WO2013018883A1 - 癌の治療及び/又は予防用医薬組成物 - Google Patents
癌の治療及び/又は予防用医薬組成物 Download PDFInfo
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- 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
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
Definitions
- the present invention relates to a novel pharmaceutical use of an antibody against CAPRIN-1 or a fragment thereof as a therapeutic and / or prophylactic agent for cancer.
- Cancer is a disease that occupies the top cause of all deaths, and the current treatment is a combination of radiation therapy and chemotherapy, mainly surgery. Despite the recent development of new surgical methods and the discovery of new anti-cancer drugs, the therapeutic results of cancer have not improved much except for some cancers. In recent years, advances in molecular biology and cancer immunology have identified antibodies that react specifically with cancer, cancer antigens that are recognized by cytotoxic T cells, genes that encode cancer antigens, There is an increasing expectation for a specific cancer therapy targeting cancer antigens (Non-patent Document 1).
- Non-Patent Documents 4 to 4 9
- clinical studies of cell therapy using immune cells that specifically react with cancer antigens and cancer-specific immunotherapy such as vaccines containing cancer antigens are being conducted targeting a part of them.
- Cytoplasmic- and propagation-associated protein 1 (CAPRIN-1) is expressed when quiescent normal cells are activated or undergo cell division, and forms RNA and intracellular stress granules within the cell to transport mRNA. It was known as an intracellular protein known to be involved in the regulation of translation, but was found to be specifically expressed on the surface of cancer cells, and as a target for antibody drugs for cancer treatment Research is ongoing (Patent Document 2).
- An object of the present invention is to produce an antibody having an antitumor activity superior to a conventional antibody targeting CAPRIN-1 that is specifically expressed on the surface of cancer cells, and to be used as a therapeutic and / or preventive agent for cancer. Is to provide.
- the present invention has the following features.
- an antibody comprising a heavy chain variable region comprising SEQ ID NOs: 5, 6 and 7 and a light chain variable region comprising SEQ ID NOs: 9, 10 and 11, and having immunological reactivity with a CAPRIN-1 protein
- the present invention provides a pharmaceutical composition for treating and / or preventing cancer, comprising a fragment thereof as an active ingredient.
- the cancer is breast cancer, renal cancer, pancreatic cancer, colon cancer, lung cancer, brain tumor, stomach cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, fibrosarcoma, obesity A cell tumor or melanoma.
- the antibody is a human antibody, a humanized antibody, a chimeric antibody, a single chain antibody, or a multispecific antibody.
- the antibody against CAPRIN-1 used in the present invention damages cancer cells. Therefore, antibodies against CAPRIN-1 are useful for the treatment and prevention of cancer.
- the antitumor activity of an antibody against the polypeptide of CAPRIN-1 used in the present invention is, as described later, by examining the suppression of tumor growth in cancer-bearing animals in vivo, or expressing the polypeptide in vitro. It can be evaluated by examining whether or not tumor cells exhibit cytotoxic activity via immune cells or complement.
- the antibody against CAPRIN-1 used in the present invention is a monoclonal antibody, and may be any kind of antibody as long as it can exhibit antitumor activity.
- a recombinant antibody such as a synthetic antibody, a multispecific antibody
- examples include humanized antibodies, chimeric antibodies, single chain antibodies (scFv), human antibodies, and antibody fragments thereof, such as Fab, F (ab ′) 2 , and Fv.
- scFv single chain antibodies
- human antibodies and antibody fragments thereof, such as Fab, F (ab ′) 2 , and Fv.
- These antibodies and fragments thereof can also be prepared by methods known to those skilled in the art.
- 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 subject to be treated and / or prevented for cancer in the present invention is a mammal such as a human, a pet animal, livestock, a sport animal, etc., and a preferred subject is a human.
- the protein or fragment thereof used as a sensitizing antigen for obtaining an antibody against CAPRIN-1 used in the present invention is used for the animal species from which it is derived, such as humans, dogs, cows, horses, mice, rats, and chickens. Not limited. However, it is preferable to select in consideration of compatibility with the parent cell used for cell fusion. In general, a protein derived from a mammal is preferable, and a protein derived from a human is particularly preferable. For example, when CAPRIN-1 is human CAPRIN-1, human CAPRIN-1 protein, a partial peptide thereof, cells expressing human CAPRIN-1 and the like can be used.
- GenBank GenBank
- FASTA Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.
- the base sequence or amino acid sequence of these ORFs or mature portions is 70% to 100%, preferably 80% to 100%, more preferably 90% to 100%, even more preferably 95% to 100%, such as 97% to 100%, 98% to 100%, 99% to 100% or 99.100%.
- a target is a nucleic acid or protein consisting of a sequence having 5% to 100% sequence identity.
- “% sequence identity” refers to an amino acid (or an alignment) when two sequences are aligned (aligned) for maximum similarity or identity with or without gaps. The percentage (%) of the same amino acid (or base) with respect to the total number of bases.
- the CAPRIN-1 protein fragment has a length less than the total length of the protein from the amino acid length of the epitope (antigenic determinant), which is the smallest unit recognized by the antibody.
- An epitope refers to a polypeptide fragment that has antigenicity or immunogenicity in a mammal, preferably a human, and its minimum unit consists of about 7 to 12 amino acids, such as 8 to 11 amino acids.
- polypeptides including human CAPRIN-1 protein and partial peptides thereof are synthesized according to chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method), for example.
- Fmoc method fluorenylmethyloxycarbonyl method
- tBoc method t-butyloxycarbonyl method
- the polynucleotide encoding the above-mentioned polypeptide can be easily prepared by a known genetic engineering technique or a conventional method using a commercially available nucleic acid synthesizer.
- DNA containing the base sequence of the human CAPRIN-1 gene can be obtained by PCR using a human chromosomal DNA or cDNA library as a template and a pair of primers designed to amplify the base sequence described in SEQ ID NO: 1.
- PCR reaction conditions can be set as appropriate. For example, using a heat-resistant DNA polymerase (eg, Taq polymerase, Pfu polymerase, etc.) and a Mg 2+ -containing PCR buffer, the reaction is performed at 94 ° C.
- a heat-resistant DNA polymerase eg, Taq polymerase, Pfu polymerase, etc.
- the reaction process consisting of seconds to 1 minute (annealing) and 2 minutes (extension) at 72 ° C. is defined as one cycle. For example, after 30 cycles, the reaction can be performed at 72 ° C. for 7 minutes. It is not limited.
- the PCR method, conditions, etc. are described in, for example, Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, A compendium of Methods from Current Protocols in Molecular Biology (1995), J ing.
- probes and primers are prepared based on information on the base sequence of the CAPRIN-1 gene and the amino acid sequence of the CAPRIN-1 protein, and a desired cDNA library is screened using the probe or primer.
- DNA can be isolated.
- the cDNA library is preferably prepared from cells, organs or tissues expressing the CAPRIN-1 protein. Examples of such cells and tissues include breast cancer, kidney cancer, pancreatic cancer, colon cancer, lung cancer, brain tumor, stomach cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, fibrosarcoma, A cell or tissue derived from a cancer or tumor such as mastocytoma or melanoma.
- 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.
- an expression vector having an origin, a promoter, a ribosome binding site, a multicloning site, a terminator, a drug resistance gene, an 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.
- 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.
- the introduction of the expression vector into the host cell may be carried out using 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.
- the target polypeptide from the host cell can be performed by combining known separation operations. For example, treatment with denaturing agents and surfactants such as urea, ultrasonic treatment, enzyme digestion, salting out and solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, etc., electric point electrophoresis, ion Examples include, but are not limited to, exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.
- denaturing agents and surfactants such as urea, ultrasonic treatment, enzyme digestion, salting out and solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, etc.
- electric point electrophoresis ion
- Examples include, but are not limited to, exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.
- 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).
- the antibody used in the present invention binds to the CAPRIN-1 protein and is a tumor such as breast cancer, kidney cancer, pancreatic cancer, colon cancer, lung cancer, brain tumor, stomach cancer, cervical cancer, ovarian cancer, prostate cancer, bladder Any kind of cancer, esophageal cancer, leukemia, lymphoma, fibrosarcoma, mastocytoma or melanoma can be used.
- the antibody is not particularly limited as long as it is a monoclonal antibody. Synthetic antibodies, multispecific antibodies (eg, diabody, triabody, etc.), human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, antibody fragments (For example, Fab, F (ab ′) 2 , Fv, etc.).
- the antibody may also be any class of immunoglobulin molecules, such as IgG, IgE, IgM, IgA, IgD and IgY, or any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and the like.
- the antibody may be further modified by acetylation, formylation, amidation, phosphorylation, or PEGylation.
- a breast cancer cell line SK-BR-3 expressing CAPRIN-1 is immunized by administering to a mouse, the spleen is extracted from the mouse, the cells are separated, and the cells are fused with mouse myeloma cells. From the obtained fused cells (hybridomas), a clone producing an antibody having a cancer cell growth inhibitory action is selected. It can be prepared by isolating a monoclonal antibody-producing hybridoma having cancer cell growth inhibitory action, culturing the hybridoma, and purifying the antibody from the culture supernatant by a general affinity purification method.
- a hybridoma producing a monoclonal antibody can also be produced, for example, as follows.
- an animal is immunized with a sensitizing antigen according to a known method.
- a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously.
- the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, or the like, and mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified.
- PBS Phosphate-Buffered Saline
- physiological saline or the like
- an ordinary adjuvant for example, Freund's complete adjuvant, if necessary, and emulsified.
- the mammal is dosed several times every 4-21 days.
- an appropriate carrier can be used during immunization with the sensitizing antigen.
- immune cells are collected from the mammal and subjected to cell fusion. Can be mentioned.
- Mammalian myeloma cells are used as the other parent cell to be fused with the immune cells.
- This myeloma cell is known in various known cell lines such as P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-511) (Margulies.
- the cell fusion between the immune cell and myeloma cell is basically performed by a known method, for example, the method of Kohler and Milstein et al. (Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46. ) And the like.
- the cell fusion is performed, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter.
- a cell fusion promoter for example, polyethylene glycol (PEG), Sendai virus (HVJ), or the like is used as the fusion promoter, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency as desired.
- the usage ratio of immune cells and myeloma cells can be arbitrarily set.
- the number of immune cells is preferably 1 to 10 times that of myeloma cells.
- the culture solution used for the cell fusion for example, RPMI1640 culture solution suitable for growth of the myeloma cell line, MEM culture solution, and other normal culture solutions used for this kind of cell culture can be used.
- Serum replacement fluid such as fetal calf serum (FCS) can be used in combination.
- a predetermined amount of the immune cells and myeloma cells are mixed well in the culture medium, and a PEG solution (for example, an average molecular weight of about 1000 to 6000) preliminarily heated to about 37 ° C. is usually 30 to 60% (
- the desired hybridoma is formed by adding at a concentration of w / v) and mixing.
- cell fusion agents and the like that are undesirable for the growth of the hybridoma are removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.
- the hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing with the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fusion cells) to die. Subsequently, the usual limiting dilution method is performed, and the hybridoma producing the target antibody is screened and single-cloned.
- a normal selective culture solution for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing with the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fusion cells) to die. Subsequently, the usual limiting dilution method is performed, and the hybridoma producing the target antibody is screened
- human lymphocytes such as human lymphocytes infected with EB virus are sensitized in vitro with proteins, protein-expressing cells or lysates thereof. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, for example, U266 (Registration No. TIB196) to obtain a hybridoma that produces a human antibody having a desired activity (for example, cell growth inhibitory activity).
- a desired activity for example, cell growth inhibitory activity
- the hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution and can be stored for a long time in liquid nitrogen.
- a desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen and immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. And can be prepared by screening monoclonal antibody-producing cells (hybridomas) by a normal screening method.
- human antibody-producing mice for example, KM mice (Kirin Pharma / Medarex) and Xeno mice (Amgen) are known (for example, International Publication Nos. WO02 / 43478, WO02 / 092812, etc.).
- fully human polyclonal antibodies can be obtained from blood.
- spleen cells can be removed from the immunized mouse and a human monoclonal antibody can be prepared by a fusion method with myeloma cells.
- the antigen can be prepared according to, for example, a method using animal cells (Japanese Patent Publication No. 2007-530068), a method using baculovirus (eg, International Publication No. WO 98/46777).
- immunization may be performed by binding to an immunogenic macromolecule such as albumin.
- a recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing it into a host, and producing it using a gene recombination technique (for example, Carl, AK Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL, ANTIBODIES, Published in the United KingdomMIMILLAN PUBLISHERS 19).
- a gene recombination technique for example, Carl, AK Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL, ANTIBODIES, Published in the United KingdomMIMILLAN PUBLISHERS 19.
- V region an antibody variable region
- DNA encoding the V region of the target antibody is obtained, it is ligated with DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector.
- DNA encoding the V region of the antibody may be incorporated into an expression vector containing DNA of the antibody C region. It is incorporated into an expression vector so as to be expressed under the control of an expression control region such as an enhancer or promoter.
- host cells can be transformed with this expression vector to express the antibody.
- the anti-CAPRIN-1 antibody of the present invention is a monoclonal antibody.
- the monoclonal antibody includes a human monoclonal antibody, a monoclonal antibody of a non-human animal (eg, mouse monoclonal antibody, rat monoclonal antibody, rabbit monoclonal antibody). , Chicken monoclonal antibodies, etc.) and chimeric monoclonal antibodies.
- Monoclonal antibodies are obtained by culturing hybridomas obtained by fusion of spleen cells and myeloma cells from non-human animals immunized with the CAPRIN-1 protein (eg, mice, human antibody-producing mice, chickens, rabbits, etc.). Can be made.
- a chimeric antibody is an antibody produced by combining sequences derived from different animals, for example, an antibody comprising a mouse antibody heavy chain, a light chain variable region and a human antibody heavy chain, a light chain constant region, and the like. is there.
- 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. In the examples described later, human-mouse chimeric monoclonal antibodies were prepared and their antitumor effects were confirmed.
- the monoclonal antibody includes a heavy chain variable (VH) region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable (VL) region having the amino acid sequence of SEQ ID NO: 12, wherein the VH region has SEQ ID NO: CDR1 represented by the amino acid sequence of 5, CDR2 represented by the amino acid sequence of SEQ ID NO: 6, and CDR3 represented by the amino acid sequence of SEQ ID NO: 7 are included, and the VL region is represented by the amino acid sequence of SEQ ID NO: 9.
- CDR1 represented by the amino acid sequence of SEQ ID NO: 10 and CDR3 represented by the amino acid sequence of SEQ ID NO: 11 are included.
- 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.
- a DNA sequence designed to link a CDR of a mouse antibody or chicken antibody and a framework region (FR) of a human antibody was prepared so as to have an overlapping portion at the end. It is synthesized by PCR from several oligonucleotides. 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).
- 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 (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine).
- basic amino acids arginine, lysine, histidine
- acidic amino acids aspartic acid, glutamic acid
- uncharged polar amino acids glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine
- modified antibody examples include antibodies bound to various molecules such as polyethylene glycol (PEG).
- PEG polyethylene glycol
- the substance to be bound is not limited. In order to obtain such a modified antibody, it can be obtained by chemically modifying the obtained antibody. These methods are already established in this field.
- “functionally equivalent” means that the target antibody has the same biological or biochemical activity as the antibody of the present invention, specifically, a function of damaging a tumor, and is applied to humans. Sometimes refers to not essentially causing rejection. Examples of such activity include cell growth inhibitory activity or binding activity.
- an antibody that recognizes the epitope of the CAPRIN-1 protein recognized by the anti-CAPRIN-1 antibody can be obtained by methods known to those skilled in the art.
- the epitope of the CAPRIN-1 protein recognized by the anti-CAPRIN-1 antibody is determined by an ordinary method (eg, epitope mapping), and an antibody is produced using a polypeptide having the amino acid sequence contained in the epitope as an immunogen.
- This method can be obtained by a method, a method of determining an epitope of an antibody prepared by a usual method, and selecting an antibody having the same epitope as the anti-CAPRIN-1 antibody.
- epitope refers to a polypeptide fragment having antigenicity or immunogenicity in a mammal, preferably a human, and its minimum unit consists of about 7 to 12 amino acids, preferably 8 to 11 amino acids.
- the affinity constant Ka (k on / k off ) of the antibody of the present invention is preferably at least 10 7 M ⁇ 1 , at least 10 8 M ⁇ 1 , at least 5 ⁇ 10 8 M ⁇ 1 , at least 10 9 M ⁇ 1 , At least 5 ⁇ 10 9 M ⁇ 1 , at least 10 10 M ⁇ 1 , at least 5 ⁇ 10 10 M ⁇ 1 , at least 10 11 M ⁇ 1 , at least 5 ⁇ 10 11 M ⁇ 1 , at least 10 12 M ⁇ 1 , or alternatively At least 10 13 M ⁇ 1 .
- the antibody of the present invention can be conjugated with an antitumor agent.
- the bond between the antibody and the antitumor agent is a group reactive with an amino group, carboxyl group, hydroxy group, thiol group, etc. (for example, succinate imidyl group, formyl group, 2-pyridyldithio group, maleimidyl group, alkoxycarbonyl group). , A hydroxy group, etc.).
- antitumor agents include the following antitumor agents known in the literature, such as paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan, piperosulfan, benzodopa (benzodopa) ), Carbocone, methredopa, uredopa, uretopa, altreamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolothramine , Camptothecin, bryostatin, calistatin ( allystatin), cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleuterbin, panclastatin, sarcodictin, spongestatin, chlorambucil, chloronaphazine, cholophosphamide, estram Ifosfamide, mechlore
- a higher therapeutic effect can be obtained by co-administering the antibody of the present invention and an antitumor agent.
- This technique can be applied to cancer patients expressing CAPRIN-1 either before or after surgery. In particular, after surgery, cancer recurrence and longer survival can be obtained for cancers expressing CAPRIN-1, which have been treated with an antitumor agent alone.
- antitumor agents used in combination administration with the antibody of the present invention include the following antitumor agents known in the literature, such as paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, Busulfan, Improsulfan, Piposulfan, Benzodopa, Carbocon, Metredopa, Uredopa, Alteramine, Triethylenemelamine, Triethylenephosphoramide, Triethylenethiophosphoramide Trimethylolomeramine, bratacin, bratacinone, camptocete , Bryostatin, callystatin, cryptophysin 1, cryptophycin 8, dolastatin, duocarmycin, eleuterbin, panclatistatin, sarcodictin, spongestatin, chloramphazine, chloronaphazine, cholophosphamide ( chlorophosphamide), estramustine, pac
- the antibodies of the present invention include radioactive substances such as 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 175 Lu, and 176 Lu, which are known in the literature. It is also possible to combine isotopes. It is desirable that the radioisotope is effective for tumor treatment and diagnosis.
- the antibody of the present invention is an antibody immunologically reactive with CAPRIN-1, or an antibody that specifically recognizes CAPRIN-1, or an antibody that specifically binds to CAPRIN-1, and which is against cancer. It is an antibody that exhibits cytotoxic activity or 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, multispecific antibodies and the like when the target animal is human.
- the heavy chain and light chain variable regions are derived from human antibodies, or the heavy chain and light chain variable regions are derived from non-human animal antibodies (CDR1, CDR2 and CDR3) and a framework region derived from a human antibody, or the variable regions of heavy and light chains are derived from a non-human animal antibody, and the constant regions of heavy and light chains are It is a recombinant antibody derived from a human antibody.
- 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 represented by a non-human animal (for example, mouse, rat, rabbit, chicken, etc.)
- the humanized antibody is prepared by linking DNA obtained by replacing the CDR coding sequence of the derived antibody with the DNA encoding the constant region of the light chain or heavy chain derived from the human antibody, respectively. Can be prepared.
- the DNA encoding the light chain or heavy chain variable region of an antibody derived from a non-human animal is derived from the light chain or heavy chain derived from a human antibody.
- a DNA encoding a chimeric antibody can be prepared by ligating with a DNA encoding the constant region.
- this antibody is an antibody in which a heavy chain variable region and a light chain variable region are linearly linked via a linker, DNA encoding the heavy chain variable region, DNA encoding the linker And a DNA encoding a light chain variable region can be combined to produce a DNA encoding a single chain antibody.
- each of the heavy chain variable region and the light chain variable region is derived from a human antibody, or only the CDR depends on the CDR of an antibody derived from an animal other than a human (eg, mouse, rat, rabbit, chicken, etc.). It is derived from a substituted human antibody.
- the linker is composed of 12 to 19 amino acids, and examples thereof include 15 amino acid (G 4 S) 3 (G.-B. Kim et al., Protein Engineering Design and Selection 2007, 20 (9): 425-432). .
- this antibody is an antibody that can specifically bind to two different epitopes, such as DNA encoding heavy chain variable region A, light chain variable region B.
- DNA, DNA encoding heavy chain variable region B, and DNA encoding light chain variable region A are joined in this order (however, DNA encoding light chain variable region B and DNA encoding heavy chain variable region B) Are linked via a DNA encoding a linker as described above), whereby a DNA encoding a bispecific antibody can be prepared.
- each of the heavy chain variable region and the light chain variable region is derived from a human antibody, or only the CDR depends on the CDR of an antibody derived from an animal other than a human (eg, mouse, rat, rabbit, chicken, etc.). It is derived from a substituted human antibody.
- 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-described method include the following antibody (a) obtained in Examples described later.
- amino acid sequences shown in SEQ ID NOs: 5, 6 and 7 are CDR1, CDR2 and CDR3 of the mouse antibody heavy chain variable region, and the amino acid sequences shown in SEQ ID NOs: 9, 10 and 12 are respectively lighter than the mouse antibody light chain. CDR1, CDR2 and CDR3 of the chain variable region.
- humanized antibody, chimeric antibody, single chain antibody or bispecific antibody of the present invention is, for example, the following antibody.
- variable region of the heavy chain comprises the amino acid sequence of SEQ ID NOs: 6, 7 and 8 and the amino acid sequence of the framework region derived from a human antibody
- variable region of the light chain is the amino acid of SEQ ID NOs: 9, 10 and 11
- An antibody comprising a sequence and an amino acid sequence of a framework region derived from a human antibody.
- variable region of the heavy chain comprises the amino acid sequence of SEQ ID NOs: 5, 6 and 7 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: 9, 10, and 11 and the framework region derived from a human antibody
- the light chain constant region comprises the amino acid sequence derived from a human antibody. antibody.
- variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 8
- the 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: 12.
- An antibody comprising a light chain constant region comprising 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.
- the heavy chain and light chain variable regions and the specific sequences of CDRs in the above antibody are merely for illustrative purposes and are not limited to specific sequences.
- a hybridoma capable of producing another human antibody against human CAPRIN-1 or a non-human animal antibody (for example, mouse antibody) is prepared, and the monoclonal antibody produced by the hybridoma is recovered, and immunological binding to human CAPRIN-1 Whether the antibody is the target antibody is determined using the cytotoxic activity as an index.
- 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 has the specificity of specifically recognizing CAPRIN-1, in particular the framework region sequence and / or the constant region sequence of each antibody of (a), one or several amino acids There may be substitutions, deletions or additions.
- substitutions, deletions or additions may be substitutions, deletions or additions.
- the present invention further provides DNA encoding the antibody of the present invention, DNA encoding the heavy chain or light chain of the antibody, or DNA encoding the variable region of the heavy chain or light chain of the antibody.
- DNA is, for example, in the case of antibody (a), a DNA encoding a heavy chain variable region comprising a base sequence encoding the amino acid sequence of SEQ ID NOs: 5, 6 and 7, and the amino acid sequences of SEQ ID NOs: 9, 10 and 11 DNA encoding a light chain variable region comprising a nucleotide sequence encoding
- complementarity determining regions (CDRs) encoded by the DNA of these sequences are regions that determine the specificity of the antibody
- sequences that encode other regions of the antibody May be a sequence derived from another antibody.
- other antibodies include antibodies derived from organisms other than humans, but those derived from humans are preferable from the viewpoint of reducing side effects. That is, in the above DNA, the regions encoding the heavy chain and light chain framework regions and the constant regions preferably include a base sequence encoding a corresponding amino acid sequence derived from a human antibody.
- DNA encoding the antibody of the present invention is, for example, a DNA encoding a heavy chain variable region comprising a base sequence encoding the amino acid sequence of SEQ ID NO: 8, and a region encoding the light chain variable region is SEQ ID NO:
- DNA containing a nucleotide sequence encoding 12 amino acid sequences is the base sequence of SEQ ID NO: 13.
- An example of the base sequence encoding the amino acid sequence of SEQ ID NO: 12 is the base sequence of SEQ ID NO: 14.
- the region encoding each heavy chain and light chain constant region includes a base sequence encoding a corresponding amino acid sequence derived from a human antibody.
- the DNA of these antibodies can be obtained, for example, by the above method or the following method.
- total RNA is prepared from a hybridoma related to the antibody of the present invention using a commercially available RNA extraction kit, and cDNA is synthesized by reverse transcriptase using a random primer or the like.
- the cDNA encoding the antibody is amplified by a PCR method using oligonucleotides having conserved sequences as primers.
- the sequence encoding the constant region can be obtained by amplifying a known sequence by the PCR method.
- the base sequence of DNA can be determined by a conventional method by incorporating it into a sequencing plasmid or phage.
- the anti-tumor effect of the anti-CAPRIN-1 antibody used in the present invention on CAPRIN-1-expressing cancer cells is considered to occur by the following mechanism.
- ADCC Effector cell antibody-dependent cytotoxicity
- CDC complement-dependent cytotoxicity
- the antitumor effect by the above mechanism correlates with the number of target molecules to which an antibody expressed on the cell surface of cancer cells binds (Niwa R., Clinical Cancer Research 2005 Mar 15; 11 ( 6): 2327-2336).
- the number of target molecules expressed on the cell surface of cancer cells can be examined using an existing measurement kit that can measure the number of molecules on the cell surface.
- the number of target molecules to which the antibody binds is caused to react with cancer cells using the antibody against the target molecule as a primary antibody, and with a calibration curve bead whose molecular number is known in advance, and with a fluorescently labeled anti-primary antibody,
- the average fluorescence intensity of the sample can be measured, and a calibration curve can be obtained to know the number of target molecules.
- the activity of the anti-CAPRIN-1 antibody used in the present invention is evaluated by the above-mentioned ADCC activity or CDC activity against cancer cells expressing CAPRIN-1 in vitro, as specifically shown in the Examples below. It can be evaluated by measuring or examining the number of CAPRIN-1 molecules expressed on the cell surface of cancer cells when the anti-CAPRIN-1 antibody of the present invention is used as a primary antibody.
- 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.
- the number of CAPRIN-1 molecules is equal to the number of CAPRIN-1 molecules per cancer cell to which the antibody binds when the anti-CAPRIN-1 antibody of the present invention is used for measurement. Is 10 4 or more, preferably 10 5 or more.
- ⁇ Binding to antigen-expressing cells The ability of an antibody to bind to CAPRIN-1 can be identified using binding assays such as those described in the Examples, such as ELISA, Western blotting, immunofluorescence and flow cytometry analysis.
- Antibodies that recognize CAPRIN-1 are inoculated with tissue obtained from patients during surgery or cell lines expressing CAPRIN-1 naturally or after transfection by immunohistochemistry in a manner well known to those skilled in the art
- tissue obtained from animals bearing selected xenografts using paraformaldehyde or acetone-fixed frozen sections or paraformaldehyde-fixed tissue sections Can do.
- an antibody reactive to CAPRIN-1 can be stained by various methods. For example, it can be visualized by reacting horseradish peroxidase-conjugated goat anti-mouse antibody, goat anti-rabbit antibody or goat anti-chicken antibody.
- the target of the pharmaceutical composition for the treatment and / or prevention of cancer of the present invention is not particularly limited as long as it is a cancer (cell) expressing the CAPRIN-1 gene.
- tumor and cancer refer to malignant neoplasms and are used interchangeably.
- the target cancer in the present invention is a cancer expressing a gene encoding CAPRIN-1 protein, preferably breast cancer, kidney cancer, pancreatic cancer, colon cancer, lung cancer, brain tumor, stomach cancer, cervical cancer Ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, fibrosarcoma, mastocytoma or melanoma.
- These specific cancers include, for example, breast cancer, complex breast cancer, malignant mixed breast tumor, intraductal papillary carcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, neuroepithelial tissue Tumor glioma, ventricular ependymoma, neuronal tumor, fetal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal type Lymphoma, digestive lymphoma, small to medium cell lymphoma, cecal cancer, ascending colon cancer, descending colon cancer, transverse colon cancer, sigmoid colon cancer, rectal cancer, ovarian epithelial cancer, germ cell tumor, stromal cell tumor, Pancreatic duct cancer, invasive pancreatic duct cancer, adenocarcinoma of pancreatic cancer, acinar cell carcinoma, adenosquamous carcinoma, giant cell tumor,
- mammals for example, mammals including primates, pet animals, domestic animals, sport animals and the like, and humans, dogs and cats are particularly preferable.
- the antibody used in the present invention when used as a pharmaceutical composition, it can be formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension.
- a pharmacologically acceptable carrier or medium specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative
- a pharmaceutical preparation by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.
- a sterile composition for injection can be formulated in accordance with normal pharmaceutical practice using a vehicle such as distilled water for injection.
- Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride.
- Suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-60 may be used in combination.
- oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent.
- oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent.
- buffer for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant.
- the prepared injection solution is usually filled into a suitable ampoule.
- Administration is oral or parenteral, preferably parenteral administration. Specific examples include injection, nasal administration, pulmonary administration, and transdermal administration. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.
- the administration method can be appropriately selected depending on the age, weight, sex, symptoms, etc. of the patient.
- the dosage of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg body weight. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values.
- the dose and administration method vary depending on the weight, age, sex, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.
- Cancer preferably breast cancer, kidney cancer, pancreatic cancer, colon cancer, lung cancer, brain tumor, stomach cancer, cervical cancer, ovarian cancer, by administering to a subject the above pharmaceutical composition containing the antibody of the present invention or a fragment thereof, Prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, fibrosarcoma, mastocytoma or melanoma can be treated and / or prevented.
- a method for treating and / or preventing cancer comprising administering the pharmaceutical composition of the present invention in combination with an antitumor agent or a pharmaceutical composition containing an antitumor agent as exemplified above to a subject.
- an antitumor agent or a pharmaceutical composition containing an antitumor agent as exemplified above to a subject.
- the antibody or fragment thereof of the present invention and the antitumor agent can be administered to a subject simultaneously or separately.
- any pharmaceutical composition may be earlier or later, and the administration interval, dosage, administration route and frequency of administration can be appropriately selected by a specialist.
- Another pharmaceutical dosage form to be administered at the same time includes, for example, a pharmaceutical composition obtained by mixing the antibody of the present invention or a fragment thereof and an antitumor agent in a pharmacologically acceptable carrier (or medium) and formulating it. Shall be included. Further, for any of the above pharmaceutical compositions and dosage forms containing an antitumor agent, the formulation, formulation, administration route, dose, cancer, etc. for the pharmaceutical composition and dosage form containing the antibody of the present invention The explanation can be applied.
- the present invention also provides a combination pharmaceutical product for treating and / or preventing cancer comprising the pharmaceutical composition of the present invention and a pharmaceutical composition comprising the antitumor agent as exemplified above.
- the present invention also provides a pharmaceutical composition for treating and / or preventing cancer comprising the antibody of the present invention or a fragment thereof and an antitumor agent together with a pharmacologically acceptable carrier.
- the present invention further provides the following polypeptides and DNAs related to the antibody (a).
- a polypeptide comprising the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 12, and a DNA encoding the polypeptide, for example, a DNA comprising the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14.
- a heavy chain CDR polypeptide selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 5, 6 and 7, and DNA encoding the polypeptide.
- a light chain CDR polypeptide selected from the amino acid sequences shown in SEQ ID NOs: 9, 10, and 11, and a DNA encoding the polypeptide.
- polypeptides and DNA can be prepared using gene recombination techniques as described above.
- ⁇ Summary of the present invention The invention described above is summarized below.
- a pharmaceutical composition for treating and / or preventing cancer comprising the antibody or fragment thereof according to (1), (2) or (3) as an active ingredient.
- the cancer is breast cancer, renal cancer, pancreatic cancer, colon cancer, lung cancer, brain tumor, stomach cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, fibrosarcoma, mastocytoma or
- the pharmaceutical composition according to (4) which is a melanoma.
- An antibody or fragment thereof according to any one of (1) to (3), a pharmaceutical composition according to (4) or (5), or a combination pharmaceutical according to (6) is administered to a subject A method for treating and / or preventing cancer.
- Example 1 Expression analysis of CAPRIN-1 gene in each tissue Expression of CAPRIN-1 gene in canine and human normal tissues and various cell lines was determined by RT-PCR according to Example 1 (4) of WO2010 / 016526. Examined. As a result, strong expression was observed in testis in healthy dog tissues, while expression was observed in canine breast cancer and adenocarcinoma tissues.
- Example 2 Production of Mouse Monoclonal Antibody against CAPRIN-1 100 ⁇ g of human CAPRIN-1 protein having the amino acid sequence of SEQ ID NO: 2 prepared by the method described in Example 3 of WO2010 / 016526 was mixed with an equal amount of MPL + TDM adjuvant (Sigma This was used as an antigen solution per mouse. The antigen solution was administered intraperitoneally to 6-week-old Balb / cc mice (manufactured by SLC, Japan), and immunization was completed 7 times every week.
- 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. 100 ⁇ l per well of (made) was seeded on 15 plates. By culturing under conditions of 37 ° C. and 5% CO 2 for 7 days, a hybridoma in which spleen cells and myeloma cells were fused was obtained.
- RPMI 1640 medium HAT selective medium
- FBS Gibco's HAT solution
- 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 a CAPRIN-1 protein solution 1 ⁇ g / ml prepared by the method described in Example 3 of WO2010 / 016526 was added per well of a 96-well plate, and the mixture was 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 a CAPRIN-1 protein solution 1 ⁇ g / ml prepared by the method described in Example 3 of WO2010 / 016526 was added per well of a 96-well plate and allowed to stand at 4 ° C. for 18 hours.
- TMB substrate solution manufactured by Thermo
- 100 ⁇ l of TMB substrate solution 100 ⁇ l was added and allowed to stand for 15 to 30 minutes for color reaction.
- 100 ⁇ l of 1N sulfuric acid was added per well to stop the reaction, and absorbance values at 450 nm and 595 nm were measured using an absorptiometer.
- 112 hybridoma lines producing monoclonal antibodies reactive to the CAPRIN-1 protein were obtained.
- the fluorescence intensity was measured with a FACS caliber from Becton Dickinson.
- the same operation as described above was performed using a serum of 6-week-old Balb / c mice that had not been treated in place of the antibody diluted 500-fold with a medium for hybridoma culture, and used as a control.
- one monoclonal antibody (# 1) having a higher fluorescence intensity than the control, ie, reacting with the breast cancer cell surface was selected.
- Example 3 Characterization of the selected monoclonal antibody
- the monoclonal antibody obtained in Example 2 was analyzed for the gene sequence encoding the variable region and its amino acid sequence according to the method described in Example 5 of WO2010 / 016526.
- monoclonal antibody # 1 consisted of the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO: 12.
- the gene sequence encoding the heavy chain variable region of the obtained monoclonal antibody # 1 is SEQ ID NO: 13, the amino acid sequence is SEQ ID NO: 8, the gene sequence encoding the light chain variable region is SEQ ID NO: 14, and the amino acid sequence is This is shown in SEQ ID NO: 12.
- monoclonal antibody # 1 consists of a heavy chain variable region of SEQ ID NO: 8 and a light chain variable region of SEQ ID NO: 12, and CDRs 1 to 3 in the heavy chain variable region are SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: It was confirmed that CDRs 1 to 3 in the light chain variable region consist of amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.
- Example 4 Preparation of human-mouse chimeric monoclonal antibody After restriction enzyme treatment on both ends of the gene amplified fragment containing the heavy chain variable region of mouse monoclonal antibody # 1 represented by SEQ ID NO: 13 obtained in Example 3 purified and inserted in a conventional manner to the already inserted already pcDNA4 / myc-His (Invitrogen Corporation) vector H chain constant region of human IgG 1 that includes a leader sequence and SEQ ID NO: 37 from the mouse antibody.
- both ends of the gene amplified fragment containing the light chain variable region of mouse monoclonal antibody # 1 represented by SEQ ID NO: 14 were purified after restriction enzyme treatment, and the mouse IgG-derived leader sequence and human IgG 1 containing SEQ ID NO: 38 were purified.
- the L chain constant region was inserted into a previously inserted pcDNA3.1 / myc-His (Invitrogen) vector according to a conventional method.
- the recombinant vector was introduced into CHO-K1 cells (obtained from Riken Cell Bank). Specifically, 2 ⁇ 10 5 CHO-K1 cells cultured in Ham's F12 medium (manufactured by Invitrogen) containing 1 ml of 10% FBS per well of a 12-well culture plate were added with PBS ( ⁇ ).
- Human-mouse chimeric monoclonal antibody # 1 having the variable region of mouse monoclonal antibody # 1 was seeded with CHO-K1 cells into which the above recombinant vector had been introduced so that there were 0.5 per well of the well plate.
- a cell line stably producing # 1) was prepared.
- the prepared cell line was cultured for 5 days in 30 ml of OptiCHO medium (manufactured by Invitrogen) at 5 ⁇ 10 5 cells / ml in a 150 cm 2 flask and containing human-mouse chimeric monoclonal antibody # 1. A supernatant was obtained.
- a mouse-derived anti-CAPRIN-1 monoclonal antibody described in WO2010 / 016526 comprising a heavy chain variable region of SEQ ID NO: 15 and a light chain variable region of SEQ ID NO: 16, SEQ ID NO: 17
- Comparative antibody 2 comprising a heavy chain variable region and a light chain variable region of SEQ ID NO: 18
- comparative antibody 3 comprising a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 20, heavy chain variable region of SEQ ID NO: 21
- a comparative antibody 4 comprising the light chain variable region of SEQ ID NO: 22, a comparative antibody 5 comprising the heavy chain variable region of SEQ ID NO: 23 and the light chain variable region of SEQ ID NO: 24, a heavy chain variable region of SEQ ID NO: 25 and SEQ ID NO: 26
- Comparative antibody 6 comprising the light chain variable region of SEQ ID NO: 27
- Comparative antibody 7 comprising the heavy chain variable region of SEQ ID NO: 27 and the light chain
- a cell line to be produced was prepared, and the prepared cell line was cultured for 5 days using 30 ml of OptiCHO medium (manufactured by Invitrogen) at 5 ⁇ 10 5 cells / ml in a 150 cm 2 flask and containing no serum. Culture supernatants containing chimeric comparative antibodies 1-11 were obtained.
- Example 5 Expression of CAPRIN-1 on the surface of various cancer cells using anti-CAPRIN-1 antibody # 1 Next, human breast cancer cell lines (ZR75-1, MCF7, T47D) in which the expression of the CAPRIN-1 gene was confirmed.
- HT29, Lovo, CaCo2, SW480, HCT116 leukemia cell line
- AML5 lymphoma cell line
- Ramos lymphoma cell line
- Each cell line was centrifuged at 5 ⁇ 10 5 cells in a 1.5 ml microcentrifuge tube. Each cell culture supernatant (100 ⁇ l) containing antibody # 1 was added and allowed to stand on ice for 1 hour. After washing with PBS, a FITC-labeled goat anti-mouse IgG (H + L) antibody (Jackson ImmunoResearch) diluted with PBS containing 0.1% FBS was added and allowed to stand at 4 ° C. for 30 minutes. After washing with PBS, the fluorescence intensity was measured with a FACS caliber from Becton Dickinson. As a negative control, a reaction with only a secondary antibody was used.
- the enhancement rate of the fluorescence intensity is represented by the increase rate of the average fluorescence intensity (MFI value) in each cell, and was calculated by the following calculation formula.
- Average fluorescence intensity increase rate (fluorescence intensity enhancement rate) (%) ((MFI value of cells reacted with anti-CAPRIN-1 antibody) ⁇ (control MFI value)) ⁇ (control MFI value) ⁇ 100
- Example 6 Anti-tumor effect of an antibody against CAPRIN-1 on cancer cells (ADCC activity) It was examined by measuring ADCC activity whether the human-mouse chimeric monoclonal antibody # 1 of the antibody against CAPRIN-1 obtained in Example 4 can damage cancer cells expressing CAPRIN-1. .
- Cell culture supernatant producing # 1 was purified using Hitrap Protein A Sepharose FF (manufactured by GE Healthcare), replaced with PBS (-) and filtered through a 0.22 ⁇ m filter (manufactured by Millipore). Used as an antibody for measurement.
- each of the purified antibody # 1 and the human-mouse chimeric comparative antibody 1 to 11 obtained in Example 4 were added thereto, and cells containing human NK cells separated from human peripheral blood lymphocyte cells by a conventional method
- the population was added at 2 ⁇ 10 5 per well and 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 cytotoxic activity against cancer cells by the anti-CAPRIN-1 antibody was calculated.
- a negative control was added with an isotype control antibody.
- the above-mentioned cells containing NK cells are obtained by isolating human peripheral blood mononuclear cells from human peripheral blood using a specific gravity separation solution Histopaque (Sigma Aldrich) for human peripheral blood mononuclear cells.
- the cells are reacted with various dye-labeled antibodies (anti-human CD3 antibody, anti-human CD20 antibody, anti-human CD19 antibody, anti-human CD11c antibody, anti-HLA-DR antibody (Becton & Dickinson)), and cell sorter (FACS Vantage SE ( Becton and Dickinson))) was used to isolate a cell population that did not stain with the antibody, or a human NK cell separation kit (Milteny).
- the cytotoxic activity when using the isotype control antibody and when using comparative antibody 1 to comparative antibody 11 was less than 5%, respectively.
- One antibody is 20%, 17% against human breast cancer cell line MCF7, human colon cancer cell line HCT-116, human pancreatic cancer cell line MIAPaCa-2, human kidney cancer cell line Caki-2, and human lung cancer cell line QG56, respectively. %, 27% and 10% cytotoxic activity.
- the cytotoxic activity is 2 ⁇ 10 3 cancer cell lines into which the antibody against CAPRIN-1 used in the present invention, lymphocyte (population containing NK cells) cells and chromium 51 are incorporated.
- the mixture was cultured for 4 hours, and the amount of chromium 51 released into the culture medium after the culture was measured, and the cytotoxic activity against the cancer cell line calculated by the following formula was shown.
- Cytotoxic activity (%) Amount of chromium 51 released from target cells upon addition of antibody against CAPRIN-1 and lymphocytes (population including NK cells) ⁇ chromium 51 from target cells added with 1N hydrochloric acid Free amount x 100
- Example 7 Antitumor effect of anti-CAPRIN-1 monoclonal antibody in vivo in mouse
- the antibody used was a column-purified culture supernatant of each cell producing antibody # 1.
- the anti-tumor effect of the anti-CAPRIN-1 antibody prepared in Example 4 on human-mouse chimeric comparative monoclonal antibodies 1 to 11 was evaluated in vivo in cancer-bearing mice.
- the antitumor effect of antibody # 1 was examined using tumor-bearing mice transplanted with human-derived cancer cell lines expressing CAPRIN-1. 2 ⁇ 10 6 human pancreatic cancer cell lines Capan-2 cells (purchased from ATCC) per mouse were transplanted subcutaneously into the back of 65 Balb / c nude mice (manufactured by Japan SLC), and the tumor was 5 mm in diameter. Grow to a size of about.
- the above tumor-bearing mice were intraperitoneally administered with antibody # 1 and human-mouse chimeric comparative antibodies 1 to 11 at a dose of 200 ⁇ g (200 ⁇ l) per mouse.
- the same amount of each antibody was administered to the peritoneal cavity of each cancer-bearing mouse 3 times in total for 2 days, and the tumor size was measured every day to observe the antitumor effect.
- the remaining 5 tumor-bearing mice were administered PBS ( ⁇ ) instead of the antibody, and this was used as a control group.
- the size of the tumor was calculated by using a calculation formula of 0.5 ⁇ (major axis ⁇ minor axis ⁇ minor axis).
- the study group administered with antibody # 1 against CAPRIN-1 was 70% on the 29th day after antibody administration, assuming that the tumor size of the control group on the same day was 100%. Tumor growth was reduced to On the other hand, about 85% of the mice were administered human-mouse chimeric antibody 1-11. From this result, it was shown that the obtained antibody # 1 against CAPRIN-1 exhibits an antitumor effect in vivo against cancer cells expressing CAPRIN-1. In addition, it was shown that antibody # 1 exerts a stronger antitumor effect in vivo compared with comparative antibodies 1-11.
- Example 8 Number of molecules of CAPRIN-1 on the surface of various cancer cells recognized by anti-CAPRIN-1 antibody # 1
- Human breast cancer cell lines ZR75-1, MCF7, T47D, SK-BR-3, MDA-MB- 157, BT-20, MDA-MB-231V, MRK-nu-1
- renal cancer cell lines Caki-1, Caki-2, A498, ACHN
- bladder cancer cell line T24
- ovarian cancer cell line SKOV3
- lung cancer cell lines QG56, A549)
- pancreatic cancer cell lines MIAPaCa-2, Capan-2
- prostate cancer cell lines PC3
- cervical cancer cell lines SW756)
- fibrosarcoma cell lines HT1080
- Brain tumor cell lines T98G, U87MG, U251, SNB19, U373)
- gastric cancer cell lines MNK28, MNK45
- colon cancer cell lines HT29, Lovo, Ca) Co2, SW480, HCT116), leukemia cell line (A
- antibody # 1 and comparative antibody 1 to comparative antibody 11 were diluted in PBS to a final concentration of 5 ⁇ g / ml in each cell line, added to each cell line, and allowed to react for 30 minutes. After washing with PBS, together with the calibration beads attached to the kit, the fluorescently labeled secondary antibody anti-mouse IgG antibody attached to the kit was added to each cell line and allowed to stand on ice for 45 minutes. Each cell line and calibration beads were washed with PBS, and the fluorescence intensity was measured with a FACS caliber manufactured by Becton Dickinson Co., Ltd. to obtain an average fluorescence intensity value (mean). Further, the same measurement was performed for the comparative antibody to obtain mean.
- a reaction with an isotype control antibody was used to obtain mean.
- the number of molecules was calculated using each average fluorescence intensity value (mean) according to the method attached to the kit.
- the number of molecules caprin-1 with antibody # 1 recognizes various cancer cell surface, in all human cancer cells examined was per cell 10 5 or more.
- the numbers of the comparative monoclonal antibodies 1 to 11 were less than 10 5 per cell.
- the antibody of the present invention is useful for the treatment and / or prevention of cancer. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
Abstract
Description
本発明では、配列番号5、6及び7を含む重鎖可変領域と配列番号9、10及び11を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体又はそのフラグメントを有効成分として含むことを特徴とする、癌の治療及び/又は予防のための医薬組成物を提供する。
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。
例えば、CAPRIN-1を発現する乳癌細胞株SK-BR-3などをマウスに投与して免疫し、同マウスより脾臓を抽出し、細胞を分離の上、該細胞とマウスミエローマ細胞とを融合させ、得られた融合細胞(ハイブリドーマ)の中から、癌細胞増殖抑制作用を持つ抗体を産生するクローンを選択する。癌細胞増殖抑制作用を持つモノクローナル抗体産生ハイブリドーマを単離し、当該ハイブリドーマを培養し、培養上清から一般的なアフィニティ精製法により抗体を精製することで、調製することが可能である。
抗体がCAPRIN-1に結合する能力は、実施例で述べられるようなたとえばELISA、ウエスタンブロット法、免疫蛍光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を認識する抗体は、当業者に周知の方法での免疫組織化学により、外科手術の間に患者から得た組織や、自然にまたはトランスフェクション後にCAPRIN-1を発現する細胞系を接種した異種移植組織を担持する動物から得た組織から、パラホルムアルデヒドまたはアセトン固定した凍結切片またはパラホルムアルデヒドで固定したパラフィン包埋した組織切片を使用して、CAPRIN-1との反応性に関して試験することができる。
本発明の癌の治療及び/又は予防のための医薬組成物の標的は、CAPRIN-1遺伝子を発現する癌(細胞)であれば特に限定されない。
本発明は更に、上記抗体(a)に関わる以下のポリペプチド及びDNAも提供する。
(i)配列番号8及び配列番号12のアミノ酸配列を含むポリペプチド、並びに該ポリペプチドをコードするDNA、例えば配列番号13及び配列番号14の塩基配列を含むDNA。
(ii)配列番号5、6及び7に示すアミノ酸配列からなる群から選択される、重鎖CDRポリペプチド、及び該ポリペプチドをコードするDNA。
(iii)配列番号9、10及び11に示すアミノ酸配列から選択される、軽鎖CDRポリペプチド、及び該ポリペプチドをコードするDNA。
上で説明した本発明を以下に要約する。
(1)配列番号5、6及び7の相補性決定領域を含む重鎖可変領域と配列番号9、10及び11の相補性決定領域を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体又はそのフラグメント。
(2)ヒト抗体、ヒト化抗体、キメラ抗体、単鎖抗体又は多重特異性抗体である、(1)に記載の抗体又はそのフラグメント。
(3)抗腫瘍剤がコンジュゲートされた(1)又は(2)に記載の抗体又はそのフラグメント。
(4)(1)、(2)又は(3)に記載の抗体又はそのフラグメントを有効成分として含むことを特徴とする、癌の治療及び/又は予防のための医薬組成物。
(5)前記癌が乳癌、腎癌、膵臓癌、大腸癌、肺癌、脳腫瘍、胃癌、子宮頸癌、卵巣癌、前立腺癌、膀胱癌、食道癌、白血病、リンパ腫、線維肉腫、肥満細胞腫又はメラノーマである、(4)に記載の医薬組成物。
(6)(4)又は(5)に記載の医薬組成物と、抗腫瘍剤を含む医薬組成物とを含んでなる、癌の治療及び/又は予防のための組み合わせ医薬品。
(7)(1)又は(2)に記載の抗体又はそのフラグメントをコードするDNA。
(8)(1)~(3)のいずれかに記載の抗体又はそのフラグメント、(4)又は(5)に記載の医薬組成物、あるいは(6)に記載の組み合わせ医薬品を、被験者に投与することを含む、癌の治療及び/又は予防方法。
CAPRIN-1遺伝子の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WO2010/016526の実施例1(4)に従ってRT-PCR法により調べた。その結果、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、ヒト組織で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、癌細胞ではヒト乳癌細胞株7種(ZR75-1、MCF7、T47D、SK-BR-3、MDA-MB-157、BT-20、MDA-MB-231V、MRK-nu-1)及び膵臓癌細胞株4種(Capan-2、MIAPaCa-2、Panc-1、BxPc-3)など、多種類の癌細胞株で発現が検出された。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、乳癌細胞株に発現していることが確認された。
WO2010/016526の実施例3に記載の方法で調製した配列番号2のアミノ酸配列を有するヒトCAPRIN-1タンパク質100μgを等量のMPL+TDMアジュバント(シグマ社製)と混合し、これをマウス1匹当たりの抗原溶液とした。抗原溶液を6週齢のBalb/ccマウス(日本SLC社製)の腹腔内に投与後、1週間毎に7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。
実施例2で得られたモノクローナル抗体について、WO2010/016526の実施例5に記載の方法に従って可変領域をコードする遺伝子配列並びにそのアミノ酸配列を解析した。その結果、モノクローナル抗体#1は配列番号8の重鎖可変領域と配列番号12の軽鎖可変領域から成るものであった。得られたモノクローナル抗体#1の重鎖可変領域をコードする遺伝子配列を配列番号13に、及びアミノ酸配列を配列番号8に、軽鎖可変領域をコードする遺伝子配列を配列番号14に及びアミノ酸配列を配列番号12に示す。
実施例3で得られた、配列番号13で示されるマウスモノクローナル抗体#1の重鎖可変領域を含む遺伝子増幅断片の両端を制限酵素処理した後精製し、マウス抗体由来のリーダー配列と配列番号37を含むヒトIgG1のH鎖定常領域を既に挿入済みのpcDNA4/myc-His(Invitrogen社製)ベクターへ常法に従って挿入した。また、配列番号14で示されるマウスモノクローナル抗体#1の軽鎖可変領域を含む遺伝子増幅断片の両端を制限酵素処理した後精製し、マウス抗体由来のリーダー配列と配列番号38を含むヒトIgG1のL鎖定常領域を既に挿入済みのpcDNA3.1/myc-His(Invitrogen社製)ベクターへ常法に従って挿入した。
次にCAPRIN-1遺伝子の発現が確認されたヒト乳癌細胞株(ZR75-1、MCF7、T47D、SK-BR-3、MDA-MB-157、BT-20、MDA-MB-231V、MRK-nu-1)、腎癌細胞株(Caki-1、Caki-2、A498、ACHN)、膀胱癌細胞株(T24)、卵巣癌細胞株(SKOV3)、肺癌細胞株(QG56、A549)、膵臓癌細胞株(Capan-2、MIAPaCa-2)、前立腺癌細胞株(PC3)、子宮頸癌細胞株(SW756)、線維肉腫細胞株(HT1080)、脳腫瘍細胞株(T98G、U87MG、U251、SNB19、U373)、胃癌細胞株(MNK28、MNK45)、大腸癌細胞株(HT29、Lovo、CaCo2、SW480、HCT116)、白血病細胞株(AML5)、リンパ腫細胞株(Ramos)について、実施例4で得られた#1を含む培養上清を用いて、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5×105細胞を1.5ml容のミクロ遠心チューブにて遠心分離した。抗体#1を含む各細胞培養上清(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で希釈したFITC標識ヤギ抗マウスIgG(H+L)抗体(Jackson ImmunoResearch社製)を添加し、4℃で30分間静置した。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。陰性コントロールには二次抗体のみを反応したものを用いた。その結果、抗体#1を添加した細胞は、陰性コントロールに比べて蛍光強度が35%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タンパク質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。
平均蛍光強度の増加率(蛍光強度の増強率)(%)=((抗CAPRIN-1抗体を反応させた細胞のMFI値)-(コントロールMFI値))÷(コントロールMFI値)×100
実施例4で得た、CAPRIN-1に対する抗体のヒト-マウスキメラモノクローナル抗体#1が、CAPRIN-1を発現する癌細胞を障害することができるかどうかを、ADCC活性を測定することによって検討した。#1を産生する細胞培養上清を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CAPRIN-1の発現が確認されているヒト乳癌細胞株MCF7、ヒト大腸癌細胞株HCT-116、ヒト膵臓癌細胞株MIAPaCa-2、ヒト腎臓癌細胞株Caki-2、ヒト肺癌細胞株QG56を50ml容の遠心チューブに集め、100μCiのクロミウム51を加え37℃で2時間インキュベートした。その後10%の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2×103個ずつ添加して標的細胞とした。これに、上記精製抗体#1ならびに実施例4で得たヒト-マウスキメラ比較抗体1~11をそれぞれ1μg添加して、ヒト末梢血リンパ球細胞から定法を用いて分離したヒトNK細胞を含む細胞集団を1穴当たり、2×105個添加して37℃、5%CO2の条件下で4時間培養した。培養後、障害を受けた腫瘍細胞から放出される培養上清中のクロミウム51の量を測定し、抗CAPRIN-1抗体による癌細胞に対する細胞障害活性を算出した。陰性コントロールにはアイソタイプコントロール抗体を添加したものを用いた。上記NK細胞を含んだ細胞は、定法に従って、ヒト末梢血からヒト末梢血単核球細胞分離用の比重分離液Histopaque(シグマアルドリッチ社)を用いて分離したヒト末梢血単核球細胞をFITC蛍光色素が標識された各種抗体(抗ヒトCD3抗体、抗ヒトCD20抗体、抗ヒトCD19抗体、抗ヒトCD11c抗体、抗HLA-DR抗体(ベクトンアンドディッキンソン社))で反応させ、セルソーター(FACS Vantage SE(ベクトンアンドディッキンソン社))を用いて、上記抗体で染まらない細胞集団を分離したものあるいは、ヒトNK細胞分離キット(ミルテニー社製)を用いて分離したものを用いた。癌細胞に対する細胞障害活性の評価の結果、アイソタイプコントロール抗体を用いた場合ならびに比較抗体1~比較抗体11を用いた場合の細胞障害活性は、それぞれ5%未満であったのに対して、抗体#1抗体は、ヒト乳癌細胞株MCF7、ヒト大腸癌細胞株HCT-116、ヒト膵臓癌細胞株MIAPaCa-2、ヒト腎臓癌細胞株Caki-2、ヒト肺癌細胞株QG56に対してそれぞれ20%、17%、27%、10%の細胞障害活性を示した。同様に、他の癌細胞、乳癌細胞株ZR75-1、T47D、Hs578T、BT-20、SK-BR-3、MDA-MB-231V、MRK-nu-1、グリオーマ細胞株T98G、U373、肺癌細胞株A549、腎臓癌細胞株Caki-1、ACHN、子宮頸癌細胞株SW756、膀胱癌細胞株T24、胃癌細胞株MKN28、MKN45、大腸癌細胞株SW480、白血病細胞株AML5及びリンパ腫細胞株Ramosについて、アイソタイプコントロール抗体を用いた場合ならびに比較抗体1~比較抗体11を用いた場合はいずれも4%未満であったのに対し、抗体#1は10%以上の細胞障害活性が認められた。以上の結果より、取得したCAPRIN-1に対するモノクローナル抗体#1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、比較抗体1~比較抗体11に比べて抗体#1は、ヒト癌細胞に対して強い細胞障害活性を示すことが明らかになった。
式:細胞障害活性(%)=CAPRIN-1に対する抗体及びリンパ球(NK細胞を含む集団)細胞を加えた際の標的細胞からのクロミウム51遊離量÷1N塩酸を加えた標的細胞からのクロミウム51遊離量×100
次に、実施例4で得た、ヒト-マウスキメラモノクローナル抗体#1の担癌マウス生体内における抗腫瘍効果を評価した。使用した抗体は抗体#1を産生する各細胞の培養上清をカラム精製したものを用いた。同様にして、実施例4で作製した抗CAPRIN-1抗体のヒト-マウスキメラ比較モノクローナル抗体1~11についても担癌マウス生体内における抗腫瘍効果を評価した。
ヒト乳癌細胞株(ZR75-1、MCF7、T47D、SK-BR-3、MDA-MB-157、BT-20、MDA-MB-231V、MRK-nu-1)、腎癌細胞株(Caki-1、Caki-2、A498、ACHN)、膀胱癌細胞株(T24)、卵巣癌細胞株(SKOV3)、肺癌細胞株(QG56、A549)、膵臓癌細胞株(MIAPaCa-2、Capan-2)、前立腺癌細胞株(PC3)、子宮頸癌細胞株(SW756)、線維肉腫細胞株(HT1080)、脳腫瘍細胞株(T98G、U87MG、U251、SNB19、U373)、胃癌細胞株(MNK28、MNK45)、大腸癌細胞株(HT29、Lovo、CaCo2、SW480、HCT116)、白血病細胞株(AML5)、リンパ腫細胞株(Ramos)について、抗CAPRIN-1抗体#1が認識する各種癌細胞表面でのCAPRIN-1分子の数を分子数測定キットQIFIKIT(DAKO社製)を用いて調べた。同様にして、実施例4で作製した抗CAPRIN-1モノクローナル抗体である比較抗体1~比較抗体11についても各種癌細胞表面でのCAPRIN-1分子の数を調べた。
本明細書で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書にとり入れるものとする。
Claims (8)
- 配列番号5、6及び7の相補性決定領域を含む重鎖可変領域と配列番号9、10及び11の相補性決定領域を含む軽鎖可変領域とを含み、かつ、CAPRIN-1タンパク質と免疫学的反応性を有する抗体又はそのフラグメント。
- ヒト抗体、ヒト化抗体、キメラ抗体、単鎖抗体又は多重特異性抗体である、請求項1に記載の抗体又はそのフラグメント。
- 抗腫瘍剤がコンジュゲートされた、請求項1又は2に記載の抗体又はそのフラグメント。
- 請求項1~3のいずれか1項に記載の抗体又はそのフラグメントを有効成分として含むことを特徴とする、癌の治療及び/又は予防のための医薬組成物。
- 前記癌が乳癌、腎癌、膵臓癌、大腸癌、肺癌、脳腫瘍、胃癌、子宮頸癌、卵巣癌、前立腺癌、膀胱癌、食道癌、白血病、リンパ腫、線維肉腫、肥満細胞腫又はメラノーマである、請求項4に記載の医薬組成物。
- 請求項4又は5に記載の医薬組成物と、抗腫瘍剤を含む医薬組成物とを含んでなる、癌の治療及び/又は予防のための組み合わせ医薬品。
- 請求項1又は2に記載の抗体又はそのフラグメントをコードするDNA。
- 請求項1~3のいずれか1項に記載の抗体又はそのフラグメント、請求項4又は5に記載の医薬組成物、あるいは請求項6に記載の組み合わせ医薬品を、被験者に投与することを含む、癌の治療及び/又は予防方法。
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BR112014002614B1 (pt) | 2022-09-20 |
ES2618026T3 (es) | 2017-06-20 |
US9180188B2 (en) | 2015-11-10 |
CA2844030A1 (en) | 2013-02-07 |
PL2740798T3 (pl) | 2017-07-31 |
CA2844030C (en) | 2019-09-03 |
KR20140054182A (ko) | 2014-05-08 |
HUE033183T2 (en) | 2017-11-28 |
AU2012290946A1 (en) | 2014-03-20 |
EP2740798B1 (en) | 2016-12-07 |
EP2740798A4 (en) | 2015-04-15 |
RU2595400C2 (ru) | 2016-08-27 |
CN103717737A (zh) | 2014-04-09 |
MX348581B (es) | 2017-06-20 |
BR112014002614A2 (pt) | 2018-02-20 |
EP2740798A1 (en) | 2014-06-11 |
RU2014108049A (ru) | 2015-09-10 |
AU2012290946B2 (en) | 2016-04-21 |
KR101968499B1 (ko) | 2019-04-12 |
PT2740798T (pt) | 2017-03-13 |
MX2014001374A (es) | 2014-03-21 |
JP6065592B2 (ja) | 2017-01-25 |
US20140193434A1 (en) | 2014-07-10 |
CN103717737B (zh) | 2015-06-10 |
DK2740798T3 (en) | 2017-03-06 |
JPWO2013018883A1 (ja) | 2015-03-05 |
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