WO2011096535A1 - 癌の治療及び/又は予防のための医薬品 - Google Patents
癌の治療及び/又は予防のための医薬品 Download PDFInfo
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- WO2011096535A1 WO2011096535A1 PCT/JP2011/052414 JP2011052414W WO2011096535A1 WO 2011096535 A1 WO2011096535 A1 WO 2011096535A1 JP 2011052414 W JP2011052414 W JP 2011052414W WO 2011096535 A1 WO2011096535 A1 WO 2011096535A1
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- 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
Definitions
- the present invention relates to a pharmaceutical for the treatment and / or prevention of cancer, characterized by combining an antibody or fragment thereof having immunological reactivity with the CAPRIN-1 protein and an antitumor agent, and use thereof , Regarding.
- Cancer is the leading cause of all deaths, and the current treatment is a combination of radiation therapy and chemotherapy, mainly surgery.
- Current treatment also treats all patients with the same type and stage of cancer in the same way. At least 40% of these patients fail primary therapy and will therefore undergo a further series of treatments. Therefore, if treatment fails further, the possibility of cancer metastasis and eventually death increases. In other words, current radiation therapy and chemotherapy cannot be tailored to various cancer types and individual cancer patients, and surgery itself is inadequate to cure cancer in most cases. .
- HERCEPTIN registered trademark
- Her2 which is a monoclonal antibody that specifically binds to Her2
- Her2 is Her2 overexpressing metastasis. It has been proven clinically effective in reducing the number of deaths in patients with recurrent or metastatic breast cancer, and there are no serious side effects other than cardiotoxicity compared to conventional chemotherapeutic agents.
- Patent Documents 1 to 3 the therapeutic effect on breast cancer in combination with chemotherapeutic agents has been proven (Patent Documents 1 to 3).
- most antigen proteins on cancer cells, which are targets of antibody drugs, including Her2 are also expressed in normal cells.
- Antigen proteins on cancer cells which are targets of antibody drugs, including Her2
- 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 1) that the CAPRIN-1 gene sequence is a membrane protein that 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. Loses 80 amino acids from the C-terminal due to a frame shift due to a single base deletion.
- 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). 2).
- 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 2).
- Patent Documents 4 and 5 include the name M11S1, CAPRIN-1 as one of the cell membrane proteins, and cancer as a target of 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). However, as reported in Non-Patent Document 2, from the time of filing of Patent Document 4 to the present day, 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 4 and 5 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 use it as a pharmaceutical for cancer treatment and / or prevention by combining an antibody and antitumor agent that are targeted to it. Is to provide.
- CAPRIN-1 protein 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 these CAPRIN-1 proteins were prepared.
- CAPRIN-1 protein is specifically expressed in cancer cells such as breast cancer, brain tumor, leukemia, lymphoma, lung cancer, cervical cancer, bladder cancer, esophageal cancer, colon cancer, stomach cancer, kidney cancer, and CAPRIN -1 part of the protein is specifically expressed on the cell surface of those cancer cells, and an antibody against the portion of CAPRIN-1 expressed on the cell surface of each cancer cell is combined with a specific antitumor agent As a result, it was found that a remarkable cancer therapeutic effect can be obtained, and the present invention has been completed.
- cancer is used interchangeably with tumor or carcinoma.
- the present invention has the following features.
- a cancer comprising an antibody or a fragment thereof immunologically reactive with a CAPRIN-1 protein and one or more antitumor agents, together or separately in combination
- a cancer comprising an antibody or a fragment thereof immunologically reactive with a CAPRIN-1 protein and one or more antitumor agents, together or separately in combination
- the antibody or fragment thereof having immunological reactivity with the CAPRIN-1 protein is an antibody or fragment thereof that specifically binds to the extracellular region of the CAPRIN-1 protein present on the surface of cancer cells.
- a method for treatment and / or prevention of cancer comprising administering the pharmaceutical agent according to any one of (1) to (9) to a subject suspected of having 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.
- 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 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.
- An amino acid residue in an amino acid sequence represented by an even number excluding SEQ ID NO: 6 and SEQ ID NO: 18 among SEQ ID NOS: 2 to 30 as a partial peptide in CAPRIN-1 protein expressed on the cell surface of cancer cells A polypeptide comprising a sequence of seven or more amino acids in the region of number (aa) 50-98 or amino acid residue number (aa) 233-305, specifically, for example, represented by SEQ ID NO: 37 Or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more of the amino acid sequence, and an antibody used in the present invention. Binds (specifically) to these peptides (or recognizes (specifically) these peptides, or immunologically Having refractory), and includes all antibodies that exhibit anti-tumor activity.
- an antibody capable of specifically binding to the CAPRIN-1 protein is desirable and is preferably a monoclonal antibody, but may be a polyclonal antibody 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.
- 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 having antigenicity or immunogenicity in a mammal, preferably a human, and its minimum unit consists of (consecutive) about 7 to 12 amino acids, for example, 8 to 11 amino acids.
- Specific examples of the partial sequence of the CAPRIN-1 protein that specifically binds to an antibody include the amino acid sequence represented by SEQ ID NO: 37, or 80% or more, preferably 85% or more, more preferably 90% with the amino acid sequence. As mentioned above, a partial sequence containing at least about 7 to 12 amino acids in the amino acid sequence having a sequence identity of 95% or more is more preferable.
- 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 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 heat-resistant DNA polymerase (eg, Taq polymerase) and a Mg2 + -containing PCR buffer, the reaction temperature is 94 ° C.
- the reaction process consisting of 2 minutes (elongation) at 72 ° C. may be defined as 1 cycle, for example, after 30 cycles, the reaction may be performed at 72 ° C. for 7 minutes, but is not limited thereto.
- PCR methods, 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 W (15th & 19th). 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 fetal kidney cell line HEK293, mouse fetal 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. At this time, 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 for example, (His) 6 to (His) 10
- FLAG tag FLAG tag
- myc tag 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 a heterotetrameric glycoprotein of about 150 kDa 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, recombinant antibodies such as synthetic antibodies, multispecific antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, etc. human antibodies, antibody fragments thereof (eg Fab, F (ab ') 2 , Fv) 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.
- any of these antibodies or fragments thereof are immunologically reactive with the CAPRIN-1 protein present on the cancer cell surface, preferably with a polypeptide in the extracellular region (preferably the protein or the polypeptide). It specifically binds to a peptide) and exhibits cytotoxic activity against cancer.
- 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 cells 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 used in the present invention is preferably a monoclonal antibody. However, it may be a polyclonal antibody, a recombinant antibody or a genetically modified antibody (for example, a chimeric antibody, a humanized antibody, a single chain antibody, a bispecific antibody, etc.).
- 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
- 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.
- 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.
- 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 amino acids, preferably 1 to 5 amino acids, 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
- the antibody may be chemically modified.
- the modified antibody include various molecules such as polyethylene glycol (PEG) and antitumor compounds (for example, antitumor agents exemplified below). Mention may be made of bound antibodies.
- PEG polyethylene glycol
- antitumor compounds for example, antitumor agents exemplified below. Mention may be made of bound antibodies.
- 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, that is, specifically binds to the epitope 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 (kon / koff) of the antibody used in 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 used in 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 examples include the following antitumor agents known in the literature, such as paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, and inprosulfan , Piposulfan, Benzodopa, Carbocon, Metredopa, Uredopa, Alteramine, Triethylenemelamine, Triethylenephosphoramide, Triethylenethiophosphoroamide, trimethylolamine), bratacin, bratacinone, camptothecin, bryostati , Calistatin, cryptophysin 1, cryptophycin 8, dolastatin, duocarmycin, eleuterbin, pancratistatin, sarcodictin, spongestatin, chlorambucil, chloronaphazine, cholophosphamide, cholophosphamide Ramustine,
- the antibodies used in the present invention include 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 SM, 212 Bi, 32 P, 175 Lu, 176 Lu, etc., which are known in the literature. It is also possible to bind the radioisotopes. It is desirable that the radioisotope is effective for tumor treatment and diagnosis.
- the antibody used in the present invention is an antibody having immunological reactivity with CAPRIN-1, an antibody that specifically recognizes CAPRIN-1, or an antibody that specifically binds to CAPRIN-1, It is an antibody showing cytotoxic activity against cancer, for example, tumor growth inhibitory action.
- 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).
- CDR1, CDR2 and CDR3 non-human animal antibody complementarity determining regions
- 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.
- DNA encoding the light chain variable region and heavy chain variable region of the antibody was prepared by RT-PCR method and the like, 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 ligated 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 is composed of 12 to 19 amino acids, and examples include 15 amino acids (G4S) 3 (G.-B. Kim et al., Protein Engineering Designing 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 comprising a heavy chain variable region CDR1 to CDR3 comprising SEQ ID NOs: 70, 71 and 72 and a light chain variable region CDR1 to CDR3 comprising SEQ ID NOs: 74, 75 and 76 (preferably a heavy chain variable of SEQ ID NO: 73
- an antibody (preferably a heavy chain variable of SEQ ID NO: 83) comprising a heavy chain variable region CDR1 to CDR3 comprising SEQ ID NOs: 80, 81 and 82 and a light chain variable region CDR1 to CDR3 comprising SEQ ID NOs: 84, 85 and 86
- an antibody comprising a heavy chain variable region CDR1 to CDR3 comprising SEQ ID NOs: 100, 101 and 102 and a light chain variable region CDR1 to CDR3 comprising SEQ ID NOs: 104, 105 and 106 (eg, heavy chain variable of SEQ ID NO: 103 An antibody comprising the region and the light chain variable region of SEQ ID NO: 107)
- an antibody comprising a heavy chain variable region CDR1 to CDR3 comprising SEQ ID NOs: 110, 111 and 112 and a light chain variable region CDR1 to CDR3 comprising SEQ ID NOs: 114, 115 and 116 (eg, heavy chain variable of SEQ ID NO: 113)
- the amino acid sequences shown in SEQ ID NOs: 120, 121 and 122 are 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,
- variable region of the heavy chain includes 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 includes 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 for example, 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
- light chain variable region comprises the amino acid sequences of SEQ ID NOs: 44, 45 and 46 and the human antibody-derived framework region amino acid sequence
- the light chain constant region comprises the human antibody-derived amino acid sequence
- variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 43
- 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 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 above antibody preferably has 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 antibody used in the present invention further encodes the DNA encoding the antibody of the present invention, the DNA encoding the heavy chain or light chain of the antibody, or the variable region of the heavy chain or light chain of the antibody. It can also be produced by genetic recombination technology using DNA.
- 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 used in the present invention is, for example, in the case of antibody (a), DNA encoding a heavy chain variable region comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43, light chain
- the region encoding the variable 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 above DNA 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.
- DNA related to the antibodies (a) to (k) are as follows.
- a polypeptide comprising the amino acid sequence 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, SEQ ID NO: 123, SEQ ID NO: 133, SEQ ID NO: 143 or SEQ ID NO: 153 DNA containing the nucleotide sequence 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 or SEQ ID NO: 128 as the encoding DNA.
- 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 the CAPRIN-1 protein in vitro, as specifically shown in the Examples below. It can be evaluated by measuring.
- the ADCC activity can be measured using a commercially available kit for measuring cytotoxic activity such as Cytotoxicity Detection Kit (Roche).
- a target cancer cell and an anti-CAPRIN-1 antibody are reacted on ice and then cultured with effector cells (eg, PBMC) for 4 hours.
- the culture supernatant is subjected to lactate dehydrogenase (LDH released into the medium).
- LDH released into the medium It can be performed by a procedure comprising measuring enzyme activity or Cr51 radioactivity.
- the CDC activity is measured by reacting the target cancer cells with anti-CAPRIN-1 antibody on ice, and then culturing with a complement solution (for example, serum) for 4 hours. This can be done by a procedure that includes measuring radioactivity.
- 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 a CAPRIN-1 protein can be identified using binding assays such as those described in the Examples, such as ELISA, Western blotting, immunofluorescence and flow cytometry analysis.
- Antibodies that recognize the CAPRIN-1 protein can be obtained from tissues obtained from patients during surgery or cell lines that express CAPRIN-1 naturally or after transfection by immunohistochemistry in a manner well known to those skilled in the art. Test for reactivity with CAPRIN-1 from tissues from animals bearing inoculated xenograft tissues using paraformaldehyde or acetone-fixed frozen sections or paraformaldehyde-embedded tissue sections be able to.
- 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 present invention is characterized by combining an anti-CAPRIN-1 antibody and an antitumor agent as exemplified above.
- the anti-CAPRIN-1 antibody and the anti-tumor agent each have anti-tumor activity, but when they are combined and administered to a cancer patient, synergistically remarkable anti-tumor effects as shown in the examples, ie, tumor-bearing animals The effect of almost completely regressing the tumor in the model is obtained.
- Such an extraordinary antitumor effect is quite surprising because even anti-CAPRIN-1 antibodies and antitumor agents whose tumor growth gradually increases over time are observed when they are used in combination. That is.
- the antitumor agent used in combination with the anti-CAPRIN-1 antibody is any chemotherapeutic agent used, used or will be used for the treatment of various cancers and tumors. Include. Such antitumor agents are antimetabolites, antibiotic anticancer agents, plant alkaloid anticancer agents, topoisomerase inhibitors, antitumor alkylating agents, etc., for example, all of the above exemplified antitumor agents known in the literature etc. It is. Non-limiting specific examples include anti-tumor agents used in the examples described later, namely anti-cancer agents such as cyclophosphamide, paclitaxel, docetaxel, vinorelbine, and their remarkable anti-tumor effects have been confirmed. ing. Therefore, in the present invention, one or more selected from cyclophosphamide, paclitaxel, docetaxel and vinorelbine and pharmaceutically acceptable salts or derivatives thereof can be used as an antitumor agent. .
- the target for the therapeutic and / or prophylactic drug for cancer of the present invention is not particularly limited as long as it is a cancer (cell) that expresses 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 CAPRIN-1 protein having an amino acid sequence having an even sequence number among SEQ ID NOs: 2 to 30, preferably breast cancer, brain tumor, Leukemia, lung cancer, lymphoma, mastocytoma, renal cancer, cervical cancer, bladder cancer, esophageal cancer, stomach 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 (glioma), ventricular ependymoma, neuronal tumor, fetal ectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, Gastrointestinal lymphoma, gastrointestinal lymphoma, small to medium cell lymphoma, cecal cancer, ascending colon cancer, descending colon cancer, transverse colon cancer, sigmoid colon cancer, rectal cancer, etc. are included, but not limited thereto .
- ⁇ Pharmaceutical composition The active ingredient constituting the medicament for the treatment and / or prevention of cancer of the present invention, that is, the above-mentioned antibody or fragment thereof and the above-mentioned antitumor agent are mixed as a pharmaceutical composition or a separate pharmaceutical composition thereof, It can be formulated by methods known to those skilled in the art.
- a pharmacologically acceptable carrier or medium specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to prepare 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 pharmaceutical composition of the present invention can also contain a pharmacologically acceptable salt.
- 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.
- parenteral administration is oral or parenteral, and examples of parenteral administration include injection, nasal administration, pulmonary administration, and transdermal administration.
- injection form it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.
- parenteral administration slow infusion administration over time is possible.
- the administration method can be appropriately selected depending on the age, weight, sex, symptoms, etc. of the patient.
- parenteral administration is preferred, whereas for pharmaceutical compositions containing antitumor agents, either oral or parenteral administration is used depending on the type and indication of the antitumor agent. Is selected.
- the dose of 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 of the antitumor agent can be selected, for example, within the range of 1 to 1000 mg / body, preferably 10 to 500 mg / body per patient, but is not necessarily limited to these values. is not.
- 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 treatment and / or prevention of cancer by the therapeutic and / or preventive agent for cancer of the present invention includes various forms in addition to the administration as the pharmaceutical composition described above.
- each active ingredient of the therapeutic and / or prophylactic agent for cancer of the present invention can be administered individually at the same time or in order.
- the second active ingredient can be administered within a time interval of up to about 3 weeks, that is, immediately after the administration of the first active ingredient to about 3 weeks.
- the surgical procedure may be performed following the surgical procedure, or the surgical procedure may be performed between the administration of the first drug and the second drug.
- the therapeutic and / or prophylactic agent for cancer of the present invention may be administered according to a plurality of administration cycles.
- a pharmaceutical composition containing both active ingredients is administered in a cycle of about 2 days to about 3 weeks. Thereafter, it can be repeated as necessary according to the judgment of the doctor in charge of the treatment cycle.
- the duration of administration of each individual drug is adjusted to cover the same period. The interval between cycles can vary from 0 to 2 months.
- the dosage of each active ingredient of the therapeutic and / or prophylactic agent for cancer of the present invention can be set in the same manner as the dosage of each active ingredient in the pharmaceutical composition.
- the medicament for the treatment and / or prevention of cancer of the present invention may be in the form of a pharmaceutical kit.
- the pharmaceutical kit is a package for using the anti-CAPRIN-1 antibody or fragment thereof, which is an active ingredient, and the antitumor agent in the form of separate pharmaceutical compositions in a method for treating or preventing cancer,
- the package includes instructions for administering each active ingredient.
- Each active ingredient of the pharmaceutical composition for treating and / or preventing cancer contained in the pharmaceutical kit is a pharmaceutical composition formulated as described above so that the active ingredients can be administered together or separately. It can be in form.
- the pharmaceutical kit includes an active ingredient in an amount sufficient for one or more doses so that each active ingredient can be administered according to the above administration method.
- the present invention further comprises administering the above-mentioned pharmaceutical product of the present invention to a subject suspected of having cancer (including having cancer).
- Methods for treatment and / or prevention are provided.
- the antibody or fragment thereof and the antitumor agent contained in the pharmaceutical product are administered to the subject simultaneously or separately.
- RNA was extracted from testicular tissue of a healthy dog by 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 the 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.
- sequence identity with the gene which codes the equine homologous factor of the acquired canine gene was the base sequence 93% and the amino acid sequence 97% in the area
- 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 nucleotide 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.
- the sequence identity between the gene encoding the human homologous factor and the gene encoding the mouse homologous factor was 89-91% of the base sequence and 95-96% of the amino acid sequence in the region translated into the protein.
- the sequence identity of the acquired canine gene with the gene encoding the chicken homologous factor was 82% for the nucleotide sequence and 87% for the amino acid sequence in the region translated into protein.
- the base 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.
- RNA expression analysis in each tissue The expression of the gene obtained by the above method in dog and human normal tissues and various cell lines was examined by RT-PCR method.
- the reverse transcription reaction was performed as follows. Specifically, total RNA was extracted from 5 mg of each tissue and 5-10 ⁇ 10 6 cells of each cell line using TRIZOL reagent (manufactured by Invitrogen) according to the attached protocol. Using this total RNA, cDNA was synthesized by Superscript First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) according to the attached protocol. The PCR reaction was performed as follows using the obtained gene-specific primers (described in SEQ ID NOs: 33 and 34).
- 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.
- 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 and allowed to stand at 4 ° C. until the tissue was sunk, and then replaced with a PBS solution containing 30% sucrose and allowed to stand at 4 ° C. until the tissue was sunk.
- 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 (manufactured by VECTASTAIN), and the dog tissue was PBS containing 10% FBS.
- 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 3 is prepared.
- the solution prepared to 10 ⁇ g / ml with a blocking solution was placed and allowed to stand overnight at 4 ° C. in a moist chamber.
- 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 sit 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 sit 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 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 was used.
- a solution prepared by adding 10 ⁇ g / ml of the monoclonal antibody # 6 reactive with the surface of cancer cells prepared in Example 4 to a PBS-T solution containing 5% FBS was placed at 4 ° C. overnight in a moist chamber.
- an appropriate amount of Peroxidase Labeled Polymer Conjugated (manufactured by DAKO) was added dropwise and allowed to stand at room temperature for 30 minutes in a moist chamber.
- DAB coloring solution manufactured by DAKO
- Example 2 Production of a 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 breast cancer tissue / cell cDNA, and two kinds of primers (described in SEQ ID NOs: 38 and 39) containing SacI and XhoI restriction enzyme cleavage sequences were each set to 0.
- Reagents and attached buffer were added to 4 ⁇ M, 0.2 mM dNTP, 1.25 U PrimeSTAR HS polymerase (Takara Shuzo) to make a total volume of 50 ⁇ l, and a thermal cycler (BIO RAD) was used at 98 ° C. This was performed by repeating the cycle of 10 seconds, 68 ° C.-2.5 minutes 30 times.
- the above two types of primers amplify the region encoding the entire amino acid sequence of SEQ ID NO: 2. After PCR, the amplified DNA was electrophoresed on a 1% agarose gel, and a DNA fragment of about 2.1 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
- 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 saline and subjected to ultrasonic crushing 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 (support: 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 50 mM hydrochloric acid buffer solution (pH 8.0) 10 times the column volume and 20 mM phosphate buffer solution (pH 8.0) containing 20 mM imidazole, and immediately, 20 mM containing 100 mM imidazole.
- Six beds were eluted with phosphate buffer (pH 8.0).
- the elution fraction of 100 mM imidazole-containing 20 mM phosphate buffer (pH 8.0) whose elution of the target protein was confirmed by Coomassie staining was applied to a strong anion exchange column (carrier: Q Sepharose (trademark) Fast Flow (GE Healthcare), column volume. 5 ml of 20 mM phosphate buffer (pH 8.0) as an equilibration buffer was added. The unadsorbed fraction was washed with 20 mM phosphate buffer (pH 7.0) 10 times the column volume and 20 mM phosphate buffer (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 3 Production of mouse and monoclonal antibody against CAPRIN-1 100 ⁇ g of the antigen protein (human CAPRIN-1) shown in SEQ ID NO: 2 prepared in Example 2 was mixed with an equal amount of MPL + TDM adjuvant (manufactured by Sigma), This was used as an antigen solution per mouse. After the antigen solution was administered intraperitoneally to a 6-week-old Balb / c mouse (manufactured by Japan SLC), the immunization was completed by further 3 or 24 administrations per week.
- MPL + TDM adjuvant manufactured by Sigma
- 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 1 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
- hybridomas were added to the plate so that the number was 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 1 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 4 Characterization of Selected Antibody (1) Cloning of Variable Region Gene of Anti-CAPRIN-1 Monoclonal Antibody mRNA was extracted from each hybridoma strain that produced each of the 11 mouse monoclonal antibodies selected in Example 3, Genes of heavy chain variable (VH) region and light chain variable (VL) region of all anti-CAPRIN-1 monoclonal antibodies by RT-PCR using primers specific to mouse FR1-derived sequence and mouse FR4-derived sequence Acquired. The genes were cloned into pCR2.1 vector (Invitrogen) for sequencing.
- VH heavy chain variable
- VL light chain variable
- mRNA is extracted from two mouse-derived hybridoma strains that produce monoclonal antibodies that react with the cell surface of breast cancer cells expressing CAPRIN-1, and primers specific to mouse FR1-derived sequences and mouse FR4-derived sequences
- the heavy chain variable (VH) region and light chain variable (VL) region genes of each antibody were obtained by RT-PCR using The genes were cloned into pCR2.1 vector (Invitrogen) for sequencing.
- the mouse antibody heavy chain gene variable region and the mouse antibody light chain gene variable region were respectively amplified by PCR using KOD-Plus-DNA Polymerase (manufactured by TOYOBO) according to a conventional method.
- 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 mouse light chain FR1 A primer specific for the sequence SEQ ID NO: 132
- a primer specific for the mouse light chain FR4 SEQ ID NO: 133
- Each PCR product obtained above was electrophoresed on an agarose gel, and the DNA bands of the VH region and VL region were excised.
- the DNA fragment was obtained using a QIAquick Gel purification kit (manufactured by QIAGEN) according to the attached protocol.
- Each purified DNA was cloned into the pCR2.1 vector using a TA cloning kit (Invitrogen).
- the ligated vector was transformed into DH5 competent cell (manufactured by TOYOBO) according to a conventional method.
- Ten clones of each transformant were cultured overnight at 37 ° C. in a medium (100 ⁇ g / ml ampicillin), and then each plasmid DNA was purified using Qiaspin Miniprep kit (manufactured by QIAGEN).
- the heavy chain variable region gene sequences of the obtained monoclonal antibodies are shown in 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, respectively, and their amino acid sequences.
- 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 the light chain variable region gene sequences are SEQ ID NO: 49, SEQ ID NO: 54, SEQ ID NO: 59, respectively.
- SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 119 and SEQ ID NO: 129, and their amino acid sequences are 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: 1 7, shown in SEQ ID NO: 117 and SEQ ID NO: 127.
- monoclonal antibody # 1 consists of a heavy chain variable region of SEQ ID NO: 43 and a light chain variable region of SEQ ID NO: 47
- # 2 consists of a heavy chain variable region of SEQ ID NO: 43 and a light chain variable region of SEQ ID NO: 53
- # 3 is composed of the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 58
- # 4 is composed of the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 63
- # 5 is the sequence The heavy chain variable region of No.
- a variable chain region and a light chain variable region of SEQ ID NO: 87, # 8 is composed of a heavy chain variable region of SEQ ID NO: 93 and a light chain variable region of SEQ ID NO: 97, and # 9 is a heavy chain variable region of SEQ ID NO: 103
- Consisting of the light chain variable region of SEQ ID NO: 107, # 1 Comprises a light chain variable region of SEQ ID NO: 117 and a heavy chain variable region of SEQ ID NO: 113, # 11 is comprised of a light chain variable region of SEQ ID NO: 127 and a heavy chain variable region of SEQ ID NO: 123.
- the same operation as described above was performed using a culture medium for hybridoma culture and used as a negative control.
- the cells to which the antibodies # 2 and # 9 were added had a fluorescence intensity that was 20% or more stronger than the control.
- 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.
- ADCC activity Antitumor effect of antibodies against CAPRIN-1 on cancer cells.
- 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). What was substituted with PBS ( ⁇ ) and filtered through a 0.22 ⁇ m filter (Millipore) was used as an antibody for activity measurement.
- the amount of chromium (Cr) 51 in the culture supernatant released from the damaged tumor cells was measured, and the ADCC activity against 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. Specifically, for example, cytotoxic activity of # 1 was 22.1%, # 2 was 29.1%, # 6 was 30.2%, and # 9 was 32.4% (see FIG. 1). ).
- # 9 is 12% or more (isotype control is 1.3%), for U373, # 9 is 16% or more (isotype control is 3%), and for A549, # 9 is 24 % Or more (isotype control is 2.6%), for QG56, # 9 is 20% or more (isotype control is 0.2%), for Caki-1, # 9 is 23% or more (isotype control is 3.0%), for ACHN, # 9 is 14% or more (isotype control is 1.5%), for SW756, # 9 is 16% or more (isotype control is 2.5%), T24 On the other hand, # 9 is 18% or more (isotype control is 2.1%), and KYSE180 is # 9 is 22% or more (isotype control) 3.0%), for MNK28, # 9 is 15% or more (isotype control is 1.7%), for MNK45, # 9 is 10% or more (isotype control is 2.3%), SW480 On the other hand, # 9 is 17% or more (isotype control is 1.
- 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 MDA-MB-231V human breast cancer cells 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 each of the monoclonal antibodies # 1 to # 13 obtained in Example 3 was added thereto, and the cells were cultured at 37 ° C. under 5% CO 2 for 4 hours. After the culture, 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. As a result, all of the monoclonal antibodies # 1 to # 11 showed CDC activity of 30% or more.
- Example 5 Identification of a peptide in CAPRIN-1 protein to which an antibody against CAPRIN-1 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.) with 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 with 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: 37 was identified as a partial sequence of CAPRIN-1 recognized by any of the monoclonal antibodies against CAPRIN-1 of # 1 to # 11.
- Example 6 Influence of CAPRIN-1 expression on cancer cell surface by antitumor agent (1) Calculation of 50% inhibitory concentration of antitumor agent on cancer cells Influence of CAPRIN-1 expression on cancer cell surface by antitumor agent In order to evaluate, 50% inhibitory concentration of an antitumor agent was calculated using cancer cells (MCF-7).
- human breast cancer cell line MCF-7 four types of antitumor agents (cyclophosphamide: “Endoxan” (registered trademark, manufactured by Shionogi & Co., Ltd.)) currently used as breast cancer therapeutic agents, paclitaxel: “Taxol” ( (Registered trademark, manufactured by Bristol-Myers), Docetaxel: “Taxotere” (registered trademark, manufactured by Sanofa Avantis), vinorelbine: "Navelvin” (registered trademark, manufactured by Kyowa Hakko Kirin Co., Ltd.) Cells were prepared to 1 ⁇ 10 5 cells / ml and cultured in a 6-well plate for 1 day under conditions of 37 ° C. and 5% CO 2.
- paclitaxel “Taxol” (Registered trademark, manufactured by Bristol-Myers)
- Docetaxel “Taxotere” (registered trademark, manufactured by Sanofa Avantis)
- vinorelbine “Nave
- each antitumor agent was added at a final concentration of 0. Treated with 0.001 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M, and 10 ⁇ M, respectively, and cultured at 37 ° C. under 5% CO 2 conditions for 2 days. Wash twice with PBS ( ⁇ ), add 0.25% Trypsin-EDTA to detach cells, adjust to 100 ⁇ l with PBS ( ⁇ ), add 10 ⁇ l of 0.4% trypan blue stock solution and mix The number of viable cells was measured using a hemocytometer, and the percentage of viable cells at the time of treatment with each chemotherapeutic agent was calculated, assuming that the number of viable cells untreated with each antitumor agent was 100%. The 50% inhibitory concentration was estimated from the results, and an antitumor agent in the vicinity of that concentration was prepared, and the same 50% inhibitory concentration was calculated by performing the same operation as described above.
- MCF-7 was prepared to 1 ⁇ 10 5 cells / mL and cultured in a 6-well plate for 1 day under conditions of 37 ° C. and 5% CO 2 .
- cyclophosphamide was treated with final concentrations of 1 ⁇ 10 ⁇ 1 ⁇ M, 5 ⁇ 10 ⁇ 2 ⁇ M, 2 ⁇ 10 ⁇ 2 ⁇ M, and 1 ⁇ 10 ⁇ 2 ⁇ M, respectively, at 37 ° C. under 5% CO 2 condition. For 2 days.
- the human breast cancer cell line MCF-7 was treated to examine the expression behavior of CAPRIN-1 protein on the cell surface.
- Cells were prepared to 1 ⁇ 10 5 cells / ml and cultured in a 6-well plate at 37 ° C. and 5% CO 2 for 1 day.
- an antitumor agent having a 50% inhibitory concentration calculated in (1) and PBS ( ⁇ ) as a control were respectively treated, and cultured at 37 ° C. under 5% CO 2 for 2 days.
- the cells were washed twice with PBS ( ⁇ ), and the cells were detached using a cell scraper. The cells were then centrifuged in a 1.5 ml microcentrifuge tube.
- mouse anti-CAPRIN-1 monoclonal antibody # 9 1 ⁇ g (5 ⁇ l) of mouse anti-CAPRIN-1 monoclonal antibody # 9, and the suspension was further suspended in PBS containing 95 ⁇ l of 0.1% fetal bovine serum and allowed to stand on ice for 1 hour. After washing with PBS, the suspension was suspended in PBS containing 5 ⁇ l of FITC-labeled goat anti-rabbit IgG antibody (manufactured by Santa Cruz) and 95 ⁇ l of 0.1% fetal bovine serum (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 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.
- Example 7 Combination therapy of anti-CAPRIN-1 antibody and antitumor agent in vivo
- Anti-CAPRIN-1 monoclonal using a mouse bearing a human breast cancer cell line MCF-7 expressing CAPRIN-1 The antitumor effect of the combined use of antibody and antitumor agent was examined. The method for examining the antitumor effect and the results using mice bearing MCF-7 are described below. 10 6 MCF-7 (purchased from ATCC) were transplanted subcutaneously in the back of 280 nude mice (manufactured by Japan SLC) and allowed to grow until the tumor was about 7 mm in diameter.
- the tumor-bearing mice transplanted with MCF-7 are the group administered only with anti-CAPRIN-1 antibody, the group administered only with anti-tumor agents (4 types) Anti-tumor agent and anti-Her2 antibody (mouse anti-human ErbB2 monoclonal antibody, isotype: IgG2b (manufactured by R & D Systems, catalog number: MAB11291)), anti-tumor agent and monoclonal antibody against CAPRIN-1 And finally, the control group (administer PBS (-)) was divided into five groups, and mouse PBMC was administered to all administration groups.
- ⁇ Experiment 1> Group receiving only anti-CAPRIN-1 antibody
- 5 mg / kg / shot of a monoclonal antibody against CAPRIN-1 of # 2 was administered intraperitoneally on the 0th, 4th, 8th, 11th, 15th, and 17th day of the test on 5 tumor-bearing mice.
- / C PBMC separated from the mouse spleen were intravenously administered at 1 ⁇ 10 7 cells / 0.2 mL (RPMI1640) per mouse on the 0th, 8th, and 15th days from the start of the test.
- Cyclophosphamide administration group For 5 tumor-bearing mice, 80 mg / kg / shot of cyclophosphamide per mouse was intraperitoneally administered on the 0th and 4th day of the test, and PBMC separated from the spleen of Balb / c mice was used per mouse. 1 ⁇ 10 7 cells / 0.2 mL (RPMI 1640) was intravenously administered on the 0th, 8th, and 15th days after the start of the test.
- paclitaxel administration group For 5 tumor-bearing mice, 15 mg / kg / shot of paclitaxel per mouse was intraperitoneally administered on the 0th and 3rd day of the test, and PBMC separated from the spleen of Balb / c mice was 1 ⁇ 10 6 per mouse. Seven cells / 0.2 mL (RPMI1640) were intravenously administered on the 0th, 8th, and 15th days after the start of the test.
- Docetaxel administration group For 5 tumor-bearing mice, 10 mg / kg / shot of docetaxel per mouse was intraperitoneally administered on the 0th and 3rd day of the test, and PBMC separated from the spleen of Balb / c mice was 1 ⁇ 10 6 per mouse. Seven cells / 0.2 mL (RPMI1640) were intravenously administered on the 0th, 8th, and 15th days after the start of the test.
- Vinorelbine administration group For 5 tumor-bearing mice, 1 mg / kg / shot of vinorelbine was intraperitoneally administered on day 0 of the test, and PBMC separated from the spleen of Balb / c mice was 1 ⁇ 10 7 cells per mouse. /0.2 mL (RPMI1640) was intravenously administered on the 0th, 8th, and 15th days after the start of the test.
- cyclophosphamide and anti-Her2 antibody administration group For 5 tumor-bearing mice, cyclophosphamide was intraperitoneally administered at a dose of 80 mg / kg / shot per mouse on the 0th and 4th days of the test, and at the same time anti-Her2 antibody was administered at 5 mg / kg / shot per mouse. Inoculated intraperitoneally at 0, 4, 8, 11, 15, and 17 days after the start of the test, and 1 ⁇ 10 7 cells / 0.2 mL (RPMI1640) of PBMC separated from the spleen of Balb / c mice were tested. On days 0, 8, and 15 after administration, intravenous administration was performed.
- paclitaxel and anti-Her2 antibody administration group For 5 tumor-bearing mice, paclitaxel was administered intraperitoneally at 15 mg / kg / shot per mouse, starting on the third day of the test, and at the same time, anti-Her2 antibody was tested at 5 mg / kg / shot per mouse. On day 0, 4, 8, 11, 15 and 17, PBMC separated from Balb / c mouse spleen was tested at 1 ⁇ 10 7 cells / 0.2 mL (RPMI 1640) per mouse, starting test 0, It was administered intravenously on days 8 and 15.
- Docetaxel and anti-Her2 antibody administration group For 5 tumor-bearing mice, 10 mg / kg / shot of docetaxel per mouse was intraperitoneally administered on the 0th and 3rd day of the test, and at the same time, anti-Her2 antibody was tested at 5 mg / kg / shot per mouse. On day 0, 4, 8, 11, 15 and 17, PBMC separated from Balb / c mouse spleen was tested at 1 ⁇ 10 7 cells / 0.2 mL (RPMI 1640) per mouse, starting test 0, It was administered intravenously on days 8 and 15.
- Vinolerubin and anti-Her2 administration group For 5 tumor-bearing mice, vinorelbine was administered intraperitoneally at 1 mg / kg / shot per mouse on day 0 of the test, and simultaneously, anti-Her2 antibody was administered at 5 mg / kg / shot per mouse at the start of test 0. 4, 8, 11, 15, 17 days, PBMC separated from the spleen of Balb / c mice were tested at 1 ⁇ 10 7 cells / 0.2 mL (RPMI 1640) per mouse, On day 15 it was administered intravenously.
- Cyclophosphamide was intraperitoneally administered to 5 tumor-bearing mice at a dose of 80 mg / kg / shot per mouse at the start of the test on the 4th day, and at the same time one monoclonal antibody against CAPRIN-1 of # 2 PBMC separated from Balb / c mouse spleen by intraperitoneal administration at 5 mg / kg / shot per day at 0, 4, 8, 11, 15, and 17 on the start of the test, 1 ⁇ 10 7 cells / 0.2 mL per mouse (RPMI1640) was administered intravenously on the 0th, 8th, and 15th days after the start of the test.
- docetaxel and anti-CAPRIN-1 antibody administration group To 5 tumor-bearing mice, docetaxel was administered intraperitoneally at 10 mg / kg / shot per mouse at the start of the test on the 3rd day, and at the same time a monoclonal antibody against # 2 CAPRIN-1 was administered at 5 mg / kg. 1 x 10 7 cells / 0.2 mL per animal (RPMI 1640) of PBMCs administered kg / shot intraperitoneally at the start of the test on days 0, 4, 8, 11, 15, and 17 and separated from the spleens of Balb / c mice Each was intravenously administered on days 0, 8, and 15 after the start of the test.
- Vinorelbine and 1 mg / kg / shot per mouse were administered intraperitoneally on the 0th day of the test to 5 tumor-bearing mice of vinorelbine and anti-CAPRIN-1 antibody, and at the same time, monoclonal antibody against CAPRIN-1 of # 2 was administered intraperitoneally at 0 mg, 4, 8, 11, 15, and 17 on the start of the test, and PBMCs separated from Balb / c mouse spleen were added at 1 ⁇ 10 7 cells / mouse.
- 0.2 mL (RPMI1640) was intravenously administered on the 0th, 8th, and 15th days after the start of the test.
- Cyclophosphamide administration group paclitaxel administration group, docetaxel administration group, vinorelbine administration group, cyclophosphamide and anti-Her2 antibody administration group, paclitaxel and anti-Her2 antibody administration group, docetaxel and anti-Her2 antibody administration group and vinorelbine
- the anti-Her2 antibody administration group was set in the same manner as in experimental group 1.
- the tumor size was measured every day and the antitumor effect was observed.
- a group in which PBS ( ⁇ ) was administered to 5 cancer-bearing mice instead of the antibody was used as a control group.
- the size of the tumor was calculated by using a formula of major axis ⁇ minor axis ⁇ minor axis ⁇ 0.5.
- the present invention is useful for the treatment and / or prevention of cancer.
- SEQ ID NO: 31 T3 primer SEQ ID NO: 32: T7 primer SEQ ID NO: 33, 34: Primer SEQ ID NO: 35, 36: GAPDH primer SEQ ID NO: 38, 39: Primer SEQ ID NO: 130 to 135: Primer
Abstract
Description
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本の重鎖及び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)鎖及び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。それぞれの重鎖及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。完全な重鎖及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及び112を含む重鎖可変領域CDR1~CDR3と配列番号114,115及び116を含む軽鎖可変領域CDR1~CDR3とを含む抗体(例えば、配列番号113の重鎖可変領域及び配列番号117の軽鎖可変領域で構成される抗体)。
抗体がCAPRIN-1タンパク質に結合する能力は、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、免疫蛍光及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タンパク質を認識する抗体は、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。
本発明は、抗CAPRIN-1抗体と、上で例示したような抗腫瘍剤とを組み合わせることを特徴とする。抗CAPRIN-1抗体と抗腫瘍剤は、それぞれ抗腫瘍活性を有するが、これらを組み合わせて癌患者に投与することによって、実施例で示される通り相乗的に顕著な抗腫瘍効果、すなわち担癌動物モデルにおいて腫瘍をほぼ完全に退縮させる効果、が得られる。このような格別な抗腫瘍効果は、腫瘍の増殖が経時的に漸増するような、抗CAPRIN-1抗体及び抗腫瘍剤であっても、それらを併用するときに認められることから、まったく驚くべきことである。
本発明の癌の治療及び/又は予防用医薬品の標的は、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。
本発明の癌の治療及び/又は予防用医薬品を構成する有効成分、すなわち上記抗体もしくはそのフラグメント及び上記抗腫瘍剤は、それらを一緒に混合した医薬組成物或いはそれらの別個の医薬組成物として、当業者に公知の方法で製剤化することが可能である。
本発明の癌の治療及び/又は予防剤による癌の治療及び/又は予防は、上記の医薬組成物として投与することの他に様々な形式を含む。例えば、本発明の癌の治療及び/又は予防剤の各有効成分は、同時に、または順序に従って個別に投与することができる。具体例として、約3週間までの時間間隔内で、すなわち1番目の有効成分を投与した直後から約3週間までに2番目の有効成分を投与することができる。その際、外科的処置に引き続いて実施しても、1番目の薬剤と2番目の薬剤の投与間に外科的処置を実施してもよい。また、本発明の癌の治療及び/又は予防剤を、複数の投与サイクルに従って投与してもよい。例えば、本発明の癌の治療及び/又は予防剤の各有効成分の同時投与を実施した場合、両方の有効成分を含む医薬組成物を約2日から約3週間を1サイクルとして投与する。その後は該治療サイクルを担当する医師の判断に従って、必要に応じて繰り返すことも可能である。同様に順序に従った処方を計画する場合、それぞれの個々の薬剤の投与期間が同じ期間に及ぶように調節する。サイクル間の間隔は0~2ヶ月まで変えることができる。本発明の癌の治療及び/又は予防剤の各有効成分の投与量は、医薬組成物での各有効成分の投与量と同様に設定することができる。
本発明の癌の治療及び/又は予防用の医薬品は、製薬キットの形態であってもよい。製薬キットとは、癌を治療または予防する方法において、有効成分である上記抗CAPRIN-1抗体もしくはそのフラグメント及び上記抗腫瘍剤を別個の医薬組成物の形態で使用するためのパッケージであり、該パッケージには各有効成分を投与するための指示書が含まれる。製薬キットに含まれる癌の治療及び/又は予防用の上記医薬組成物の各有効成分は、各有効成分を一緒に又は別々に投与できるようにそれぞれが上記の通り製剤化された医薬組成物の形態でありうる。また、製薬キットには、各有効成分を上記投与方法に従って投与できるよう、1つまたは複数回の用量に十分な量の有効成分が含まれる。
(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5mg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。
(5)-1 マウス及びイヌ正常組織におけるCAPRIN-1の発現
マウス(Balb/c、雌)及びイヌ(ビーグル犬、雌)をエーテル麻酔下及びケタミン/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3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及び実施例3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3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及び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3で作製したモノクローナル抗体#2または#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(5)-3と同様の方法で実施例3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3で作製したモノクローナル抗体#2または#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(5)-3と同様の方法で実施例3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3で作製したモノクローナル抗体#2または#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各種癌組織アレイ(BIOMAX社製)の検体を用いて、上述と同様の方法によって、実施例3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は食道癌、結腸癌、直腸癌、肺癌、腎癌、膀胱癌及び子宮頸癌で強い発現が認められた。なお、本結果は、モノクローナル抗体#2または#9を用いた場合も同様であった。
(1)組換えタンパク質の作製
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、乳癌組織・細胞cDNAよりRT-PCR法による発現が確認できたcDNAを1μl、SacI及び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及び及び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2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/c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回すなわち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3で選抜した11個のマウス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由来配列及びマウス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)領域及び軽鎖可変(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。及びまた、CAPRIN-1を発現する乳癌細胞の細胞表面に反応するモノクローナル抗体を産生するマウス由来ハイブリドーマ株2株から、mRNAを抽出し、マウスFR1由来配列及びマウス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、各抗体の重鎖可変(VH)領域及び軽鎖可変(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(Invitrogen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、High Pure RNA Isolation Kit(Roche社製)を用いて全RNAを抽出した後、PrimeScriptII 1st strand cDNA Synthesis Kit(Takara社製)を用いてcDNAを合成した。これら操作は各キットの添付プロトコルに従って行った。
上記で得られた各プラスミド中のVH領域及び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)及び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)。
(2)抗CAPRIN-1抗体#2及び#9を用いた各種癌細胞表面でのCAPRIN-1の発現
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,MDA-MB-231T)及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5種(T98G,SNB19,U251,U87MG,U373)、腎臓癌細胞株4種(Caki-1,Caki-2,A498,ACHN)、胃癌細胞株2種(MNK28,MKN45)、大腸癌細胞株5種(HT29,LoVo,Caco2,SW480,HCT116)、肺癌細胞株3種(A549、QG56、PC8)、白血病細胞株4種(AML5,Namalwa、BDCM、RPI1788)、リンパ腫細胞株1種(Ramos)、子宮頸癌細胞株1種(SW756)、膀胱癌細胞株1種(T24)及び食道癌細胞株1種(KYSE180)について、実施例3で得られた#2及び#9を産生するハイブリドーマの培養上清を用いて、各細胞の細胞表面上でのCAPRIN-1タンパク質の発現を調べた。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。実施例3で得られた#2及び#9を産生するハイブリドーマの培養上清(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標識ヤギ抗ヒトIgG(H+L)抗体(SouthernBiotech社製)及びFITC標識抗マウスIgG(H+L)抗体(Invitrogen社製)を添加し、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、ハイブリドーマ培養用の培地を用いて行い、陰性コントロールとした。その結果、#2及び#9の抗体を添加した細胞は、コントロールに比べて、いずれも蛍光強度が20%以上強かった。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。
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(Cr)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(Cr)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1参照)。一方、実施例2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1参照)。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。同様にして、他のヒト癌細胞である、グリオーマ細胞株T98G、U373、肺癌細胞株A549、QG56、腎臓癌細胞株Caki-1、ACHN、子宮頸癌細胞株SW756、膀胱癌細胞株T24、食道癌細胞株KYSE180、胃癌細胞株MNK28、MNK45、大腸癌細胞株SW480、白血病細胞株AML5及びリンパ腫細胞株Ramosに対する抗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、アイソタイプコントロールに比べて高いADCC活性を示した。具体的には、例えばT98Gに対して、#9は12%以上(アイソタイプコントロールは1.3%)、U373に対して、#9は16%以上(アイソタイプコントロールは3%)、A549に対して、#9は24%以上(アイソタイプコントロールは2.6%)、QG56に対して、#9は20%以上(アイソタイプコントロールは0.2%)、Caki-1に対して、#9は23%以上(アイソタイプコントロールは3.0%)、ACHNに対して、#9は14%以上(アイソタイプコントロールは1.5%)、SW756に対して、#9は16%以上(アイソタイプコントロールは2.5%)、T24に対して、#9は18%以上(アイソタイプコントロールは2.1%)、KYSE180に対して、#9は22%以上(アイソタイプコントロールは3.0%)、MNK28に対して、#9は15%以上(アイソタイプコントロールは1.7%)、MNK45に対して、#9は10%以上(アイソタイプコントロールは2.3%)、SW480に対して、#9は17%以上(アイソタイプコントロールは1.3%)、AML5に対して、#9は10%以上(アイソタイプコントロールは3.0%)及びRamosに対して、#9は10%以上の活性を示した(アイソタイプコントロールは4.1%)。以上の結果より、取得した抗CAPRIN-1抗体(#1~#11)は、CAPRIN-1を発現する各種ヒト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。これに実施例3で得た#1~#13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2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。
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。
(1)癌細胞に対する抗腫瘍剤の50%阻害濃度の算出
抗腫瘍剤による癌細胞表面上のCAPRIN-1発現の影響を評価するため、癌細胞(MCF-7)を用いて、抗腫瘍剤の50%阻害濃度の算出を行った。ヒト乳癌細胞株MCF-7について、現在乳癌治療薬として用いられている抗腫瘍剤4種(シクロホスファミド:“エンドキサン”(登録商標、塩野義製薬株式会社製)、パクリタキセル:“タキソール”(登録商標、ブリストル・マイヤーズ社製)、ドセタキセル:“タキソテール”(登録商標、サノファ・アバンティス社製)、ビノレルビン:“ナベルビン”(登録商標、協和発酵キリン株式会社製)の50%阻害濃度を調べた。細胞を1×105cells/mlになるように調製し、6穴プレートにて37℃、5%CO2条件下1日培養を行った。次に、各抗腫瘍剤を最終濃度0.001μM,0.01μM,0.1μM,1.0μM,10μMそれぞれ処理し、37℃、5%CO2条件下で2日培養を行った。培養液を除去した後、PBS(-)で2回洗浄を行い、0.25%Trypsin-EDTAを添加して細胞を剥がし、PBS(-)で100μlに調製した後、0.4%トリパンブルーストック溶液を10μl加えて混合し、血球計算盤を用いて生細胞数を測定した。各抗腫瘍剤未処理の生細胞数を100%としたときの各化学療法剤処理時の生細胞の割合を算出し、本検討の結果から50%阻害濃度を概算して、さらにその濃度付近の抗腫瘍剤を調製し、上述と同様の操作を行うことにより詳細な50%阻害濃度を算出することとした。
(1)で算出した50%阻害濃度の各抗腫瘍剤を癌細胞株(MCF-7)に処理して、その細胞表面上のCAPRIN-1タンパク質の発現挙動について調べた。
(抗CAPRIN-1抗体のみ投与する群)
5匹の担癌マウスに対して#2のCAPRIN-1に対するモノクローナル抗体を1匹あたり5mg/kg/shotずつ、試験開始0,4,8,11,15,17日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
5匹の担癌マウスに対して、シクロホスファミドを1匹あたり80mg/kg/shotずつ、試験開始0、4日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈投与した。
5匹の担癌マウスに対して、パクリタキセルを1匹あたり15mg/kg/shotずつ、試験開始0、3日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
5匹の担癌マウスに対して、ドセタキセルを1匹あたり10mg/kg/shotずつ、試験開始0、3日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
5匹の担癌マウスに対して、ビノレルビンを1匹あたり1mg/kg/shotずつ、試験開始0日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
5匹の担癌マウスに対して、シクロホスファミドを1匹あたり80mg/kg/shotずつ、試験開始0、4日に腹腔内投与し、同時に抗Her2抗体を1匹あたり5mg/kg/shotずつ、試験開始0、4,8,11,15,17日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
5匹の担癌マウスに対して、パクリタキセルを1匹あたり15mg/kg/shotずつ、試験開始0、3日に腹腔内投与し、同時に抗Her2抗体を1匹あたり5mg/kg/shotずつ、試験開始0、4,8,11,15,17日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
5匹の担癌マウスに対して、ドセタキセルを1匹あたり10mg/kg/shotずつ、試験開始0、3日に腹腔内投与し、同時に抗Her2抗体を1匹あたり5mg/kg/shotずつ、試験開始0、4,8,11,15,17日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
5匹の担癌マウスに対して、ビノレルビンを1匹あたり1mg/kg/shotずつ、試験開始0日に腹腔内投与し、同時に抗Her2抗体を1匹あたり5mg/kg/shotずつ、試験開始0、4,8,11,15,17日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
5匹の担癌マウスに対して、シクロホスファミドを1匹あたり80mg/kg/shotずつ、試験開始0、4日に腹腔内投与し、同時に#2のCAPRIN-1に対するモノクローナル抗体を1匹あたり5mg/kg/shotずつ、試験開始0、4,8,11,15,17日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
5匹の担癌マウスに対して、パクリタキセルを1匹あたり15mg/kg/shotずつ、試験開始0、3日に腹腔内投与し、同時に#2のCAPRIN-1に対するモノクローナル抗体を1匹あたり5mg/kg/shotずつ、試験開始0、4,8,11,15,17日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
5匹の担癌マウスに対して、ドセタキセルを1匹あたり10mg/kg/shotずつ、試験開始0、3日に腹腔内投与し、同時に#2のCAPRIN-1に対するモノクローナル抗体を1匹あたり5mg/kg/shotずつ、試験開始0、4,8,11,15,17日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
ビノレルビンと抗CAPRIN-1抗体の5匹の担癌マウスに対して、ビノレルビンを1匹あたり1mg/kg/shotずつ、試験開始0日に腹腔内投与し、同時に#2のCAPRIN-1に対するモノクローナル抗体を1匹あたり5mg/kg/shotずつ、試験開始0、4,8,11,15,17日に腹腔内投与し、Balb/cマウス脾臓から分離したPBMCを1匹あたり1×107cells/0.2mL(RPMI1640)ずつ、試験開始0,8,15日に静脈内投与した。
抗CAPRIN-1抗体のみ投与する群、シクロホスファミドと抗CAPRIN-1抗体の投与群、パクリタキセルと抗CAPRIN-1抗体の投与群、ドセタキセルと抗CAPRIN-1抗体の投与群及びビノレルビンと抗CAPRIN-1抗体の投与群については、抗CAPRIN-1抗体として#9のCAPRIN-1に対するモノクローナル抗体を投与する以外は、実験区1と同様に設定した。
また、実験区2では、PBS(-)を投与したコントロール群の試験開始から26日目の腫瘍体積を100%とした場合に、各抗腫瘍剤投与群では約68%程度、抗CAPRIN-1抗体のみ投与する群では約45%程度、各抗腫瘍剤と抗Her2抗体の投与群では約55%にまで退縮した。一方、各抗腫瘍剤と抗CAPRIN-1抗体の投与群では、14日目には数10%にまで腫瘍が退縮し、22日目以降には腫瘍はほぼ完全に退縮した。(図7~図10参照)
配列番号32: T7プライマー
配列番号33、34: プライマー
配列番号35、36: GAPDHプライマー
配列番号38、39: プライマー
配列番号130~135: プライマー
Claims (11)
- CAPRIN-1タンパク質と免疫学的反応性を有する抗体又はそのフラグメントと、1種類又は2種類以上の抗腫瘍剤とを、一緒に又は別々に、組み合わせ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品。
- 前記CAPRIN-1タンパク質と免疫学的反応性を有する抗体又はそのフラグメントが、癌細胞表面上に存在するCAPRIN-1タンパク質の細胞外領域と特異的に結合する抗体又はそのフラグメントであることを特徴とする、請求項1に記載の医薬品。
- 前記CAPRIN-1タンパク質と免疫学的反応性を有する抗体又はそのフラグメントが、癌細胞表面上に存在するCAPRIN-1タンパク質の細胞外領域の内、配列番号37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特異的に結合する抗体又はそのフラグメントであることを特徴とする、請求項1又は2に記載の医薬品。
- 前記CAPRIN-1タンパク質がヒト由来である、請求項1~3のいずれか1項に記載の医薬品。
- 前記抗腫瘍剤が明細書に記載の抗腫瘍剤である、請求項1~4のいずれか1項に記載の医薬品。
- 前記抗腫瘍剤が、シクロホスファミド、パクリタキセル、ドセタキセル、ビノレルビン並びにそれらの薬学的に許容可能な塩及び誘導体からなる群から選択される、請求項5に記載の医薬品。
- 前記癌が乳癌、脳腫瘍、白血病、リンパ腫、肺癌、肥満細胞腫、腎癌、子宮頸癌、膀胱癌、食道癌、胃癌もしくは大腸癌である、請求項1~6のいずれかに記載の医薬品。
- 前記抗体が、モノクローナル抗体、ポリクローナル抗体又は組換え抗体である、請求項1~7のいずれか1項に記載の医薬品。
- 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1~8のいずれか1項に記載の医薬品。
- 請求項1~9のいずれか1項に記載の医薬品を、癌をもつと疑われる被験者に投与することを含む、癌の治療及び/又は予防のための方法。
- 前記医薬品に含まれる、抗体又はそのフラグメント及び抗腫瘍剤を、同時に又は別々に、前記被験者に投与することを含む、請求項10に記載の方法。
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DK11739883.4T DK2532367T3 (en) | 2010-02-04 | 2011-02-04 | PHARMACEUTICAL AGENT FOR TREATMENT AND / OR CANCER PREVENTION |
ES11739883.4T ES2691738T3 (es) | 2010-02-04 | 2011-02-04 | Agente farmacéutico para el tratamiento y/o la prevención de cáncer |
AU2011211700A AU2011211700B2 (en) | 2010-02-04 | 2011-02-04 | Medicament for treating and/or preventing cancer |
RU2012137503A RU2624029C2 (ru) | 2010-02-04 | 2011-02-04 | Лекарственный препарат для лечения и/или профилактики рака |
EP11739883.4A EP2532367B1 (en) | 2010-02-04 | 2011-02-04 | Pharmaceutical agent for treatment and/or prevention of cancer |
KR1020127022763A KR101843807B1 (ko) | 2010-02-04 | 2011-02-04 | 암의 치료 및/또는 예방을 위한 의약품 |
CN201910271408.5A CN109925511B (zh) | 2010-02-04 | 2011-02-04 | 用于癌的治疗和/或预防的药物 |
CA2788720A CA2788720C (en) | 2010-02-04 | 2011-02-04 | Medicament for treating and/or preventing cancer |
CN2011800172337A CN102844048A (zh) | 2010-02-04 | 2011-02-04 | 用于癌的治疗和/或预防的药物 |
JP2011510197A JP5923984B2 (ja) | 2010-02-04 | 2011-02-04 | 癌の治療及び/又は予防のための医薬品 |
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MX2012008991A MX2012008991A (es) | 2010-02-04 | 2011-02-04 | Medicamento para tratar y/o prevenir el cancer. |
PL11739883T PL2532367T3 (pl) | 2010-02-04 | 2011-02-04 | Środek farmaceutyczny do leczenia i/lub zapobiegania nowotworu |
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RU2012137503A (ru) | 2014-03-10 |
CN109925511B (zh) | 2024-03-19 |
AU2011211700A1 (en) | 2012-08-23 |
US9180187B2 (en) | 2015-11-10 |
CA2788720C (en) | 2019-08-20 |
HUE040012T2 (hu) | 2019-02-28 |
EP2532367A1 (en) | 2012-12-12 |
US20120294860A1 (en) | 2012-11-22 |
KR101843807B1 (ko) | 2018-03-30 |
DK2532367T3 (en) | 2018-11-19 |
MX2012008991A (es) | 2012-09-07 |
CA2788720A1 (en) | 2011-08-11 |
CN109925511A (zh) | 2019-06-25 |
RU2624029C2 (ru) | 2017-06-30 |
JP5923984B2 (ja) | 2016-05-25 |
KR20120139720A (ko) | 2012-12-27 |
ES2691738T3 (es) | 2018-11-28 |
EP2532367A4 (en) | 2014-05-28 |
EP2532367B1 (en) | 2018-08-29 |
AU2011211700B2 (en) | 2015-06-11 |
CN102844048A (zh) | 2012-12-26 |
JPWO2011096535A1 (ja) | 2013-06-13 |
BR112012018943A8 (pt) | 2017-12-19 |
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