (MFI value) in each cell, and was calculated by the following calculation formula.
- Average fluorescence intensity increase rate ((MFI value of cells reacted with anti-human CAPRIN-1 antibody) ⁇ (control MFI value)) ⁇ (control MFI value) ⁇ 100 (3)
- Antitumor effect of antibodies against CAPRIN-1 on cancer cells (ADCC activity)
- the cytotoxic activity (ADCC activity) against cancer cells of the monoclonal antibodies against CAPRIN-1 of # 1 to # 11 selected above was evaluated.
- Each hybridoma producing the monoclonal antibodies # 1 to # 11 was cultured using a hybridoma SFM (manufactured by Invitrogen) medium, and the resulting supernatant was purified using Hitrap Protein A Sepharose FF (manufactured by GE Healthcare).
- the amount of chromium 51 in the culture supernatant released from the damaged tumor cells was measured, and the ADCC activity against the cancer cells by the anti-CAPRIN-1 antibody was calculated.
- all of the monoclonal antibodies # 1 to # 11 showed ADCC activity of 20% or more against MDA-MB-157.
- the cytotoxic activity of # 1 was 22.1%
- # 2 was 29.1%
- # 6 was 30.2%
- # 9 was 32.4% (see FIG. 4). ).
- CDC activity Antitumor effect of antibodies against CAPRIN-1 on cancer cells
- the cytotoxic activity (CDC activity) against the cancer cells of the monoclonal antibodies against # 1 to # 11 CAPRIN-1 selected above was evaluated.
- Blood collected from a rabbit was placed in an Eppendorf tube, allowed to stand at room temperature for 60 minutes, and then centrifuged at 3000 rpm for 5 minutes to prepare serum for measuring CDC activity.
- 10 5 human breast cancer cells, MDA-MB-231V were collected in a 50 ml centrifuge tube, added with 100 ⁇ Ci of chromium 51, incubated at 37 ° C. for 2 hours, and then washed 3 times with RPMI medium containing 10% FBS. .
- the suspension was suspended in RPMI medium containing 50% of the rabbit serum prepared above, and 10 3 pieces were added per well of a 96-well V-bottom plate.
- 1 ⁇ g of each of the monoclonal antibodies # 1 to # 11 obtained in the above (3) was added thereto and cultured at 37 ° C. under 5% CO 2 for 4 hours.
- the amount of chromium 51 in the culture supernatant released from the damaged tumor cells was measured, and the CDC activity against MDA-MB-231V by the anti-CAPRIN-1 monoclonal antibody in the hybridoma supernatant was calculated.
- all of the monoclonal antibodies # 1 to # 11 showed CDC activity of 30% or more.
- Example 6 Anti-Tumor Effect of Anti-CAPRIN-1 Monoclonal Antibody in Mice In Vivo Next, the anti-tumor effect of the obtained monoclonal antibodies against # 1 to # 11 CAPRIN-1 in the tumor-bearing mouse in vivo was evaluated.
- the antibody used was obtained by column purification of the culture supernatant of each hybridoma in the same manner as described above.
- Antitumor effects of monoclonal antibodies against # 1 to # 11 CAPRIN-1 were examined using tumor-bearing mice transplanted with cancer cell lines derived from mice expressing CAPRIN-1. Subcutaneously into the back of 70 Balb / c mice (manufactured by Nippon SLC Co., Ltd.), were implanted with 10 6 CT26 cells per mouse (purchased from ATCC), tumors were allowed to grow to a size of about 7mm diameter . Among them, 60 cancer-bearing mice react with the monoclonal antibodies against CAPRIN-1 of # 1 to # 11 and the CAPRIN-1 protein itself prepared in Example 4, but on the cell surface of cancer cells.
- One non-reacting monoclonal antibody was intraperitoneally administered to 300 mice (300 ⁇ l) per mouse, 5 mice per antibody. Thereafter, the same amount of each antibody was administered to the abdominal cavity of each cancer-bearing mouse 3 times in total for 2 days, and the tumor size was measured every day to observe the antitumor effect. On the other hand, the remaining 10 tumor-bearing mice were administered PBS ( ⁇ ) instead of the antibody, and this was used as a control group. As a result of the observation of the antitumor effect, the study group administered with the monoclonal antibody against CAPRIN-1 of # 1 to # 11 had a tumor volume of 50% on the 4th day when the tumor volume at the start of antibody administration was 100%.
- the tumor regressed to about 10% on the 6th day, about 10% on the 6th day, to several% on the 8th day, and the tumor regressed almost completely by the 11th to 14th days (see FIG. 5).
- tumors increased to about 260%, 350%, 550%, and 800% on the 4th day, 6th day, 8th day, and 11th day, respectively (see FIG. 5).
- the antitumor effect was not obtained, and the tumor increased as in the control group.
- the obtained monoclonal antibodies against CAPRIN-1 of # 1 to # 11 exhibited a strong antitumor effect in vivo against cancer cells expressing CAPRIN-1.
- the size of the tumor calculated the volume using the calculation formula of major axis x minor axis x minor axis x 0.5.
- Example 7 Identification of a peptide in CAPRIN-1 protein to which an antibody against CAPRIN-1 that reacts with the cell surface of cancer cells binds CAPRIN-1 of # 1 to # 11 that reacts with the cell surface of cancer cells obtained above Using monoclonal antibodies against, the partial sequences in the CAPRIN-1 proteins that they recognize were identified.
- DTT (manufactured by Fluka) is added to 100 ⁇ l of a recombinant CAPRIN-1 protein solution dissolved in PBS to a concentration of 1 ⁇ g / ⁇ l so as to have a final concentration of 10 mM. Reduction of the disulfide bond in one protein was performed, and then iodoacetamide (manufactured by Wako Pure Chemical Industries, Ltd.) having a final concentration of 20 mM was added, and an alkylation reaction of a thiol group was performed for 30 minutes at 37 ° C. under light shielding conditions.
- trypsin manufactured by Promega
- 1% BSA manufactured by Sigma
- Protein A-glass beads blocked with PBS containing, washed with PBS, 1 mM calcium carbonate, NP-40 buffer (20 mM phosphate buffer (pH 7.4), 5 mM EDTA, 150 mM NaCl, 1% NP-40) and reacted for 30 minutes each.
- the antigen-antibody complex was eluted using 100 ⁇ l of 0.1% formic acid, and Q-TOF Premier (manufactured by Waters-MicroMass) was used as the eluate. LC-MS analysis was performed using this. The analysis followed the protocol attached to the instrument.
- polypeptide of SEQ ID NO: 136 was identified as a partial sequence of CAPRIN-1 recognized by any of the monoclonal antibodies against CAPRIN-1 of # 1 to # 11. Furthermore, the peptide of SEQ ID NO: 137 was identified as a partial sequence in the polypeptide of SEQ ID NO: 136 recognized by the monoclonal antibodies # 2 to # 5, # 6 to # 8 and # 10, and this partial sequence peptide It was found that monoclonal antibodies # 2 to # 5 recognize the peptide of SEQ ID NO: 138.
- the antibody of the present invention is useful for the treatment and / or prevention of cancer.
Abstract
Description
(a)配列番号40、41および42を含む重鎖可変領域と配列番号44、45および46を含む軽鎖可変領域とを含む抗体。
本発明で用いられるCAPRIN-1に対する抗体を取得するための感作抗原として使用されるタンパク質又はその断片は、ヒト、イヌ、ウシ、ウマ、マウス、ラット、ニワトリなど、その由来となる動物種に制限されない。しかし細胞融合に使用する親細胞との適合性を考慮して選択することが好ましく、一般的には、哺乳動物由来のタンパク質が好ましく、特にヒト由来のタンパク質が好ましい。例えば、CAPRIN-1がヒトCAPRIN-1の場合、ヒトCAPRIN-1タンパク質やその部分ペプチド、ヒトCAPRIN-1を発現する細胞などを用いることができる。
抗体は通常少なくとも2本の重鎖および2本の軽鎖を含むヘテロ多量体糖タンパク質である。IgMは別として、2本の同一の軽(L)鎖および2本の同一の重(H)鎖で構成される約150kDaのヘテロ四量体糖タンパク質である。典型的には、それぞれの軽鎖は1つのジスルフィド共有結合により重鎖に連結されているが、種々の免疫グロブリンアイソタイプの重鎖間のジスルフィド結合の数は変動する。それぞれの重鎖および軽鎖はまた鎖内ジスルフィド結合も有する。それぞれの重鎖は一方の端に可変ドメイン(VH領域)を有し、それにいくつかの定常領域が続く。それぞれ軽鎖は可変ドメイン(VL領域)を有し、その反対の端に1つの定常領域を有する。軽鎖の定常領域は重鎖の最初の定常領域と整列しており、かつ軽鎖可変ドメインは重鎖の可変ドメインと整列している。抗体の可変ドメインは特定の領域が相補性決定領域(CDR)と呼ばれる特定の可変性を示して抗体に結合特異性を付与する。可変領域の相対的に保存されている部分はフレームワーク領域(FR)と呼ばれている。完全な重鎖および軽鎖の可変ドメインはそれぞれ3つのCDRにより連結された4つのFRを含む。3つのCDRは重鎖ではそのN末から順にCDRH1,CDRH2,CDRH3、同様に軽鎖ではCDRL1,CDRL2,CDRL3と呼ばれている。抗体の抗原への結合特異性には、CDRH3が最も重要である。また、各鎖のCDRはFR領域によって近接した状態で一緒に保持され、他方の鎖からのCDRと共に抗体の抗原結合部位の形成に寄与する。定常領域は抗体が抗原に結合することに直接寄与しないが、種々のエフェクター機能、例えば、抗体依存性細胞性細胞障害活性(ADCC)への関与、Fcγ受容体への結合を介した食作用、新生児Fc受容体(FcRn)を介した半減期/クリアランス速度、補体カスケードのC1q構成要素を介した補体依存性細胞障害(CDC)を示す。
本発明における抗CAPRIN-1抗体とは、CAPRIN-1タンパク質の全長又はその断片と免疫学的反応性を有する抗体を意味する。
(j)配列番号110,111および112を含む重鎖可変領域と配列番号114,115および116を含む軽鎖可変領域とを含む抗体(好ましくは配列番号113の重鎖可変領域および配列番号117の軽鎖可変領域で構成される抗体)。
抗体がCAPRIN-1に結合する能力は、実施例で述べられるようなたとえば ELISA、ウエスタンブロット法、免疫蛍光およびフローサイトメトリー分析などを用いた結合アッセイを利用して特定することができる。
CAPRIN-1を認識する抗体は、当業者に周知の方法での免疫組織化学により、外科手術の間に患者から得た組織や、自然にまたはトランスフェクション後にCAPRIN-1を発現する細胞系を接種した異種移植組織を担持する動物から得た組織から、パラホルムアルデヒドまたはアセトン固定した凍結切片またはパラホルムアルデヒドで固定したパラフィン包埋した組織切片を使用して、CAPRIN-1との反応性に関して試験することができる。
本発明の癌の治療及び/又は予防のための医薬組成物の標的は、CAPRIN-1遺伝子を発現する癌(細胞)であれば特に限定されない。
本発明は更に、上記抗体(a)~(k)に関わる以下のポリペプチド及びDNAも提供する。
(1)cDNAライブラリーの作製
健常な犬の精巣組織から酸グアニジウム-フェノール-クロロホルム法(Acid guanidium-Phenol-Chloroform法)により全RNAを抽出し、Oligotex-dT30 mRNA purification Kit(宝酒造社製)を用いてキット添付のプロトコールに従ってポリA RNAを精製した。
上記作製したイヌ精巣cDNAファージライブラリーを用いて、イムノスクリーニングを行った。具体的にはΦ90×15mmのNZYアガロースプレートに2210クローンとなるように宿主大腸菌(XL1-Blue MRF’)に感染させ、42℃、3~4時間培養し、溶菌斑(プラーク)を作らせ、IPTG(イソプロピル-β-D-チオガラクトシド)を浸透させたニトロセルロースメンブレン(Hybond C Extra: GE Healthcare Bio-Scinece社製)でプレートを37℃で4時間覆うことによりタンパク質を誘導・発現させ、メンブレンにタンパク質を転写した。その後メンブレンを回収し0.5%脱脂粉乳を含むTBS(10mM Tris-HCl, 150mM NaCl pH7.5)に浸し4℃で一晩振盪することによって非特異反応を抑制した。このフィルターを500倍希釈した患犬血清と室温で2~3時間反応させた。
上記方法により単離した5個の陽性クローンを塩基配列解析に供するため、ファージベクターからプラスミドベクターに転換する操作を行った。具体的には宿主大腸菌(XL1-Blue MRF’)を吸光度OD600が1.0となるよう調製した溶液200μlと、精製したファージ溶液250μlさらにExAssist helper phage(STRATAGENE社製)1μlを混合した後37℃で15分間反応後、LB培地を3ml添加し37℃で2.5~3時間培養を行い、直ちに70℃の水浴にて20分間保温した後、4℃、1000×g、15分間遠心分離を行い上清をファージミド溶液として回収した。次いでファージミド宿主大腸菌(SOLR)を吸光度OD600が1.0となるよう調製した溶液200μlと、精製したファージ溶液10μlを混合した後37℃で15分間反応させ、50μlをアンピシリン(終濃度50μg/ml)含有LB寒天培地に播き37℃一晩培養した。トランスフォームしたSOLRのシングルコロニーを採取し、アンピシリン(終濃度50μg/ml)含有LB培地37℃にて培養後、QIAGEN plasmid Miniprep Kit(キアゲン社製)を使って目的のインサートを持つプラスミドDNAを精製した。
上記方法により得られた遺伝子に対しイヌ及びヒトの正常組織及び各種細胞株における発現をRT-PCR法により調べた。逆転写反応は以下の通り行なった。すなわち、各組織50~100mg及び各細胞株5~10×106個の細胞からTRIZOL試薬(invitrogen社製)を用いて添付のプロトコールに従い全RNAを抽出した。この全RNAを用いてSuperscript First-Strand Synthesis System for RT-PCR(invitrogen社製)により添付のプロトコールに従いcDNAを合成した。PCR反応は、取得した遺伝子特異的なプライマー(配列番号33及び34に記載)を用いて以下の通り行った。すなわち、逆転写反応により調製したサンプル0.25μl、上記プライマーを各2μM、0.2mM各dNTP、0.65UのExTaqポリメラーゼ(宝酒造社製)となるように各試薬と添付バッファーを加え全量を25μlとし、Thermal Cycler(BIO RAD社製)を用いて、94℃-30秒、60℃-30秒、72℃-30秒のサイクルを30回繰り返して行った。なお、上記遺伝子特異的プライマーは、配列番号5の塩基配列(イヌCAPRIN-1遺伝子)中の206番~632番及び配列番号1の塩基配列(ヒトCAPRIN-1遺伝子)中の698番~1124番塩基の領域を増幅するものであった。比較対照のため、GAPDH特異的なプライマー(配列番号35及び36に記載)も同時に用いた。その結果、図1に示すように、健常なイヌ組織では精巣に強い発現が見られ、一方イヌ乳癌及び腺癌組織で発現が見られた。さらに、取得した遺伝子のヒト相同因子の発現を併せて確認したところ、イヌCAPRIN-1遺伝子と同様、正常組織で発現が確認できたのは精巣のみだったが、癌細胞では乳癌、脳腫瘍、白血病、肺癌、食道癌細胞株など、多種類の癌細胞株で発現が検出され、特に多くの乳癌細胞株で発現が確認された。この結果から、CAPRIN-1は精巣以外の正常組織では発現が見られず、一方、多くの癌細胞で発現しており、特に乳癌細胞株に発現していることが確認された。
CAPRIN-1に結合する抗体を得るために、CAPRIN-1由来ペプチド(Arg-Asn-Leu-Glu-Lys-Lys-Lys-Gly-Lys-Leu-Asp-Asp-Tyr-Gln(配列番号37))を合成した。このペプチド1mgを抗原として、等容量の不完全フロイントアジュバント(IFA)溶液と混合し、これを2週間毎に4回、ウサギの皮下に投与を行った。その後血液を採取し、ポリクローナル抗体を含む抗血清を得た。さらにこの抗血清をプロテインG担体(GEヘルスケアバイオサイエンス社製)を用いて精製し、CAPRIN-1由来ペプチドに対するポリクローナル抗体を得た。また、抗原を投与していないウサギの血清を上記と同様にしてプロテインG担体を用いて精製したものをコントロール抗体とした。
次にCAPRIN-1遺伝子の発現が多く確認された乳癌細胞株7種(MDA-MB-157, T47D, MRK-nu-1, MDA-MB-231V, BT20, SK-BR-3, MDA-MB-231T)について、その細胞表面上にCAPRIN-1タンパク質が発現しているかどうかを調べた。上記で遺伝子発現が認められた各ヒト乳癌細胞株それぞれ106細胞を1.5mlのミクロ遠心チューブにて遠心分離した。これに上記(5)で調製したCAPRIN-1由来ペプチドに対するポリクローナル抗体2μg(5μl)を添加し、さらに95μlの0.1%牛胎児血清を含むPBSで懸濁後、氷上で1時間静置した。PBSで洗浄した後、5μlのFITC標識ヤギ抗ラビットIgG抗体(サンタクルズ社製)及び95μlの0.1% 牛胎児血清(FBS)を含むPBSで懸濁し、氷上で1時間静置した。PBSで洗浄後、ベクトンディッキンソン株式会社のFACSキャリバーにて蛍光強度を測定した。一方、上記と同様の操作を、CAPRIN-1由来ペプチドに対するポリクローナル抗体の代わりに上記(5)で調製したコントロール抗体を用いて行い、コントロールとした。その結果、抗ヒトCAPRIN-1抗体を添加された細胞は、コントロールに比べて、いずれも蛍光強度が30%以上強かった。具体的には、MDA-MB-157が184%、T47Dが221%、MRK-nu-1が115%、MDA-MB-231Vが82%、BT20が32%、SK-BR-3が279%、MDA-MB-231Tが80%の蛍光強度の増強を示した。このことから、上記ヒト癌細胞株の細胞膜表面上にCAPRIN-1タンパクが発現していることが確認された。なお、上記蛍光強度の増強率は、各細胞における平均蛍光強度(MFI値)の増加率にて表され、以下の計算式により算出した。
(7)免疫組織化学染色
(7)-1 マウスおよびイヌ正常組織におけるCAPRIN-1の発現
マウス(Balb/c、雌)およびイヌ(ビーグル犬、雌)をエーテル麻酔下およびケタミン/イソフルラン麻酔下で放血させ、開腹後、各臓器(胃、肝臓、眼球、胸腺、筋肉、骨髄、子宮、小腸、食道、心臓、腎臓、唾液腺、大腸、乳腺、脳、肺、皮膚、副腎、卵巣、膵臓、脾臓、膀胱)をそれぞれPBSの入った10cmディッシュに移した。PBS中で各臓器を切り開き、4% paraformaldehyde(PFA)を含む0.1M リン酸緩衝液(pH7.4)で一晩還流固定した。還流液を捨て、PBSで各臓器の組織表面をすすぎ、10%ショ糖を含むPBS溶液を50ml容の遠心チューブに入れ、その中に各組織を入れて4℃で2時間ローターを用いて振とうした。20%ショ糖を含むPBS溶液に入れ替え、4℃で組織が沈むまで静置後、30%ショ糖を含むPBS溶液に入れ替え、4℃で組織が沈むまで静置した。組織を取り出し、必要な部分を手術用メスで切りだした。次に、OCTコンパウンド(Tissue Tek社製)をかけて組織表面になじませた後、クライオモルドに組織を配置した。ドライアイスの上にクライオモルドをおいて急速凍結させた後、クライオスタット(LEICA社製)を用いて10~20μmに薄切し、スライドガラスごとヘアードライアーで30分間風乾し、薄切組織がのったスライドガラス作製した。次にPBS-T(0.05% Tween20を含む生理食塩水)を満たした染色瓶に入れて5分ごとにPBS-Tを入れ替える操作を3回行った。切片周囲の余分な水分をキムワイプでふき取り、DAKOPEN(DAKO社製)で囲んだ後、ブロッキング液として、マウス組織はMOMマウスIgブロッキング試薬(VECTASTAIN社製)を、イヌ組織は10%FBSを含むPBS-T溶液をそれぞれのせ、モイストチャンバー上で室温で1時間静置した。次に、実施例4で作製した癌細胞表面に反応する、配列番号:73の重鎖可変領域と配列番号:77の軽鎖可変領域を有するCAPRIN-1に対するモノクローナル抗体(モノクローナル抗体#6)をブロッキング液で10μg/mlに調製した溶液をのせ、モイストチャンバー内で4℃下で一晩静置した。PBS-Tで10分間3回洗浄を行った後、ブロッキング液で250倍に希釈したMOMビオチン標識抗IgG抗体(VECTASTAIN社製)をのせ、モイストチャンバー内で室温で1時間静置した。PBS-Tで10分間3回洗浄を行った後、アビジンービオチンABC試薬(VECTASTAIN社製)をのせ、モイストチャンバー内で室温で5分間静置した。PBS-Tで10分間3回洗浄を行った後、DAB発色液(DAB 10mg+30% H2O2 10μl/0.05M Tris-HCl(pH7.6)50ml)をのせ、モイストチャンバー内で室温で30分間静置した。蒸留水でリンスし、ヘマトキシリン試薬(DAKO社製)を載せて室温で1分間静置後、蒸留水でリンスした。70%、80%、90%、95%、100%の各エタノール溶液に順番に1分間ずつ入れた後、キシレン中で一晩静置した。スライドガラスを取り出し、Glycergel Mounting Medium(DAKO社製)で封入後、観察を行った。その結果、CAPRIN-1は、唾液腺、腎臓、結腸、胃の各組織において細胞内で僅かに発現が認められたが、細胞表面での発現は認められず、また、その他の臓器由来の組織では全く発現が認められなかった。なお、本結果は、配列番号:103の重鎖可変領域と配列番号:107の軽鎖可変領域を有するCAPRIN-1に対するモノクローナル抗体(モノクローナル抗体#9)を用いた場合も同様であった。
病理診断で悪性乳癌と診断されたイヌの凍結された乳癌組織108検体を用いて、上述と同様の方法で凍結切片スライド作製および実施例4で作製したモノクローナル抗体#6を用いた免疫組織化学染色を行った。その結果、CAPRIN-1は108検体中100検体(92.5%)で発現が確認され、特に異型度の高い癌細胞表面に強く発現していた。なお、本結果は、実施例4で作製したモノクローナル抗体#9を用いた場合も同様であった。
パラフィン包埋されたヒト乳癌組織アレイ(BIOMAX社製)の乳癌組織188検体を用いて、免疫組織化学染色を行った。ヒト乳癌組織アレイを60℃で3時間処理後、キシレンを満たした染色瓶に入れて5分ごとにキシレンを入れ替える操作を3回行った。次にキシレンの代わりにエタノールおよびPBS-Tで同様の操作を行った。0.05% Tween20を含む10mM クエン酸緩衝液(pH6.0)を満たした染色瓶にヒト乳癌組織アレイを入れ、125℃で5分間処理後、室温で40分以上静置した。切片周囲の余分な水分をキムワイプでふき取り、DAKOPENで囲み、Peroxidase Block(DAKO社製)を適量滴下した。室温で5分間静置後、PBS-Tを満たした染色瓶に入れて5分ごとにPBS-Tを入れ替える操作を3回行った。ブロッキング液として、10%FBSを含むPBS-T溶液をのせ、モイストチャンバー内で室温で1時間静置した。次に実施例4で作製した癌細胞表面に反応するモノクローナル抗体#6を5%FBSを含むPBS-T溶液で10μg/mlに調製した溶液をのせ、モイストチャンバー内で4℃で一晩静置し、PBS-Tで10分間3回洗浄を行った後、Peroxidase Labelled Polymer Conjugated(DAKO社製)適量滴下し、モイストチャンバー内で室温で30分間静置した。PBS-Tで10分間3回洗浄を行った後、DAB発色液(DAKO社製)をのせ、室温で10分程度静置した後、発色液を捨て、PBS-Tで10分間3回洗浄を行った後、蒸留水でリンスし、70%、80%、90%、95%、100%の各エタノール溶液に順番に1分間ずつ入れた後、キシレン中で一晩静置した。スライドガラスを取り出し、Glycergel Mounting Medium(DAKO社製)で封入後、観察を行った。その結果、CAPRIN-1は全乳癌組織188検体の内、138検体(73%)で強い発現が認められた。なお、本結果は、実施例4で作製したモノクローナル抗体#9を用いた場合も同様であった。
パラフィン包埋されたヒト悪性脳腫瘍組織アレイ(BIOMAX社製)の悪性脳腫瘍組織247検体を用いて、上述(7)-3と同様の方法で実施例4で作製したモノクローナル抗体#6を用いた免疫組織化学染色を行った。その結果、CAPRIN-1は全悪性脳腫瘍組織247検体の内、227検体(92%)で強い発現が認められた。なお、本結果は、実施例4で作製したモノクローナル抗体#9を用いた場合も同様であった。
パラフィン包埋されたヒト乳癌転移リンパ節組織アレイ(BIOMAX社製)の乳癌転移リンパ節組織150検体を用いて、上述(7)-3と同様の方法で実施例4で作製したモノクローナル抗体#6を用いた免疫組織化学染色を行った。その結果、CAPRIN-1は全乳癌転移リンパ節組織150検体の内、136検体(90%)で強い発現が認められた。すなわち、乳癌から転移した癌組織においてもCAPRIN-1は強く発現することが判った。なお、本結果は、実施例4で作製したモノクローナル抗体#9を用いた場合も同様であった。
次にCAPRIN-1に対する抗体が、CAPRIN-1を発現する腫瘍細胞を障害することができるかどうかを検討した。実施例1で調製したヒトCAPRIN-1由来ペプチドに対するポリクローナル抗体を用いて評価を行った。CAPRIN-1の発現が確認されている2種類のヒト乳癌細胞株、T47D及びMDA-MB-157をそれぞれ106個50ml容の遠心チューブに集め、100μCiのクロミウム51を加え37℃で2時間インキュベートした。その後10%牛胎児血清を含むRPMI1640培地で3回洗浄し、96穴V底プレート1穴あたり103個ずつ添加した。これに、上記ヒトCAPRIN-1由来ペプチドに対するポリクローナル抗体をそれぞれ1μgずつ添加し、さらにウサギの末梢血から分離したリンパ球をそれぞれ2×105個ずつ添加して、37℃、5% CO2の条件下で4時間培養した。培養後、障害を受けた腫瘍細胞から放出される培養上清中のクロミウム(Cr)51の量を測定し、ヒトCAPRIN-1由来ペプチドに対するポリクローナル抗体による各癌細胞に対するADCC活性を算出した。その結果、T47D及びMDA-MB-157それぞれに対して、それぞれ15.4%及び17.3%のADCC活性が確認された(図2及び図3参照)。一方、抗原が免疫されていないウサギの末梢血から調製したコントロール抗体(実施例1(5))を用いて同様の操作を行った場合、及び抗体を添加しなかった場合には、活性はほとんど認められなかった(図2及び3参照)。従って、CAPRIN-1に対する抗体を用いたADCC活性により、CAPRIN-1を発現する腫瘍細胞を障害することができることが明らかになった。
(1)組換えタンパク質の作製
実施例1で取得した配列番号1の遺伝子を基に、以下の方法にてヒト相同遺伝子の組換えタンパク質を作製した。PCRは、実施例1で作製した各種組織・細胞cDNAよりRT-PCR法による発現が確認できたcDNAを1μl、SacIおよびXhoI制限酵素切断配列を含む2種類のプライマー(配列番号38および39に記載)を各0.4μM, 0.2mM dNTP, 1.25UのPrimeSTAR HSポリメラーゼ(宝酒造社製)となるように各試薬と添付バッファーを加え全量を50μlとし、Thermal Cycler(BIO RAD社製)を用いて、98℃-10秒、68℃-2.5分のサイクルを30回繰り返すことにより行った。なお、上記2種類のプライマーは、配列番号2のアミノ酸配列全長をコードする領域を増幅するものであった。PCR後、増幅されたDNAを1%アガロースゲルにて電気泳動し、QIAquick Gel Extraction Kit(QIAGEN社製)を用いて約2.1kbpのDNA断片を精製した。
(2)組換えタンパク質の精製
上記で得られた、配列番号1の遺伝子を発現するそれぞれの組換え大腸菌を30μg/ml カナマイシン含有LB培地にて600nmでの吸光度が0.7付近になるまで37℃で培養後、イソプロピル-β-D-1-チオガラクトピラノシド終濃度が1mMとなるよう添加し、37℃で4時間培養した。その後4800rpmで10分間遠心し集菌した。この菌体ペレットをリン酸緩衝化生理食塩水に懸濁し、さらに4800rpmで10分間遠心し菌体の洗浄を行った。
実施例3で調製した配列番号2に示される、抗原タンパク質(ヒトCAPRIN-1)100μgを等量のMPL+TDMアジュバント(シグマ社製)と混合し、これをマウス1匹当たりの抗原溶液とした。抗原溶液を6週齢のBalb/ccマウス(日本SLC社製)の腹腔内に投与後、1週間毎にさらに3回および24回投与を行い免疫を完了した。最後の免疫から3日後に摘出したそれぞれの脾臓を滅菌した2枚のスライドガラスに挟んで擦り潰し、PBS(-)(日水社製)を用いて洗浄し1500rpmで10分間遠心して上清を除去する操作を3回繰り返して脾臓細胞を得た。得られた脾臓細胞とマウスミエローマ細胞SP2/0(ATCCから購入)とを10:1の比率にて混和し、そこに37℃に加温した10% FBSを含むRPMI1640培地200μlとPEG1500(ベーリンガー社製)800μlを混和して調製したPEG溶液を加えて5分間静置して細胞融合を行った。1700rpmで5分間遠心し、上清を除去後、Gibco社製のHAT溶液を2%当量加えた15% FBSを含むRPMI1640培地(HAT選択培地)150mlで細胞を懸濁し、96穴プレート(ヌンク社製)の1ウェル当たり100μlずつ、プレート15枚に播種した。7日間、37℃、5% CO2の条件で培養することで、脾臓細胞とミエローマ細胞が融合したハイブリドーマを得た。
(1)抗CAPRIN-1モノクローナル抗体の可変領域遺伝子のクローニング
実施例4で選抜した11個のモノクローナル抗体をそれぞれ産生する各ハイブリドーマ株から、mRNAを抽出し、マウスFR1由来配列およびマウスFR4由来の配列に特異的なプライマーを使用したRT-PCR法により、全ての抗CAPRIN-1モノクローナル抗体の重鎖可変(VH)領域および軽鎖可変(VL)領域の遺伝子を取得した。配列決定のために、それら遺伝子をpCR2.1ベクター(インビトロジェン社製)にクローニングした。
106個の各ハイブリドーマ株から、mRNA micro purification kit(GEヘルスケア社製)を用いてmRNAを調製し、SuperScriptII 1st strand synthesis kit(インビトロジェン社製)を用いて、得られたmRNAを逆転写してcDNAを合成した。これら操作は各キットの添付プロトコールに従って行った。
上記で得られた各PCR産物を用いてアガロースゲルにて電気泳動を行い、VH領域およびVL領域それぞれのDNAバンドを切り出した。DNA断片はQIAquick Gel purification kit(キアゲン社製)を用いてその添付プロトコールに従って行った。精製した各DNAはTAクローニングキット(インビトロジェン社製)を用いてpCR2.1ベクターにクローニングした。連結したベクターをDH5aコンピテントセル(TOYOBO社製)に定法に従い形質転換を行った。各形質転換体それぞれ10クローンを培地(100μg/mlアンピシリン)で37℃一晩培養後、各プラスミドDNAをQiaspin Miniprep kit(キアゲン社製)を用いて精製した。
上記で得られた各プラスミド中のVH領域およびVL領域の遺伝子配列解析は、M13フォワードプライマー(配列番号134)およびM13リバースプライマー(配列番号135)を用いて、蛍光シーケンサー(ABI社製DNAシーケンサー3130XL)により、ABI社製のビッグダイターミネーターVer3.1サイクルシーケンシングキットを用いて、その添付プロトコールに従い行った。その結果、各々の遺伝子配列が決定された(各々10クローンで一致)。
次にCAPRIN-1遺伝子の発現が確認された乳癌細胞株7種(MDA-MB-157,T47D,MRK-nu-1,MDA-MB-231V,BT20,SK-BR-3,DA-MB-231T)およびその他の乳癌細胞株3種(MDA-MB-231C,MCF-7,ZR75-1)、グリオーマ細胞株6種(T98G,SNB19,U251,U87G)、腎臓癌細胞株3種(Caki-1,Caki2,A498)、胃癌細胞株1種(MKN45)、大腸癌細胞株1種(Caco2)、肺癌細胞株3種(A549、QG56、PC8)、白血病細胞株3種(Namalwa、BDCM、RPI1788)について、その細胞表面上にCAPRIN-1タンパク質が発現しているかどうかを調べた。各細胞株それぞれ106細胞を1.5ml容のミクロ遠心チューブにて遠心分離した。これに実施例4で作製した癌細胞表面に反応する、#1から#11のCAPRIN-1に対するモノクローナル抗体を含むハイブリドーマ上清(100μl)を添加し、氷上で1時間静置した。PBSで洗浄した後、0.1% FBSを含むPBSで500倍希釈したFITC標識ヤギ抗マウスIgG抗体(インビトロジェン社製)で懸濁し、氷上で1時間静置した。PBSで洗浄後、ベクトンディッキンソン株式会社のFACSキャリバーにて蛍光強度を測定した。一方、上記と同様の操作を、#1から#11のCAPRIN-1に対するモノクローナル抗体を含むハイブリドーマ上清の代わりに上記(5)で調製したコントロール抗体を用いて行い、コントロールとした。その結果、#1~#11のモノクローナル抗体を添加された細胞は、コントロールに比べて、いずれも蛍光強度が30%以上強かった。具体的例を挙げると、#9のモノクローナル抗体を用いた場合、MDA-MB-157が211%、T47Dが145%、MRK-nu-1が123%、MDA-MB-231Vが251%、BT20が168%、MDA-MB-231Tが94%の蛍光強度の増強を示した。このことから、上記ヒト癌細胞株の細胞膜表面上にCAPRIN-1タンパクが発現していることが確認された。なお、上記蛍光強度の増強率は、各細胞における平均蛍光強度(MFI値)の増加率にて表され、以下の計算式により算出した。
(3)CAPRIN-1に対する抗体の癌細胞に対する抗腫瘍効果(ADCC活性)
上記で選抜した#1~#11のCAPRIN-1に対するモノクローナル抗体の癌細胞に対する細胞障害活性(ADCC活性)を評価した。#1~#11のモノクローナル抗体を産生する各ハイブリドーマをハイブリドーマSFM(インビトロジェン社製)培地を用いて培養し、得られた上清をHitrap ProteinA SepharoseFF(GEヘルスケア社製)を用いて精製し、PBS(-)に置換して0.22μmのフィルター(ミリポア社製)で濾過したものを活性測定用の抗体として用いた。106個のヒト乳癌細胞株MDA-MB-157を50ml容の遠心チューブに集め、100μCiのクロミウム51を加え37℃で2時間インキュベートした。その後10%FBSを含むRPMI1640培地で3回洗浄し、96穴V底プレート1穴あたり103個ずつ添加して標的細胞とした。これに、上記精製抗体をそれぞれ1μg添加し、さらにマウス脾臓から分離したマウスリンパ球を2×105個添加して、37℃、5%CO2の条件下で4時間培養した。培養後、障害を受けた腫瘍細胞から放出される培養上清中のクロミウム51の量を測定し、抗CAPRIN-1抗体による癌細胞に対するADCC活性を算出した。その結果、#1~#11の全てのモノクローナル抗体が、MDA-MB-157に対して、20%以上のADCC活性を示した。具体的には、例えば、#1は22.1%、#2は29.1%、#6は30.2%、#9は32.4%の細胞障害活性が得られた(図4参照)。一方、実施例4で作製した、CAPRIN-1タンパク自体には反応するが、癌細胞の細胞表面に反応しないモノクローナル抗体を用いて同様の操作を行った場合、細胞障害活性は認められなかった(図4参照)。以上の結果より、取得した抗CAPRIN-1モノクローナル抗体(#1~#11は、ADCC活性によってCAPRIN-1を発現する癌細胞を障害することが示された。
上記で選抜した#1~#11のCAPRIN-1に対するモノクローナル抗体の癌細胞に対する細胞障害活性(CDC活性)を評価した。ウサギから採血した血液をエッペンドルフチューブに入れ、室温で60分間、静置した後3000rpmで5分間、遠心分離することで、CDC活性測定用の血清を調製した。ヒト乳癌細胞であるMDA-MB-231Vを105個を50ml容の遠心チューブに集め、100μCiのクロミウム51を加え37℃で2時間インキュベートした後、10%FBSを含むRPMI培地で3回洗浄した。その後上記で調製したウサギ血清を50%含むRPMI培地で懸濁し、96穴V底プレート1穴あたり103個ずつ添加した。これに上述(3)で得た#1~#11のモノクローナル抗体をそれぞれ1μgずつ添加して、37℃、5%CO2の条件下で4時間培養した。培養後、障害を受けた腫瘍細胞から放出される培養上清中のクロミウム51の量を測定し、ハイブリドーマ上清中の抗CAPRIN-1モノクローナル抗体によるMDA-MB-231Vに対するCDC活性を算出した。その結果、#1~#11の全てのモノクローナル抗体が、30%以上のCDC活性を示した。一方、実施例4で作製した、CAPRIN-1タンパク自体には反応するが、癌細胞の細胞表面に反応しないモノクローナル抗体を用いて同様の操作を行った場合、細胞障害活性は認められなかった。従って、CAPRIN-1に対するモノクローナル抗体(#1~#11)は、CDC活性によっても、CAPRIN-1を発現する腫瘍細胞を障害することができることが明らかになった。
次に、取得した#1~#11のCAPRIN-1に対するモノクローナル抗体の担癌マウス生体内における抗腫瘍効果を評価した。使用した抗体は上記と同様に各ハイブリドーマの培養上清をカラム精製したものを用いた。
上記で取得した、癌細胞の細胞表面に反応する#1~#11のCAPRIN-1に対するモノクローナル抗体を用いて、それらが認識するCAPRIN-1タンパク質中の部分配列の同定を行った。
Claims (22)
- 配列番号2~30のうち偶数の配列番号で表されるいずれかのアミノ酸配列、又は該アミノ酸配列と80%以上の配列同一性を有するアミノ酸配列、を有するCAPRIN-1タンパク質、又は連続する7以上のアミノ酸を含むその断片、と免疫学的反応性を有する抗体又はそのフラグメントを有効成分として含むことを特徴とする、癌の治療及び/又は予防のための医薬組成物。
- 前記CAPRIN-1タンパク質の断片が、配列番号2~30のうち配列番号6および配列番号18を除く偶数の配列番号で表されるいずれかのアミノ酸配列中のアミノ酸残基番号50-98又はアミノ酸残基番号233-305の領域内の連続する7個以上のアミノ酸配列からなるポリペプチド、又は該ポリペプチドを部分配列として含むポリペプチド、と免疫学的反応性を有する抗体又はそのフラグメントを有効成分として含むことを特徴とする、請求項1に記載の医薬組成物。
- 配列番号37又は配列番号136で表されるアミノ酸配列、又は該アミノ酸配列と80%以上の配列同一性を有するアミノ酸配列、を有するCAPRIN-1の部分ポリペプチド、又は連続する7以上のアミノ酸を含むその断片、と免疫学的反応性を有する抗体又はそのフラグメントを有効成分として含むことを特徴とする、請求項1に記載の医薬組成物。
- 前記癌が乳癌、脳腫瘍、白血病、リンパ腫、肺癌、食道癌もしくは大腸癌である、請求項1~3のいずれか1項に記載の組成物。
- 前記抗体が、モノクローナル抗体又はポリクローナル抗体である、請求項1~4のいずれか1項に記載の医薬組成物。
- 前記抗体が、ヒト抗体、ヒト化抗体、キメラ抗体、単鎖抗体又は二重特異性抗体である、請求項1~4のいずれか1項に記載の医薬組成物。
- 配列番号37又は配列番号136で表されるアミノ酸配列、又は該アミノ酸配列と80%以上の配列同一性を有するアミノ酸配列、を有するポリペプチドと免疫学的反応性を有する抗体。
- 配列番号40、41及び42を含む重鎖可変領域と配列番号44、45及び46を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- 配列番号40,41及び42を含む重鎖可変領域と配列番号50,51及び52を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- 配列番号40,41及び42を含む重鎖可変領域と配列番号55,56及び57を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- 配列番号40,41及び42を含む重鎖可変領域と配列番号60,61及び62を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- 配列番号40,41及び42を含む重鎖可変領域と配列番号65,66及び67を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- 配列番号70,71及び72を含む重鎖可変領域と配列番号74,75及び76を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- 配列番号80,81及び82を含む重鎖可変領域と配列番号84,85及び86を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- 配列番号90,91及び92を含む重鎖可変領域と配列番号94,95及び96を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- 配列番号100,101及び102を含む重鎖可変領域と配列番号104,105及び106を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- 配列番号110,111及び112を含む重鎖可変領域と配列番号114,115及び116を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- 配列番号120,121及び122を含む重鎖可変領域と配列番号124,125及び126を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体。
- ヒト抗体、ヒト化抗体、キメラ抗体、単鎖抗体又は二重特異性抗体である、請求項7~17のいずれか1項に記載の抗体。
- 請求項7~18のいずれか1項に記載の抗体又はそのフラグメントを有効成分として含むことを特徴とする、癌の治療及び/又は予防のための医薬組成物。
- 配列番号2~30のうち偶数の配列番号で表されるアミノ酸配列、又は該アミノ酸配列と80%以上の配列同一性を有するアミノ酸配列、を有するCAPRIN-1タンパク質、又は連続する7以上のアミノ酸を含むその断片、と免疫学的反応性を有する抗体又はそのフラグメントを用いた、癌の治療及び/又は予防方法。
- 請求項7~18のいずれか1項に記載の抗体又はそのフラグメントを用いた、癌の治療及び/又は予防方法。
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EP09805009.9A EP2322221B1 (en) | 2008-08-05 | 2009-08-05 | Pharmaceutical composition for treatment and prevention of cancer |
CN2009801389599A CN102170907A (zh) | 2008-08-05 | 2009-08-05 | 用于治疗和预防癌症的药物组合物 |
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CA2733223A CA2733223C (en) | 2008-08-05 | 2009-08-05 | Pharmaceutical composition comprising anti-caprin-1 antibody for treatment and prevention of cancers |
RU2011108260/10A RU2498819C2 (ru) | 2008-08-05 | 2009-08-05 | Фармацевтическая композиция для лечения и профилактики злокачественных опухолей |
ES09805009.9T ES2502940T3 (es) | 2008-08-05 | 2009-08-05 | Composición farmacéutica para el tratamiento y la prevención del cáncer |
US13/057,709 US9416192B2 (en) | 2008-08-05 | 2009-08-05 | Pharmaceutical composition for treatment and prevention of cancers |
PL09805009T PL2322221T3 (pl) | 2008-08-05 | 2009-08-05 | Kompozycja farmaceutyczna do leczenia i zapobiegania raka |
JP2009546598A JP5691172B2 (ja) | 2008-08-05 | 2009-08-05 | 癌の治療及び予防用医薬組成物 |
AU2009278386A AU2009278386B2 (en) | 2008-08-05 | 2009-08-05 | Pharmaceutical composition for treatment and prevention of cancers |
KR1020167017895A KR101667319B1 (ko) | 2008-08-05 | 2009-08-05 | 암의 치료 및 예방용 의약 조성물 |
DK09805009.9T DK2322221T3 (da) | 2008-08-05 | 2009-08-05 | Farmaceutisk sammensætning til behandling og forebyggelse af cancer |
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AU2009278386B2 (en) | 2015-05-21 |
AU2009278386A1 (en) | 2010-02-11 |
BRPI0911926A2 (pt) | 2020-08-18 |
MX341883B (es) | 2016-09-07 |
KR20160085372A (ko) | 2016-07-15 |
ES2502940T3 (es) | 2014-10-06 |
PL2322221T3 (pl) | 2015-01-30 |
KR101638490B1 (ko) | 2016-07-11 |
JP5691172B2 (ja) | 2015-04-01 |
CA2733223C (en) | 2018-02-27 |
PT2322221E (pt) | 2014-09-19 |
RU2011108260A (ru) | 2012-09-10 |
US9982059B2 (en) | 2018-05-29 |
EP2322221A1 (en) | 2011-05-18 |
JPWO2010016526A1 (ja) | 2012-01-26 |
RU2498819C2 (ru) | 2013-11-20 |
CN102170907A (zh) | 2011-08-31 |
EP2322221B1 (en) | 2014-07-09 |
EP2322221A4 (en) | 2013-02-27 |
KR101667319B1 (ko) | 2016-10-18 |
US9416192B2 (en) | 2016-08-16 |
KR20110039475A (ko) | 2011-04-18 |
DK2322221T3 (da) | 2014-09-01 |
CN106039307A (zh) | 2016-10-26 |
JP5930004B2 (ja) | 2016-06-08 |
US20110256144A1 (en) | 2011-10-20 |
US20210188998A1 (en) | 2021-06-24 |
US20180312603A1 (en) | 2018-11-01 |
US20160208014A1 (en) | 2016-07-21 |
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