MONOCLONAL ANTIBODY, PHARMACEUTICAL COMPOSITION, PHARMACEUTICAL COMBINATION, USE OF AN ANTIBODY, USE

专利摘要:
pharmaceutical composition, antibody, pharmaceutical combination and method for the treatment and / or prevention of a cancer in accordance with the present invention, a cancer antigen protein has been identified that is specifically expressed on the surfaces of cancer cells and, therefore, the use of an antibody directed against the cancer antigen protein as an agent for the treatment and / or prevention of cancer. specifically, the present invention provides a pharmaceutical composition for the treatment and / or prevention of cancer, which comprises an antibody or fragment thereof as an active ingredient having immunological reactivity with a partial polypeptide of the caprin-1 protein, wherein caprin- 1 is represented by any of the numbered even numbered sequences of sequences: 2 to 30, and wherein the polypeptide comprises the amino acid sequence represented by sequence: 37 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence.
公开号:BR112012018951B1
申请号:R112012018951-4
申请日:2011-02-04
公开日:2020-05-12
发明作者:Shinichi Kobayashi；Okano Fumiyoshi；Saito Takanori
申请人:Toray Industries, Inc.；
IPC主号:

专利说明:

“MONOCLONAL ANTIBODY, PHARMACEUTICAL COMPOSITION, PHARMACEUTICAL COMBINATION, USE OF AN ANTIBODY, USE OF A PHARMACEUTICAL COMPOSITION AND USE OF A PHARMACEUTICAL COMBINATION”
Field of the Invention [001] The present invention relates to a new pharmaceutical use of an antibody against CAPRIN-1 or a fragment thereof, such as an agent to treat and / or prevent cancer.
Background to the Invention [002] Cancer is the leading cause of death. Currently performed therapy mainly comprises surgical therapy in combination with radiotherapy and chemotherapy. Despite the development of new surgical procedures and the discovery of new anticancer agents in recent years, with the exception of some types of cancers, treatment results have not improved. Recent advances in molecular biology or cancer immunology have led to the identification of antibodies that specifically react with cancer, cancer antigens that are recognized by cytotoxic T cells, genes that encode cancer antigens and the like. The demand for specific therapies directed against cancer antigens is increasing (non-patent literature 1).
[003] In cancer therapy, it is desirable that peptides, polypeptides or proteins recognized as antigens are almost absent in normal cells, but present specifically in cancer cells, in order to alleviate side effects. In 1991, Boon, et al. (Ludwig Institute for Cancer Research in Belgium) isolated a human MAGE1 melanoma antigen recognized by CD8-positive T cells by a cDNA expression cloning method using a cancer autologous cell line and cancer-reactive T cells (Literature not patent 2).
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Subsequently, the SEREX method (serological identification of antigens by cloning recombinant expression) was described, which included the identification of tumor antigens recognized by antibodies that are produced in vivo in response to autologous cancer of a subject's own cancer by a cloning technique. of gene expression (non-patent literature 3 and patent literature 1). Using this method, some cancer antigens, which are almost never expressed in normal cells, but which are specifically expressed in cancer cells, have been isolated (Non-patent literature 4-9). In addition, clinical trials based on cell therapies directed against some cancer antigens have been carried out using immunocytes specifically reactive to cancer antigens or with cancer-specific immunotherapies with vaccines or similar containing cancer antigens.
[004] On the other hand, in the last few years, several antibody drugs have appeared around the world that target antigenic proteins in cancer cells for the treatment of cancer. Antibody drugs exhibit some pharmacological effects as cancer-specific therapeutic agents and thus attract attention. However, most of the antigen proteins used as a target for the antibody are also expressed in normal cells, so that not only cancer cells, but also normal cells that express such antigens are damaged as a result of administration of the antibodies. The resulting side effects are a cause for concern. Therefore, it is expected that the identification of cancer antigens that are specifically expressed on the surface of a cancer cell and the use of antibodies directed against cancer antigens as pharmaceutical products will carry out treatment with antibody drugs with lower side effects.
[005] The protein associated with cytoplasmic proliferation 1
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3/82 (CAPRIN-1) is expressed when normal cells in the resting phase are activated or subjected to cell division, and it is an intracellular protein known to form intracellular stress granules with RNA inside the cells, in order to be involved mRNA transport and translation regulation. However, there are many other names that represent CAPRIN1, such as the membrane protein anchored to GPI 1 or surface marker protein of membrane component 1 (M11S1), as if such proteins were known to be cell membrane proteins. These names originated from a report that the CAPRIN1 gene sequence is a membrane protein that has a GPI-binding region and is expressed in colorectal cancer cells (Non-patent literature 10). However, the CAPRIN-1 genetic sequence provided in this report was later revealed to be wrong. Then it was reported that the deletion of a single nucleotide in the sequence of the CAPRIN-1 gene registered in the GenBank or similar bank causes a change in the picture, so that 80 amino acids are lost from the C-terminal end, which results in the generation of an artifact (74 amino acids), which corresponds to the GPI-binding portion in the previous report, and, in addition, another error is also present 5 'from the gene sequence, so that 53 amino acids were lost from the N-terminal end (Non-patent literature 11). It has also recently been reported that the protein encoded by the CAPRIN-1 gene sequence registered with GenBank or a similar bank is not a cell membrane protein (Non-patent literature 11).
[006] In addition, based on the report in the non-patent literature 10 that CAPRIN-1 is a cell membrane protein, patent literature 2 and 3 describe CAPRIN-1 (as a cell membrane protein) under the name M11S1, can be used as an antibody drug target in cancer therapy, although they do not describe practical examples
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4/82 of treatment using an antibody against the protein. However, as reported in non-patent literature 11, it is believed from the deposit of patent literature 2 to the present date that CAPRIN-1 is not expressed on the surface of a cell. The content of patent literature 2 and 3, based only on the incorrect information that CAPRIN-1 is a cell membrane protein, should not be clearly understood as common general knowledge for those skilled in the art.
State of the Art Literature
Patent Literature [007] Patent Literature 1: US Patent 5,698,396 [008] Patent Literature 2: US2008 / 0075722 [009] Patent Literature 3: WO2005 / 100998.
Non-patent literature [0010] Non-patent literature 1: Tsuyoshi Akiyoshi, “Gan To Kagaku-Ryoho (Cancer and Chemotherapy),” 1997, Vol. 24, pages 551-519 (Cancer and Chemotherapy Publishers, Inc., Japan).
[0011] Non-Patent Literature 2: Bruggen P et al., Science, 254: 1643-1647 (1991).
[0012] Non-Patent Literature 3: Proc. Natl. Acad. Sci. USA, 92: 11810-11813 (1995).
[0013] Literature Not Patent 4: Int. J. Cancer, 72: 965-971 (1997).[0014] Literature Not Patent 5: Cancer Res., 58: 1034-1041 (1998).[0015] Literature Not Patent 6: Int. J. Cancer, 29: 652-658 (1998).[0016] Literature Not Patent 7: Int. J. Oncol., 14: 703-708
(1999).
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5/82 [0017] Non-Patent Literature 8: Cancer Res., 56: 4766-4772 (1996).
[0018] Non-Patent Literature 9: Hum. Mol. Genet 6: 33-39 (1997).
[0019] Non-Patent Literature 10: J. Biol Chem., 270: 2071720723 (1995).
[0020] Non-Patent Literature 11: J. Immunol., 172: 2389-2400, 2004.
Brief Description of the Invention
Problems to be solved by the invention [0021] The objects of the present invention are to identify a cancer antigen protein that is specifically expressed on the surface of a cancer cell and to provide the use of an antibody directed against the cancer antigen protein as a agent to treat and / or prevent cancer.
Ways to Solve Problems [0022] As a result of intensive studies, the present inventors have now obtained a cDNA that encodes a protein that binds to an antibody in the serum of dogs with breast cancer by the SEREX method using two cDNA libraries prepared from from dog testis tissues and serum from dogs with breast cancer. The inventors additionally prepared CAPRIN-1 proteins having the even numbered amino acid sequences of SEQ ID NOS: 2 to 30 and antibodies against such CAPRIN-1 proteins based on the dog gene obtained and the corresponding homologous genes in human, bovine, horse , mouse and chicken. Thus, the inventors of the present invention have now discovered that CAPRIN-1 is specifically expressed in breast cancer, brain tumor, leukemia, lymphoma, lung cancer, cervical cancer, bladder cancer, breast cancer
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6/82 esophagus, colorectal cancer, gastric cancer, kidney cancer, ovarian cancer, prostate cancer and fibrosarcoma, and that a portion of the CAPRIN-1 protein is specifically expressed on the surface of each cancer cell. The inventors have now found that an antibody or antibodies against the portion of CAPRIN-1 expressed on the surface of each cancer cell is / are cytotoxic to cancer cells expressing CAPRIN-1. Based on this finding, the present invention as described below has been completed.
[0023] The present invention has the following characteristics.
[0024] The present invention provides a pharmaceutical composition for treating and / or preventing cancer, comprising an antibody or a fragment thereof as an active ingredient that has immunological reactivity with a partial CAPRIN-1 polypeptide, in which CAPRIN-1 is represented by one of the even numbered sequences of SEQ ID NOS: 2 to 30, and wherein the partial polypeptide comprises the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 37.
[0025] In one embodiment example, the above cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, cervical cancer, bladder cancer, esophageal cancer, colorectal cancer, gastric cancer, cancer kidney, ovarian cancer, prostate cancer or fibrosarcoma.
[0026] In another embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.
[0027] In another embodiment, the antibody is a human antibody, a humanized antibody, chimeric antibody, single chain antibody or bispecific antibody.
[0028] This description includes all or part of the content as disclosed in the description and / or drawings of the patent applications
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Japanese 7/82 Nos 2010-023453 and 2010-183161, from which this application claims priority.
Effect of the Invention [0029] The antibody against CAPRIN-1 used in the present invention is cytotoxic to cancer cells. Thus, the antibody against CAPRIN-1 is useful to treat or prevent cancers.
Brief Description of the Figures [0030] Fig. 1 shows the expression patterns of genes encoding CAPRIN-1 proteins in normal tissues and in tumor cell lines. Reference No. 1 indicates the expression patterns of genes encoding CAPRIN-1 proteins, and Reference No. 2 indicates the expression patterns of GAPDH genes.
[0031] Fig. 2 shows the cytotoxicity for the breast cancer cell line MDA-MB-157 that expresses CAPRIN-1 by polyclonal anti-CAPRIN-1 antibodies that are reactive with the surfaces of cancer cells. Reference No. 3 indicates the activity displayed when the polyclonal anti-CAPRIN-1 # 1 antibody was added. Reference No. 4 indicates the activity displayed when a control antibody from a rabbit not immunized with antigen was added. Reference No. 5 indicates the activity displayed when PBS was added instead of antibodies.
Detailed Description of the Invention [0032] The antitumor activity of an antibody against a polypeptide represented by any of the even numbering sequences of SEQ ID NOS: 2 to 30 used in the present invention can be evaluated by examining the suppression of tumor growth in vivo, in animals with cancer, or, assessing whether the antibody exhibits cytotoxicity via immunocytes or complement to tumor cells expressing the polypeptide in vitro, as described below.
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8/82 [0033] In context, the nucleotide sequences of protein-encoding polynucleotides comprising the even numbered amino acid sequences (i.e. SEQ ID NOS: 2, 4, 6, ..., 28, 30) of SEQ ID NOS: 2 to 30 are represented by the odd numbered strings (ie SEQ ID NOS: 1, 3, 5, ..., 27, 29) of SEQ ID NOS: 1 to 29.
[0034] The amino acid sequences that are represented by SEQ ID NOS: 6, 8, 10, 12 and 14 in the Sequence Listing disclosed in the present invention are the CAPRIN-1 amino acid sequences isolated as polypeptides that specifically bind to antibodies existing in the serum of a dog with cancer, using the SEREX method using a cDNA library from dog testicular tissue and the serum of a dog with breast cancer. The amino acid sequences represented by SEQ ID NOS: 2 and 4 are the CAPRIN-1 amino acid sequences isolated as human homologues. The amino acid sequence represented by SEQ ID NO: 16 is the CAPRIN-1 amino acid sequence isolated in the form of a livestock homolog. The amino acid sequence represented by SEQ ID NO: 18 is the CAPRIN-1 amino acid sequence isolated in the form of a horse homolog. The amino acid sequences represented by SEQ ID NOS: 20 to 28 are the CAPRIN-1 amino acid sequences isolated as mouse homologs. The amino acid sequence represented by SEQ ID NO: 30 is the CAPRIN-1 amino acid sequence isolated as a chicken homolog (see Example 1 described below). CAPRIN-1 is known to be expressed when normal cells in the resting phase are activated or cause cell division.
[0035] It was known that CAPRIN-1 was not expressed on cell surfaces. However, as a result of examination by the inventors of the present invention, it has now been revealed that a portion of the CAPRIN-1 protein is expressed on the cell surfaces of various types of cancer. So it was
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9/82 it is now disclosed that an antibody that recognizes a partial CAPRIN-1 protein polypeptide, comprising the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence that is 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more of sequence identity with the amino acid sequence of SEQ ID NO: 37, exhibits antitumor activity. Examples of the antibody of the present invention include all antibodies that bind to a fragment of the CAPRIN-1 protein above and exhibit anti-tumor activity.
[0036] The anti-CAPRIN-1 antibody described above used in the present invention can be any type of antibody, as long as it can exhibit anti-tumor activity. Examples of such antibodies include monoclonal antibodies, polyclonal antibodies, recombinant antibodies, such as synthetic antibodies, multispecific antibodies, humanized antibodies, chimeric antibodies, and single chain antibodies (scFv), human antibodies, and fragments thereof, such as Fab, F (ab ') 2, and Fv. These antibodies and fragments thereof can be prepared by methods known to those skilled in the art. In the present invention, antibodies that have immunological reactivity with the CAPRIN-1 proteins or partial polypeptides of them (that is, binding to CAPRIN-1 proteins through the antigen-antibody reaction) and, preferably, antibodies capable of binding specifically to CAPRIN-1 proteins. Preferably, they are monoclonal antibodies. Polyclonal antibodies can also be used, as long as homogeneous antibodies can be produced stably. In addition, when a subject (subject) is a human being, human antibodies or humanized antibodies are desired in order to avoid or suppress rejection. The term "specifically binds to a CAPRIN-1 protein", as used herein, means that the antibody specifically binds to a CAPRIN-1 protein, but does not substantially bind to proteins other than
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10/82 CAPRIN-1 protein.
[0037] The antitumor activity of an antibody that can be used in the present invention can be evaluated, as described below, by in vivo analysis of tumor growth suppression in animals with cancer, or, evaluating whether or not said antibody exhibits an activity of in vitro cytotoxicity, which is mediated by immunocytes or complement, in tumor cells that express the polypeptide.
[0038] In addition, examples of subject to treat and / or prevent a cancer of the present invention, include mammals, such as humans, pets, domestic animals and animals for competition. A preferred subject is a human.
[0039] The preparation of antigens, antibodies and pharmaceutical compositions related to the present invention are described below.
Preparation of Antigens for the Preparation of Antibody [0040] The proteins or fragments thereof to be used as sensitizing antigens for obtaining the antiCAPRIN-1 antibodies used in the present invention can be derived from any animal species, without particular limitation, such as humans, dogs, cattle, horses, mice, rats and chickens. However, the proteins or fragments thereof are preferably selected taking into account the compatibility with the parental cells used for cell fusion. In general, proteins derived from mammals are preferred and, in particular, proteins derived from humans are preferred. For example, when CAPRIN-1 is human CAPRIN-1, they can be used; the human CAPRIN-1 protein, a partial peptide thereof, or cells expressing human CAPRIN-1.
[0041] The nucleotide and amino acid sequences of human CAPRIN-1 and homologues thereof can be obtained
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11/82 accessing GenBank (NCBI, USA) and using an algorithm such as BLAST or FASTA (Karlin and Altschul, Proc. Natl. Acad. Sci USA, 90: 5873-5877 1993; Altschul et al, Nucleic Acids Res, 25 : 3389-3402, 1997).
[0042] In the present invention, based on the nucleotide sequence (SEQ ID NO: 1 or 3), or the amino acid sequence (SEQ ID NO: 2 or 4) of human CAPRIN-1, a target nucleic acid or target protein comprises a sequence of about 70% to 100%, preferably from 80% to 100%, more preferably from 90% to 100%, even more preferably from 95% to 100% (for example, 97% to 100%, 98% 100%, 99% to 100%, or 99.5% to 100%) sequence identity to the ORF nucleotide sequence or amino acid sequence or mature portion of human CAPRIN-1. As used herein, the term "% sequence identity" refers to a percentage (%) of amino acids (or nucleotides) that are identical to the total number of amino acids (or nucleotides) when two sequences are aligned to achieve the greatest similarity with or without the introduction of gaps (gaps).
[0043] The length of a CAPRIN-1 protein fragment varies from the amino acid length of an epitope (antigenic determinant), which is the minimum unit recognized by an antibody, to a length less than the total length of the protein. The term "epitope" refers to a polypeptide fragment having antigenicity or immunogenicity in mammals, preferably humans, and the minimal unit of the epitope consists of about 7 to 12 amino acids, for example, 8 to 11 amino acids. Therefore, the antibody of the present invention is characterized by the recognition of a fragment containing at least one epitope consisting of about 7 to 12 continuous amino acids (for example, 8 to 11 continuous amino acids) in the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence that is 80% or more,
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12/82 preferably 85% or more, more preferably 90% or more, more preferably 95% or more of sequence identity with the amino acid sequence of SEQ ID NO: 37. Thus, the antibody is characterized by binding (preferably, specific link) to such a partial sequence (fragment).
[0044] Polypeptides comprising the human CAPRIN-1 protein or the partial protein peptides mentioned above can be synthesized by a chemical synthesis method, such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method) ( Edited by the Japanese Society for Biochemistry, Seikagaku Jikken Koza (Biochemical Experimental Lecture Series) 1, Protein Chemistry IV, Chemical Modification and Peptide Synthesis, TOKYO KAGAKU DOZIN (Japan), 1981). Alternatively, the polypeptides mentioned above can also be synthesized by conventional methods using various commercially available peptide synthesizers. Moreover, with the use of known techniques of genetic engineering (for example, Sambrook et al., Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology (1989), Cold Spring Harbor Laboratory Press, Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, A compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons), a polynucleotide encoding the above polypeptide is prepared and then incorporated into an expression vector, which is subsequently introduced into a host cell in order to produce a polypeptide of interest in the host cell, and then the polypeptide is recovered.
[0045] The polynucleotides encoding the above polypeptides can be easily prepared by techniques known from genetic engineering or by conventional techniques using a commercially available nucleic acid synthesizer. For example, DNA comprising the nucleotide sequence of SEQ ID NO: 1 can be prepared by PCR, using DNA
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13/82 human chromosome or cDNA library as a template, and a pair of primers designed to be able to amplify the nucleotide sequence represented by SEQ ID NO: 1. PCR conditions can be determined appropriately. For example, PCR conditions comprise performing 30 cycles of the reaction cycle: denaturation at 94 ° C for 30 seconds; annealing at 55 ° C for 30 seconds to 1 minute, and extension at 72 ° C for 2 minutes, using a thermostable DNA polymerase (for example, Taq polymerase or Pfu polymerase) and PCR buffer containing Mg2 +, followed by the reaction at 72 ° C for 7 minutes. However, PCR conditions are not limited to the example above. PCR techniques, conditions, etc., are described in Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, A Compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons (especially Chapter 15).
[0046] In addition, based on the nucleotide sequence and amino acid sequence information represented by SEQ ID NOS: 1 to 30 in the Sequence Listing described in the present invention, appropriate probes or primers are prepared, and then a library of a human or similar cDNA is screened using the same, so that the desired DNA can be isolated. A cDNA library is preferably constructed from cells, organs or tissues that express proteins having the even number sequences of SEQ ID NOS: 2 to 30. Examples of such cells or tissues include cells or tissues derived from the testis, cancers or tumors, such as leukemia, breast cancer, lymphomas, brain tumors, lung cancer, colorectal cancer, and the like. Procedures such as the preparation of probes or primers, the construction of a cDNA library, the screening of a cDNA library and cloning of target genes are known to a person skilled in the art and such procedures can be performed using the methods described in Sambrook et al. Molecular Cloning, 2nd Edition, Current
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Protocols in Molecular Biology (1989), Ausbel et al., (Above), and etc. The DNA encoding the human CAPRIN-1 protein or a partial peptide can be obtained from the DNA obtained in this way.
[0047] Host cells can be of any cell type, as long as they can express the polypeptide mentioned above. Examples of prokaryotic cells include, but are not limited to, E. coli and the like. Examples of eukaryotic cells include, but are not limited to, mammalian cells, such as monkey kidney cells (COS1) and Chinese hamster ovary cells (CHO), the human fetal kidney cell line (HEK293), lineage of mouse fetal skin cells (NIH3T3), yeast cells such as budding yeast and yeast, silkworm cells, and Xenopus oocytes.
[0048] When prokaryotic cells are used as host cells, an expression vector used in the present invention includes a replicable origin within prokaryotic cells, a promoter, a ribosome binding site, a multiple cloning site, a terminator, a drug resistance gene, a complementary auxotrophic gene, and the like. Examples of expression vector for Escherichia coli include a vector based on pUC, pBluescript II, a pET expression system, and a pGEX expression system. DNA encoding the above polypeptide is incorporated into such an expression vector, prokaryotic host cells are transformed with the vector, the transformed cells thus obtained are cultured, and in this way, the polypeptide encoded by the DNA can be expressed in prokaryotic host cells. At this point, the polypeptide can also be expressed as a fusion protein with another protein.
[0049] When eukaryotic cells are used as host cells, an expression vector used in the present invention is an expression vector for eukaryotic cells, which contains a promoter, a region of
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15/82 splicing, a poly (A) addition site, and the like. Examples of such expression vectors include pKa1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, and pYES2. In a manner similar to that described above, the DNA encoding the polypeptide above is incorporated into such an expression vector, eukaryotic host cells are transformed with the vector, transformed cells obtained in this way are cultured, and thus the polypeptide encoded by DNA can be expressed in eukaryotic host cells. When pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or similar is used as an expression vector, the above polypeptide can be expressed as a fusion protein to which a marker, such as the His marker (e.g., (His) 6- (His) 10), FLAG marker, myc marker, HA or GFP marker, was added.
[0050] For the introduction of an expression vector in host cells, a known method, such as electroporation, the calcium phosphate method, the liposome method, DEAE dextran method, microinjection, viral infection, lipofection can be employed. , and binding to a cell membrane-permeable peptide.
[0051] The polypeptide of interest can be isolated and purified from host cells by a combination of known separation processes. Examples of such known separation processes include, but are not limited to treatment with a denaturing agent, such as urea or a surfactant; ultrasonication; enzymatic digestion; salification-out precipitation or fractionation and solvent precipitation; dialysis; centrifugation; ultrafiltration; gel filtration; SDS-PAGE; isoelectric focusing; ion exchange chromatography; hydrophobic interaction chromatography, affinity chromatography and reverse phase chromatography.
Antibody Structure [0052] An antibody is a heteromultimeric glycoprotein that
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16/82 generally contains at least two heavy chains and two light chains. Antibodies that are not of the IgM class are heterotetrameric glycoproteins of about 150-kDa, composed of two identical light (L) chains and two identical heavy (H) chains. Normally, each light chain is connected to a heavy chain by means of a simple covalent disulfide bond, however, the number of disulfide bonds between the heavy chains varies among the different immunoglobulin isotypes. Each heavy chain or light chain also has an intrachain disulfide bond. Each heavy chain has a variable domain (VH region) at one end followed by several constant regions. Each light chain has a variable domain (VL region), and has a constant region at one end opposite the other end The constant region of a light chain is aligned with the first constant region of a heavy chain, and a light chain variable domain is aligned with a heavy chain variable domain. The specific region of an antibody variable domain exhibits specific variability that is referred to as the complementarity determining region (CDR), so that it confers specificity of binding to the antibody. A portion of a variable region, which is relatively conserved, is referred to as a framework region (FR) (or structural region - framework). The complete heavy chain and the light chain variable domains separately contain four FRs linked by means of three CDRs. The three CDRs of a heavy chain are referred to as CDRH1, CDRH2, and CDRH3, in that order from the N-terminus. Likewise, in the case of a light chain, CDRLs are referred to as CDRL1, CDRL2 and CDRL3. CDRH3 is the most important for the specificity of binding an antibody to an antigen. In addition, the CDRs of each chain are kept together in an adjacent state due to the FR regions, contributing to the formation of the antibody antigen binding site along with the CDRs of the other chain. The constant region does not contribute
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17/82 directly with the binding of an antibody to an antigen, but has several effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis through binding to an Fcy receptor, the rate of half-life / clearance through the neonatal Fc receptor (FcRn) and complement-dependent cytotoxicity (CDC), through a C1q component of the complement system cascade.
Antibody Preparation [0053] The term "anti-CAPRIN-1 antibody", as used herein, refers to an antibody with immunological reactivity to a full-length CAPRIN-1 protein or a fragment thereof.
[0054] As used in the present invention, the term "immunological reaction" refers to the in vivo binding property of an antibody to a CAPRIN-1 antigen. Through such an in vivo link, the function of damaging the tumor (for example, death, suppression or degeneration) is displayed. Specifically, an antibody used in the present invention can be any type of antibody, as long as it binds to a CAPRIN-1 protein in order to be able to damage the tumor, such as leukemia, lymphoma, breast cancer, brain tumor, lung cancer, esophageal cancer, gastric cancer, kidney cancer, colorectal cancer, ovarian cancer, prostate cancer or fibrosarcoma.
[0055] Examples of antibodies include a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, a single chain antibody, and an antibody fragment (for example, Fab and F (ab ') 2). In addition, the antibody can be an immunoglobulin molecule of any class, such as IgG, IgE, IgM, IgA, IgD or IgY, or of any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.
[0056] The antibody can be further modified by glycosylation, acetylation, formylation, amidation, phosphorylation, pegylation (PEG)
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18/82 and / or the like.
[0057] Several examples of antibody preparation are as described below.
[0058] When the antibody is a monoclonal antibody, for example, the breast cancer cell line SK-BR-3 expressing CAPRIN-1 is administered to a mouse for immunization, the spleen of the mouse is removed, the cells are separated and then the mouse myeloma cells and cells are fused. Among the fusion cells thus obtained (hybridomas), a clone producing an antibody that has the effect of suppressing the proliferation of cancer cells is selected. A hybridoma that produces a monoclonal antibody that has the effect of suppressing the proliferation of cancer cells is isolated, the hybridoma is cultured, and then an antibody is purified from the culture supernatant by general affinity purification, so that the antibody can be prepared.
[0059] The hybridoma that produces a monoclonal antibody can also be prepared as described below, for example. First, an animal is immunized with a sensitizing antigen according to a known method. A general method is carried out by injecting a sensitizing antigen to a mammal intraperitoneally or subcutaneously. Specifically, a sensitizing antigen is diluted with PBS (phosphate-buffered saline), saline, or the like in an appropriate amount, followed by suspension. The resultant is then mixed with an appropriate amount of a general adjuvant, as needed, such as Freund's complete adjuvant. After emulsification, the solution was administered to a mammal several times every 4 to 21 days. In addition, a suitable vehicle can also be used after immunization with a sensitizing antigen.
[0060] A mammal is immunized as described above. After
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19/82 confirmation of an increase in the level of the desired antibody in the serum, the immunized cells are collected from the mammal and then subjected to cell fusion. Preferred immunized cells are particularly splenocytes.
[0061] Mammalian myeloma cells are used as other parental cells to be fused with the immunized cells. As myeloma cells, several known cell lines are preferably employed, 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, 511-519), MPC-11 (Margulies. DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (deSt. Groth, SF et al., J. Immunol. Methods (1980) 35, 1-21), S194 ( Trowbridge, ISJ Exp. Med. (1978) 148, 313-323), and R210 (Galfre, G. et al., Nature (1979) 277, 131-133).
[0062] The fusion between the immunized cell and the myeloma cell can be carried out basically according to a known method, for example, such as the Kohler and Milstein technique (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).
[0063] More specifically, the above cell fusion is carried out, for example, in the presence of a cell fusion accelerator in a conventional nutrient medium. As a fusion accelerator, polyethylene glycol (PEG), Sendai virus (HVJ), or similar is used. If desired, an auxiliary agent, such as dimethyl sulfoxide, can be added and used in order to increase the melting efficiency.
[0064] The relationship of immunized cells and myeloma cells to be used in the present invention can be established arbitrarily. For example, the number of immunized cells that are preferably used is one to ten times the number of myeloma cells. As a culture medium,
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20/82 can be used for the cell fusion mentioned above, an RPMI1640 culture medium suitable for the proliferation of the aforementioned myeloma cell line, a MEM culture medium, and other culture media normally used for this type of culture can be used. cell culture. In addition, the liquid that is supplementary to the serum, such as fetal bovine serum (SBF), can be used in conjunction with it.
[0065] Cell fusion can be accomplished by mixing predetermined amounts of the above immunized cells and myeloma cells in the said culture medium, and a PEG solution (for example, having an average molecular weight ranging from about 1000 to 6000), preheated to about 37 ° C is added normally and a concentration of 30% to 60% (w / v) and mixed, thus forming a culture containing hybridomas of interest. Then, an appropriate culture medium is successively added to the culture thus obtained, which is then centrifuged to remove the supernatant, and this process is repeated to remove the cell fusion agent or the like, which is not preferable for the growth of hybridomas.
[0066] The hybridomas thus obtained are cultured by selection in a usual selection culture medium (for example, a HAT culture medium containing hypoxanthine, aminopterin and thymidine). The culture in that HAT culture medium continues for a sufficient period of time (usually several days to several weeks), so that cells (non-fused cells) that are not of the desired hybridoma die. Subsequently, screening and simple cloning of the hybridoma that produces an antibody of interest using the general limiting dilution method is performed.
[0067] The above hybridomas are obtained by immunizing a non-human animal with an antigen. In addition to this method, hybridomas that produce a human antibody having desired activity (for example, cell proliferation suppression activity) can also be obtained in vitro.
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21/82 by sensitizing human lymphocytes, such as human lymphocytes that have been infected with the EB virus, with a protein, a cell expressing protein, or a lysate thereof, followed by the fusion of the sensitized lymphocytes with myeloma cells of human origin having a capacity to divide permanently, such as U266 (record no .: TIB196).
[0068] The hybridoma so prepared that produces a monoclonal antibody of interest can be spiked in general culture medium and can be stored in liquid nitrogen for a long period of time.
[0069] Specifically, a hybridoma can be prepared by immunization by a general immunization method using, as a sensitizing antigen, a desired antigen or a cell that expresses the desired antigen, fusing the immunized cell thus obtained with a parental cell known to a general cell fusion method, and then a monoclonal antibody-producing cell (for example, a hybridoma) is screened by a general screening method.
[0070] Another example of an antibody that can be used in the present invention is a polyclonal antibody. A polyclonal antibody can be obtained as described below, for example.
[0071] A small animal, such as a mouse, a mouse producing human antibodies, or a rabbit, is immunized with a natural CAPRIN-1 protein, a recombinant CAPRIN-1 protein expressed in a microorganism such as Escherichia coli in the form of a GST or similar fusion protein, or a partial peptide thereof, and then the serum is obtained. The serum is purified by precipitation with protein ammonium sulfate, protein A column, protein G column, DEAE ion exchange chromatography, affinity column to which a CAPRIN-1 protein or synthetic peptide has been coupled, or similar, so that an antibody
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22/82 polyclonal can be prepared.
[0072] As a mouse producing human antibodies, a KM mouse (Kirin Pharma / Medarex) and a Xeno mouse (Amgen) are known (for example, International Patent Application Publications WO 02/43478 and WO 02/092812), by example. When such a mouse is immunized with the CAPRIN-1 protein or a fragment thereof, a complete human polyclonal antibody can be obtained from the blood. In addition, splenocytes are collected from the immunized mouse, and then a human-type monoclonal antibody can be prepared using a method for fusing with myeloma cells.
[0073] An antigen can be prepared according to a method using animal cells (JP Patent Publication (Kohyo) No. 2007-530068) or baculovirus (for example, International Publication WO98 / 46777), for example. When an antigen has low immunogenicity, the antigen can be linked to a macromolecule that has immunogenicity, such as albumin, and then immunization is performed.
[0074] In addition, an antibody gene is cloned from said hybridoma and then incorporated into an appropriate vector. The vector is then introduced into a host, and then the genetically recombined antibody produced by using gene recombination techniques can be used (for example, see Carl, AK Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, the cDNA of a variable region (V region) of an antibody is synthesized from the hybridoma mRNA using reverse transcriptase. When the DNA encoding the V region of an antibody of interest can be obtained, the DNA is linked to the DNA encoding the constant region (C region) of the desired antibody, and then the resulting fusion product is incorporated into a vector of expression.
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Alternatively, the DNA encoding the V region of an antibody can be incorporated into an expression vector containing the DNA for the C region of an antibody. At this point, the DNA can be incorporated into an expression vector so that it is expressed under the control of the expression control regions, such as an enhancer and promoter. Then, the host cells are transformed with the expression vector, so that the antibody can be expressed.
[0075] The anti-CAPRIN-1 antibody of the present invention is preferably a monoclonal antibody. However, the anti-CAPRIN1 antibody can also be a polyclonal antibody or a genetically modified antibody (for example, a chimeric antibody or humanized antibody), for example.
[0076] Examples of a human monoclonal antibody include monoclonal antibodies, monoclonal antibodies of non-human animals (for example, a mouse monoclonal antibody, a mouse monoclonal antibody, a rabbit monoclonal antibody and a chicken monoclonal antibody), and antibodies chimeric monoclonal. A monoclonal antibody can be prepared by culturing a hybridoma obtained by fusing spleen cells (splenocytes) from a non-human mammal (for example, a mouse, a human antibody-producing mouse, a chicken or rabbit) immunized with a CAPRIN-1 protein, with a myeloma cell. A chimeric antibody is prepared by combining sequences from different animals, such as an antibody heavy chain comprising and variable regions of a mouse antibody's heavy and light chain and the heavy and light chain constant regions of an antibody human. A chimeric antibody can be prepared using a known method. For example, a chimeric antibody can be obtained by binding the DNA encoding an antibody V region to a coding DNA
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24/82 of a human antibody C region, incorporating the resulting fusion product into an expression vector and then introducing the vector into a host for the production of the chimeric antibody.
[0077] In the examples described below, monoclonal antibodies having immunological reactivity with a partial CAPRIN-1 polypeptide have been prepared, wherein CAPRIN-1 is represented by any of the even numbered SEQ ID sequences from 2 to 30, and wherein the partial polypeptide comprises the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 37. The anti-tumor effects of antibodies monoclonal compounds have been confirmed. These monoclonal antibodies comprise a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NOS: 43, 47, or 63 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID No: 51 or 67, where: the VH region comprises CDR1 represented by the amino acid sequence of SEQ ID NO: 40, 44, or 60, CDR2 represented by the amino acid sequence of SEQ ID NO: 41, 45, or 61, and CDR3 represented by the amino acid sequence of SEQ ID NO: 42, 46, or 62, and the VL region comprises CDR1 represented by the amino acid sequence of SEQ ID NO: 48 or 64, CDR2 represented by the amino acid sequence of SEQ ID NO : 49 or 65, and CDR3 represented by the amino acid sequence of SEQ ID NO: 50 or 66.
[0078] Examples of a polyclonal antibody include an antibody obtained by immunizing an animal producing human antibodies (for example, a mouse) with a CAPRIN-1 protein.
[0079] A humanized antibody is a modified antibody that is also referred to as a remodeled human antibody. A humanized antibody can be constructed by transplanting CDRs from a
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25/82 antibody from an immunized animal to the complementarity determining regions of a human antibody. General techniques of gene recombination are also known.
[0080] Specifically, DNA sequences designed to have each of the CDRs of a mouse or chicken antibody linked to each of the regions of the framework (FRs) of a human antibody are synthesized by the PCR method from several oligonucleotides, which are prepared in order to have overlapping portions in their terminal portions, for example. A humanized antibody can be obtained by linking the DNA obtained in this way to a DNA encoding the constant region of a human antibody, incorporating the resulting fusion product into an expression vector, introducing the vector into a host and thereby causing the host produces the gene product (see European Patent Publication No. 239,400 and International Publication WO 96/02576). As FRs of a human antibody, which are linked by means of CDRs, the FRs that allow the formation of an antigen binding site with good complementarity determining regions are selected. If necessary, for the formation of an antigen binding site having the appropriate complementarity determining regions of a remodeled human antibody, the amino acids of the framework regions of a variable region of the antibody can be substituted (Sato, K. et al. , Cancer Research 1993, 53: 851-856). In addition, FR amino acids can be replaced with those from the framework regions of different human antibodies (see International Patent Publication WO 99/51743).
[0081] As the regions of the frameworks (frameworks - FRs) of a human antibody, which are linked by means of CDRs, FRs are selected that allow the formation of an antigen binding site with good complementarity determining regions. If necessary, for the
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26/82 formation of an antigen binding site having the appropriate complementarity determining regions of a remodeled human antibody, the amino acids of the framework regions of a variable region of the antibody can be substituted (Sato, K. et al., Cancer Research 1993, 53: 851-856).
[0082] After the preparation of a chimeric antibody or a humanized antibody, the amino acids in the variable regions (for example, FR) or in the constant region can be replaced by other amino acids.
[0083] Amino acid substitution is the substitution of, for example, less than 15, less than 10, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less amino acids and is preferably a substitution of 1 to 5 amino acids, and more preferably 1 or 2 amino acids. A substituted antibody must be functionally equivalent to an unsubstituted antibody. The substitution is, desirably, a conservative substitution of amino acid (s) between amino acids that have analogous properties, such as electric charge, side chain, polarity, aromaticity. Amino acids having analogous properties can be classified, for example, into basic amino acids (lysine, arginine and histidine), acidic amino acids (aspartic acid and glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine and tyrosine ), nonpolar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan and methionine), branched chain amino acids (threonine, valine and isoleucine) and aromatic amino acids (phenylalanine, tyrosine, tryptophan and histidine).
[0084] Examples of a modified antibody product include antibodies bound to several molecules, such as polyethylene glycol (PEG). The substances to be bound in the modified antibody product of the present invention are not limited. Such a modified antibody product can be obtained by subjecting the antibody thus obtained to chemical modification. Methods for
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27/82 this has already been established in the state of the art.
[0085] As used herein, the "functional equivalent" refers to an antibody that has a biological or biochemical activity similar to that of the antibody of the present invention and, specifically, refers to a subject antibody that has the function of impairing the tumor essentially without causing rejection after its application to a human, for example. An example of such an activity includes the activity of suppressing cell proliferation or binding activity.
[0086] As a method well known to those skilled in the art of preparing a polypeptide functionally equivalent to a polypeptide, a method for introducing mutations into a polypeptide is known. For example, those skilled in the art can prepare an antibody functionally equivalent to the antibody of the present invention by appropriately introducing a mutation into the antibody using site-directed mutagenesis (Hashimoto-Gotoh, T et al., (1995) Gene 152, 271 -275; Zoller, MJ., And Smith, M. (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al., (1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W and Fritz, HJ., (1987) Methods Enzymol. 154, 350-367; Kunkel, TA., (1985) Proc. Natl. Acad. Sci. USA 82, 488492; Kunkel (1988) Methods Enzymol. 85, 2763-2766 ).
[0087] An antibody that recognizes an epitope of a CAPRIN-1 protein recognized by the anti-CAPRIN-1 antibody above can be obtained by a method known to those skilled in the art. For example, such an antibody can be obtained using a method that involves determining an epitope of a CAPRIN-1 protein recognized by an anti-CAPRIN1 antibody, by a general method (for example, epitope mapping) and then preparation of an antibody using a polypeptide having an amino acid sequence contained in the epitope as an immunogen, or a method that involves determining an epitope of such a prepared antibody
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28/82 by a general method, and then an antibody is selected having the epitope identical to that of an anti-CAPRIN-1 antibody. As used herein, the term "epitope" refers to, in a mammal and preferably in a human, a polypeptide fragment with antigenicity or immunogenicity. The unit of minimum size thereof consists of about 7 to 12 amino acids, and preferably 8 to 11 amino acids.
The Ka (Kon / Koff) affinity constant of the antibody of the present invention is preferably at least 107 M-1, at least 108 M-1, at least 5x108 M-1, at least 109 M-1, at least minus 5x109 M-1, at least 1010 M-1, at least 5x1010 M-1, at least 1011 M-1, at least 5x1011 M-1, at least 1012 M-1 or at least 1013 M-1.
[0089] The antibody of the present invention can be conjugated to an anti-tumor agent. Conjugation of the antibody with an antitumor agent can be accomplished by means of a spacer having a reactive group with an amine group, a carboxyl group, a hydroxyl group, a thiol group or the like (for example, a succinimidyl succinate group, a formyl group, a 2-pyridylthio group, a maleimidyl group, an alkoxy carbonyl group and a hydroxyl group).
[0090] Examples of antitumor agents include the following antitumor agents known in the literature and the like, such as paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa, carbohydrone, meturedopa, uuredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethyleneethiophosphoramide, trimethylolomelamine, bulatacin, bulatacinone, camptothecin, briostatin, calistatin, cryptoficin, styptophine, chloroquine, chloroquine, hematoxychine meclorethamine hydrochloride oxide, melphalan, novembicin,
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29/82 phenesterin, prednimustine, trophosphamide, uracil mustards carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, caliceamicin, dinemicin, clodronate, speramycin, aclacinomycin, actinomycin, carcinicin, carcinicin, carcinomycin, carcinomycin, carcinomycin, carcinicin, carcinomycin, carcinicin, carcinomycin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin, carcinicin. chromomycin, dactinomycin, detorbicin, 6-diazo-5-oxo-L-norleucine, adriamycin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, rodomycin, quinoline streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, tiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofuridine, eoxidine, detoxified, andaroxide , calusterone, dromostanolone propionate, epitiostanol, mepitiostane and testolactone), aminog lutetimide, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisanthrene, edatraxate, defofamine, demecolcin, diaziquone, elfornitine, ephynitine, acetaminophen, acetaminophen, acetaminophen , mitoguazone, mitoxantrone, mopidanmol, nitraerin, pentostatin, fenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxin, scizofilano, spirogeranium, tenuazonic acid, triazine, triazine, triazine dacarbazine, manomustine, mitobronitol, mitolactol, pipobroman, gacitosin, docetaxel, chlorambucil, gemcitabine, 6thioguanine, mercaptopurine, cisplatin, oxaliplatin, carboplatin, vinblastine, etoposide, ifosfamide, vinegar, mitoxanine, aminxanitanine, aminxanthrone, vinegar xeloda, ibandronate, irinotecan, topoisomerase inhibitor, difluoromethylorniti na (DMFO), retinoic acid and capecitabine, and pharmaceutically acceptable salts and derivatives thereof.
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[0091] By administering the antibody of the present invention in combination with an anti-tumor agent, even greater therapeutic effects can be obtained. This technique is applicable before and after surgery of a subject with cancer with CAPRIN-1 expression. Especially after surgery, more effective prevention of cancer recurrences or a prolonged survival period can be achieved against cancer that expresses CAPRIN-1, which has been treated conventionally with an antitumor agent alone.
[0092] Examples of anti-tumor agents to be administered in combination with the antibody of the present invention include the following anti-tumor agents known in the previous literature or the like, such as paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5fluorouracil, thiotepa, busulfan, improsulfan, piposulfan , benzodopa, carboquone, meturedopa, uredopa, altretamine, triethyleneemelamine, triethylenephosphoramide, triethyleneethiophosphoramide, trimethylolomelamine, bulatacin, bulatacinone, camptothecin, briostatin, calistatin, chlorophycine, chrysotoxin, panthycin, panthycin , colofosfamide, estramustina, ifosfamida, mecloretamina, mecloretamine hydrochloride oxide, melphalan, novembicine, phenesterina, prednimustine, trophosphamide, uracil mustards carmustine, chlorzotocin, fotemustine, lomustine, nimustine, dinimicine, ranimustine, ranimustine, ranimustine, icin, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophylline, chromomycin, dactinomycin, detorbicin, 6-diazo-5oxo-L-norleucine, adriamycin, epirubicin, esorubicin, esorubicin, esorubicin, esorubicin, esorubicin, esoromycin, mycophenolic, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopine, 6-mercaptopine
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31/82 tiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, amititone, testosterone, tritone, acid aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisanthrene, edathraxate, defofamine, demecolcine, diaziquone, elfornitine, eliptinium acetate, epothilone, etoglucine, lentin, mitanine, mitoidone, mitidamine, monidamine, lonidamine, lonidamine fenamet, pyrarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxin, scizophilane, spirogermanium, tenuazonic acid, triaziquone, roridin A, anguidine, urethane, vindesine, mitobroncholine, mitacobinol, gombobol, , docetaxel, chlorambucil, gemcitabine, 6thioguanine, mercaptopurine, cisplatin, oxalipla thine, carboplatin, vinblastine, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan, topoisomerase inhibitor, difluoromethylnitine and acid (retinorethylnitine); ) pharmaceutically acceptable or (known) derivatives thereof. Of the examples mentioned above, particularly cyclophosphamide, paclitaxel, docetaxel and vinorelbine are preferably used.
Alternatively, a radioactive isotope known in the prior or similar literature, such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Lu175 or Lu176, can be linked to the antibody of the present invention. A desired radioisotope is effective for treating or diagnosing a tumor.
[0094] The antibody of the present invention is an antibody that has immunological reactivity with CAPRIN-1, an antibody that specifically recognizes CAPRIN-1, or an antibody that specifically binds to
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CAPRIN-1, which exhibits a cytotoxic activity against cancer or suppression effect on tumor growth. The antibody must have a structure so that rejection is almost or completely prevented in an animal subject to which the antibody is administered. Examples of such an antibody include, when the subject animal is a human, a human antibody, a humanized antibody, a chimeric antibody (e.g., human mouse chimeric antibody), a single chain antibody, and a bispecific antibody. These antibodies are: recombinant antibodies having the heavy and light chain variable regions from a human antibody; recombinant antibodies having the heavy and light chain variable regions composed of the complementarity determining regions (CDRs) (CDR1, CDR2 and CDR3) of an antibody from a non-human animal and the framework regions of a human antibody; or recombinant antibodies having the heavy and light chain variable regions from a non-human animal antibody; wherein said recombinant antibodies also have the heavy chain and light chain constant regions of a human antibody. Preferred antibodies are the first two antibodies mentioned.
[0095] These recombinant antibodies can be prepared as follows by cloning the DNA encoding an anti-CAPRIN-1 human monoclonal antibody (e.g., a human monoclonal antibody, a mouse monoclonal antibody, a mouse monoclonal antibody, an antibody rabbit monoclonal, or chicken monoclonal antibody) from an antibody producing cell, such as a hybridoma, by preparing the DNA encoding a light chain variable region and an antibody heavy chain variable region by an RT- PCR using the same as template, and then determining the sequence of each variable region of light chain and heavy chain or each sequence of CDR1, CDR2 and CDR3
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33/82 based on the EU numbering system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
[0096] In addition, the DNA encoding each of the variable regions or DNA encoding each CDR is prepared using gene recombination techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) or a DNA synthesizer. In the present invention, the human monoclonal antibody-producing hybridoma can be prepared by immunizing a human antibody-producing animal (for example, a mouse) with human CAPRIN-1, followed by fusing the spleen cells taken from the animal previously immunized with myeloma cells. Alternatively, DNAs encoding a light chain variable region and a heavy chain constant region of a human antibody are prepared as needed, using gene recombination techniques or using a DNA synthesizer.
[0097] In the case of humanized antibody, DNA is prepared by replacing a CDR coding sequence in DNA that encodes a variable region of light chain or heavy chain derived from a human antibody, with a CDR coding sequence corresponding to that of a antibody derived from a non-human animal (for example, a mouse, rat, or chicken) and then linking the DNA obtained in this way to a DNA encoding a light chain or heavy chain constant region derived from a human antibody . Thus, the DNA encoding the humanized antibody can be prepared.
[0098] In the case of the chimeric antibody, the DNA encoding a chimeric antibody can be prepared by ligating the DNA encoding a variable region of light or heavy chain of an antibody of a non-human animal (for example, a mouse, a mouse, or chicken) to DNA
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34/82 encoding a human antibody light chain or heavy chain constant region.
[0099] In the case of the single chain antibody, this antibody is an antibody prepared by linearly linking the variable region of the heavy chain to a variable region of the light chain by means of a linker. Thus, DNA encoding a single chain antibody can be prepared by ligating the DNA encoding a heavy chain variable region, and a DNA encoding a ligand, and a DNA encoding a light chain variable region. In the present invention, a heavy chain variable region and a light chain variable region are both from a human antibody, or, only the CDRs are replaced by CDRs from an antibody derived from a non-human animal (for example, a mouse, a rat, and chicken), although the other regions originate from a human antibody. In addition, the linker has 12 to 19 amino acids, and examples of it include (G4S) 3 having 15 amino acids (Kim, GB. Et al., Protein Engineering Design and Selection 2007, 20 (9): 425- 432).
[00100] In the case of bispecific antibody (diabody), this antibody is able to specifically bind to two different epitopes. For example, DNA encoding a bispecific antibody can be prepared by binding the DNA encoding a variable region of heavy chain "A", a DNA encoding a variable region of light chain "B", a DNA encoding a variable region heavy chain “B”, and DNA encoding a variable region of light chain “A”, in that order (in this case, the DNA encoding a variable region of light chain “B” is linked to the DNA encoding a variable region heavy chain “B” via the DNA encoding the linker (linker) above). In this case, a heavy chain variable region and a light chain variable region are both from a human antibody, or, a human antibody in which only the CDRs have been replaced.
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35/82 with CDRs of an antibody from a non-human animal (for example, a mouse, a rat, or a chicken).
[00101] The recombinant DNA prepared above is incorporated into one or a variety of vectors, these vectors are introduced into host cells (for example, mammalian cells, yeast or insect cells), then (co) expression is caused , so that the recombinant antibody can be prepared (PJ Delves, ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES, 1997 WILEY; P Shepherd and C. Dean., Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; JW Goding., Monoclonal Antibodies: principles and practice, 1993 ACADEMIC PRESS).
[00102] Examples of the antibody of the present invention prepared by the method described above include the following antibody (a), (b) or (c) obtained in the Examples below:
(a) an antibody (for example, the antibody composed of the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 51) which comprises a heavy chain variable region comprising the SEQ ID NOS: 40, 41 and 42 and a light chain variable region comprising SEQ ID NOS: 48, 49 and 50; and (b) an antibody (for example, the antibody composed of the heavy chain variable region of SEQ ID NO: 47 and the light chain variable region of SEQ ID NO: 51) which comprises a heavy chain variable region comprising the SEQ ID NOS: 44, 45 and 46 and a light chain variable region comprising SEQ ID NOS: 48, 49 and 50; and (c) an antibody (for example, the antibody composed of the heavy chain variable region of SEQ ID NO: 63 and the light chain variable region of SEQ ID NO: 67) which comprises a heavy chain variable region comprising the SEQ ID NOS: 60, 61 and 62 and a light chain variable region comprising SEQ ID NOS: 64, 65 and 66.
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36/82 [00103] The amino acid sequences represented by SEQ ID NOS: 40, 41, and 42, SEQ ID NOS: 44, 45, and 46, and SEQ ID NOS: 60, 61, and 62 are CDR1, CDR2 and CDR3, respectively, of the mouse antibody heavy chain variable regions. In addition, the amino acid sequences represented by SEQ ID NOS: 48, 49, and 50, and SEQ ID NOS: 64, 65, and 66 are CDR1, CDR2 and CDR3, respectively, of the mouse antibody light chain variable regions, respectively. .
[00104] In addition, the humanized antibody, chimeric antibody, single chain antibody or bispecific antibody of the present invention is the following antibody (exemplified as "antibody (a)"), for example:
(i) an antibody in which the heavy chain variable region comprises the amino acid sequence of SEQ ID NOS: 40, 41, and 42 and the amino acid sequences of the framework regions of a human antibody, and, a region The light chain variable comprises the amino acid sequences of SEQ ID NOS: 48, 49, and 50 and the amino acid sequences of the framework regions of a human antibody (preferably, the antibody in which the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 43, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 51); and (ii) an antibody in which the heavy chain variable region comprises the amino acid sequence of SEQ ID NOS: 40, 41, and 42, and the amino acid sequences of the framework regions of a human antibody; and a heavy chain constant region comprises an amino acid sequence of a human antibody; and a light chain variable region comprising the amino acid sequences of SEQ ID NOS: 48, 49, and 50 and the amino acid sequences of the human antibody framework regions and a light chain constant region comprising a sequence of amino acid of a human antibody (preferably, the
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37/82 antibody, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 43, and the heavy chain constant region comprises the amino acid sequence of a human antibody, as well as a light chain variable region comprises the amino acid sequence of SEQ ID NO: 51, and a light chain constant region comprising an amino acid sequence of a human antibody).
[00105] In addition, human antibody heavy chain sequences and light chain constant regions and variable regions, can be obtained from the NCBI (for example, in the USA: GenBank, Unigene), for example. For example, the sequence with Accession number J00228 can be referred to a human IgG1 heavy chain constant region, the sequence with Accession number J00230 can be referred to a human IgG2 heavy chain constant region, the sequence with accession number X03604 can be referred to a human IgG3 heavy chain constant region, accession sequence K01316 can be referred to a human IgG4 heavy chain constant region, sequences with accession numbers V00557, X64135, X64133, and the like can be referred to human κ light chain constant regions, and sequences with accession numbers X64132, X64134, and the like, can be referred to human λ light chain constant regions.
[00106] The antibodies referred to above preferably have a cytotoxic activity and, therefore, can exhibit anti-tumor effects.
[00107] In addition, the specific sequences of heavy and light chain variable regions or CDRs in the above antibodies are given for illustrative purposes only, and therefore are not clearly limited to those specific sequences. A hybridoma capable of producing another human antibody or antibody of a non-human animal (for example, an antibody of
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38/82 mouse) against human CAPRIN-1 is prepared, a monoclonal antibody that is produced by the hybridoma is collected, and then it is determined whether the antibody is the non-desired antibody having as indicators the immunological binding property with human CAPRIN-1 and cytotoxic activity. After identifying a hybridoma that produces the target monoclonal antibody in this way, the DNA encoding the heavy chain and light chain variable region of the target antibody is prepared from the hybridoma as described above, and sequencing is performed, and then , DNA is used for the preparation of another antibody.
[00108] Furthermore, in relation to the above antibody, the sequence of each of the antibodies from (a) to (c) mentioned above, and particularly the sequence of the framework region and / or the sequence of the constant region of each of the antibodies may have a substitution, a deletion, or an addition of one or more amino acids, as long as the specific recognition specificity of CAPRIN-1 is maintained. In this case, the term "various" refers preferably to 2 to 5, and more preferably 2 or 3.
[00109] The present invention further provides a DNA encoding the antibody of the present invention cited above, or, a DNA encoding the heavy chain or light chain of the above antibody, or a DNA encoding the heavy chain or light chain variable region of the above antibody. . Examples of such DNAs include, in the case of antibody (a), the DNA encoding a heavy chain variable region comprising the nucleotide sequences encoding the amino acid sequences of SEQ ID NOS: 40, 41, and 42, and the encoding DNA a light chain variable region comprising the nucleotide sequences encoding the amino acid sequences of SEQ ID NOS: 48, 49, and 50.
[00110] The complementarity determining regions (CDRs) encoded by the DNA sequences are regions for determining the
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39/82 specificity of the antibody. Therefore, the coding sequences for regions in an antibody other than CDRs (more specifically, a constant region and a region of the framework) may be of other antibodies. In the present invention, examples of such "other antibodies" include antibodies derived from non-human organisms, and are preferably derived from humans in order to reduce side effects. Thus, in the case of the above DNA, the coding regions of each region of the framework and each contact region of heavy and light chains preferably comprise the nucleotide sequences that encode corresponding amino acid sequences of a human antibody.
[00111] Other examples of alternative DNA encoding the antibody of the present invention include, in the case of antibody (a), DNA encoding a heavy chain variable region comprising the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43 and a DNA encoding a light chain variable region comprising the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 51. In this case, an example of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43 is the nucleotide sequence of SEQ ID NO: 52. In addition, an example of the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 51 is the nucleotide sequence of SEQ ID NO: 53. In these DNAs, the coding regions each heavy and light chain constant region preferably comprises nucleotide sequences encoding the corresponding amino acid sequences of a human antibody.
[00112] The DNAs of these antibodies can be obtained by the methods above or, for example, by the following method. First, the total RNA is prepared from a hybridoma related to the antibody of the present invention using a commercial RNA extraction kit, then the cDNA and
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40/82 is synthesized with a reverse transcriptase using random primers or the like. Subsequently, the cDNA encoding an antibody is amplified by a PCR method using primers of the conserved sequence oligonucleotides in each variable region of known mouse antibody heavy and light chains. The coding sequence for a constant region can be obtained by amplifying a sequence known by the PCR method. The sequence of DNA nucleotides can be determined by a conventional method such as insertion of it into a plasmid or phage for sequencing.
[00113] An anti-CAPRIN-1 antibody to be used in the present invention is considered an antibody that exhibits antitumor effects against cancer cells that express CAPRIN-1 through the following mechanism:
[00114] Antibody-dependent effector cell-mediated cytotoxicity (ADCC) of cells expressing CAPRIN-1 and complement-dependent cytotoxicity (CDC) of cells expressing CAPRIN-1.
[00115] For this reason, the activity of an anti-CAPRIN1 antibody to be used in the present invention can be assessed, as specifically described in the Examples below, by measuring the ex vivo activity of ADCC or CDC against cancer cells expressing CAPRIN- 1.
[00116] An anti-CAPRIN-1 antibody to be used in the present invention, binds to a CAPRIN-1 protein in a cancer cell and exhibits anti-tumor effects due to the activity mentioned above, and is therefore useful to treat or prevent cancer. Specifically, the present invention provides a pharmaceutical composition for treating and / or preventing cancer, which comprises an anti-CAPRIN-1 antibody as an active ingredient. When the antiCAPRIN-1 antibody is used for administration to a human body (antibody therapy), it is preferably a human antibody or humanized antibody, in order to decrease immunogenicity.
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41/82 [00117] In addition, the greater the binding affinity between an anti-CAPRIN-1 antibody and a CAPRIN-1 protein on the surfaces of cancer cells, the stronger anti-tumor activity of the antiCAPRIN-1 antibody can be obtained. Therefore, when an anti-CAPRIN-1 antibody with high binding affinity for the CAPRIN-1 protein can be acquired, strong anti-tumor effects can be expected, so that the application of the antibody as a pharmaceutical composition for the purpose of treating and / or preventing cancer becomes possible. The high binding affinity is desirably as follows. As described above, the binding constant (affinity constants) Ka (kon / koff) is preferably at least 107 M-1, at least 108 M-1, at least 5 * 108 M-1, at least 109 M -1, at least 5 * 109 M-1, at least 1010 M-1, at least 5 * 1010 M-1, at least 1011 M1, at least 5 * 1011 M-1, at least 1012 M-1 or at least least 1013 M-1.
Binding to Cells Expressing the Antigen [00118] The ability of an antibody to bind CAPRIN-1 can be specified by the binding assay using the ELISA method, a Western blot method, immunofluorescence and flow cytometric analysis, or similar techniques , such as those described in the Examples.
Immunohistochemistry staining [00119] An antibody that recognizes CAPRIN-1 can be tested for reactivity to CAPRIN-1 by means of an immunohistochemistry method known to those skilled in the art, using frozen tissue sections fixed in acetone or sections of fixed tissue embedded in paraffin, which is prepared from tissue samples obtained from a subject during surgery, or tissue samples obtained from an animal having a heterotransplant inoculated with a cell line that expresses CAPRIN1, naturally or after transfection.
[00120] An antibody reactive to CAPRIN-1 can be stained by
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42/82 several immunohistochemistry staining methods. For example, a goat anti-mouse antibody or goat anti-chicken antibody conjugated to horseradish peroxidase can perform the reaction, and a target antibody can be visualized.
Pharmaceutical Composition [00121] A target of the pharmaceutical composition to treat and / or prevent a cancer of the present invention is not particularly limited, as long as it is the cancer (cell) that expresses a CAPRIN-1 gene.
[00122] The terms "tumor" and "cancer", used in the present invention, refer to malignant neoplasms and are used interchangeably.
[00123] The cancer to be submitted in the present invention is the cancer that expresses genes that encode CAPRIN-1 proteins having the amino acid sequences NE numbered even from SEQ ID NOS: 2 to 30. Examples of such cancer preferably include breast cancer, tumor brain, leukemia, lymphoma, lung cancer, mast cell tumor, kidney cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer, colorectal cancer, ovarian cancer, prostate cancer or fibrosarcoma.
[00124] Examples of such specific cancer include, but are not limited to, breast adenocarcinoma, compound type breast adenocarcinoma, malignant mixed mammary gland tumor, intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma , large cell carcinoma, glioma that is a tumor of neural epithelial tissue, ependymoma, neurocytoma, fetal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, digestive lymphoma, small and medium cell lymphoma, cecum, ascending colon, descending colon, transverse colon cancer, sigmoid colon, rectal cancer, ovarian epithelial cancer, germ cell tumor and interstitial cell tumor.
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In addition, preferred individuals are mammals, including primates, pets, domestic animals, bred animals, and the like and are particularly preferably humans, dogs and cats.
[00126] When the antibody used in the present invention is used as a pharmaceutical composition, it can be formulated by a method known to those skilled in the art. For example, the antibody can be used parenterally in the form of an injectable preparation, such as an aseptic solution or suspension prepared with water or a pharmaceutically acceptable solution other than water. For example, it can be formulated by mixing a unit dosage form required by generally accepted pharmaceutical practice, in suitable combination with a vehicle the pharmacologically acceptable medium, specifically, sterile water or saline, vegetable oil, an emulsifier, a suspension, a surfactant, a stabilizer, a flavoring compound, an excipient, a vehicle, an antiseptic, a binding agent, and the like. The amounts of active ingredients in these preparations are determined so that an adequate dose within the indicated range can be obtained.
[00127] A composition for aseptic injection can be prescribed according to general pharmaceutical practice, using a vehicle such as distilled water for injection.
[00128] Examples of an aqueous solution for injection include saline, isotonic solution containing dextrose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These examples can be used in combination with a suitable solubilizing agent such as alcohol, especially ethanol and polyalcohol (eg propylene glycol and polyethylene glycol), and a nonionic surfactant (surfactant) (eg polysorbate 80® and HCO -60).
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44/82 [00129] Examples of oils include sesame oil and soy oil, which can be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol. In addition, buffering agents, such as phosphate buffer or sodium acetate buffer, agents with calming action such as procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, or antioxidants can be mixed with it. A suitable ampoule is usually filled with the injection solution thus prepared.
[00130] Administration is oral or parenteral administration and is preferably parenteral administration. Specific examples of the route of administration include injection, transnasal administration, pulmonary administration and transdermal administration. Examples of injection include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection, so that systemic or local administration is possible.
[00131] In addition, administration methods can be appropriately selected depending on the subject's age, body weight, sex, symptoms, among others. The dosage by administering a pharmaceutical composition containing an antibody or polynucleotide that encodes the antibody can be selected from the range between 0.0001 mg and 1000 mg per kg of body weight, for example. Alternatively, the dosage can be selected, for example, from the range between 0.001 mg / body and 100000 mg / body per subject. However, the dosage range is not always limited to these numerical values. The dosage and method of administration vary depending on the subject's body weight, age, sex, symptoms, and the like, but can be appropriately selected by those skilled in the art.
[00132] The pharmaceutical composition above containing the antibody or a fragment thereof of the present invention is administered to a subject, so that the cancer, preferably breast cancer, brain tumor,
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45/82 leukemia, lung cancer, lymphoma, mastocytoma, kidney cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer, colorectal cancer, can be treated and / or prevented.
[00133] The present invention further encompasses a method for treating and / or preventing cancer, comprising administering to a subject the pharmaceutical composition of the present invention in combination with the antitumor agent exemplified above or a pharmaceutical composition containing said antitumor agent. The antibody or fragment thereof and the antitumor agent can be administered simultaneously or separately to a subject. They can be administered separately, regardless of the order of administration. Administration intervals, dosage, route of administration, and frequency of administration can be appropriately selected by a specialist. Examples of another pharmaceutical formulation to be administered simultaneously include pharmaceutical compositions obtained by mixing the antibody or fragment thereof of the present invention with an antitumor agent in a pharmaceutically acceptable carrier (or medium) followed by the formulation. In addition, it is applicable for the above pharmaceutical composition containing an antitumor agent or formulation, explanations relating to the prescription, formulation, route of administration, dose, cancer to be treated and the like for the administration of a pharmaceutical composition containing the antibody of the present invention; and for the formulation.
[00134] Therefore, the present invention also provides a pharmaceutical combination for treating and / or preventing cancer, comprising the pharmaceutical composition of the present invention and the pharmaceutical composition exemplified above containing an anti-tumor agent. In addition, the present invention provides a pharmaceutical composition for treating and / or preventing cancer, comprising the antibody or fragment thereof.
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46/82 invention and an antitumor agent together with a pharmacologically acceptable carrier.
POLIPEPTIDE AND DNA [00135] The present invention also provides the following polypeptides and DNAs related to antibody (a), (b) or (c) above:
(i) a polypeptide comprising the amino acid sequence of SEQ ID NOS: 43, 47 and 63, as well as the DNA encoding the polypeptide, wherein the DNA comprises the nucleotide sequences of SEQ ID NOS: 52, 70, and 68 ;
(ii) a polypeptide comprising the amino acid sequence of SEQ ID NOS: 51, 67 and 63, and the DNA encoding the polypeptide, wherein the DNA comprises the nucleotide sequences of SEQ ID NOS: 53 and 69;
(iii) A heavy chain CDR polypeptide selected from the group consisting of the amino acid sequences represented by SEQ ID NOS: 40, 41 and 42, SEQ ID NOS: 44, 45 and 46, and SEQ ID NOS: 60, 61 and 62, and the DNA encoding the polypeptide;
(iv) A light chain CDR polypeptide selected from the amino acid sequences represented by SEQ ID NOS: 48, 49 and 50, and SEQ ID NOS: 64, 65 and 66, and the DNA encoding the polypeptide.
[00136] These polypeptides and DNAs can be prepared using the techniques of gene recombination as described above.
Brief Description of the Invention [00137] The present invention explained above is summarized as follows:
(1) A pharmaceutical composition to treat and / or prevent cancer, comprising an antibody or a fragment thereof as an active ingredient that has immunological reactivity with a partial polypeptide
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47/82 of CAPRIN-1, where CAPRIN-1 is represented by any of the even numbering sequences of SEQ ID NOS: 2 to 30, and where the polypeptide comprises the partial amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 37;
(2) The pharmaceutical composition according to item (1) in which the cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mast cell tumor, kidney cancer, cervical cancer, bladder cancer, cancer of esophagus, gastric cancer or colorectal cancer;
(3) The pharmaceutical composition according to item (1) or (2) wherein said antibody is a monoclonal antibody or polyclonal antibody;
(4) The pharmaceutical composition according to any of items from (1) to (3), characterized by the fact that said antibody is a human antibody, humanized antibody, chimeric antibody, single chain antibody or a bispecific antibody;
(5) An antibody having immunological reactivity with a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence having 80% or more of sequence identity with the amino acid sequence of SEQ ID NO: 37;
(6) The antibody according to item (5) above, which has a cytotoxic activity against the cancer cell that expresses a CAPRIN-1 protein;
(7) An antibody that comprises a heavy chain variable region comprising SEQ ID NOS: 40, 41, and 42 and a light chain variable region comprising SEQ ID NOS: 48, 49 and 50 and has immunological reactivity with a CAPRIN- 1;
(8) An antibody that comprises a variable region of
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48/82 heavy chain comprising SEQ ID NOS: 44, 45, and 46 and a light chain variable region comprising SEQ ID NOS: 48, 49 and 50 and has immunological reactivity with a CAPRIN-1 protein;
(9) An antibody that comprises a heavy chain variable region comprising SEQ ID NOS: 60, 61, and 62 and a light chain variable region comprising SEQ ID NOS: 64, 65, and 66 and has immunological reactivity with a CAPRIN protein -1;
(10) The antibody according to any of the items from (5) to (9) above, characterized by the fact that it is a human antibody, humanized antibody, chimeric antibody, single chain antibody or bispecific antibody;
(11) A pharmaceutical composition for treating and / or preventing cancer, comprising the antibody or fragment thereof from any of items (5) to (10) above as an active ingredient;
(12) The pharmaceutical composition according to item (11) in which the cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mast cell tumor, kidney cancer, cervical cancer, bladder cancer, cancer of esophagus, gastric cancer or colorectal cancer;
(13) A pharmaceutical combination to treat and / or prevent cancer, which comprises the pharmaceutical composition of any of items from (1) to (4) above, or the pharmaceutical composition of item (11) or (12) above, and a pharmaceutical composition containing an anti-tumor agent;
(14) A pharmaceutical composition for treating and / or preventing cancer, comprising administering to a subject the antibody or fragment thereof from any of items (5) to (10) or the pharmaceutical composition according to item (11 ) or (12) above; and (15) A method for treating and / or preventing cancer, comprising using pharmaceutical compositions of the pharmaceutical combination
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49/82 of item (13) above, in combination, in a subject.
Examples [00138] The present invention is described more specifically on the basis of examples, but the scope of the present invention is not limited by these specific examples.
Example 1 Identification of New Cancer Antigen Proteins by the SEREX method (1) Preparation of cDNA library [00139] Total RNA was extracted from a testis tissue of a healthy dog by a guanidine-phenol-chloroform method. The polyA RNA was purified according to the protocols provided with an “Oligotex-dT30 mRNA” purification kit (Takara Shuzo Co., Ltd.) using the kit.
A dog testis cDNA phage library was synthesized using the mRNA thus obtained (5 pg). For the preparation of the cDNA phage library, a cDNA synthesis kit, a “ZAPcDNA” synthesis kit, and a “ZAP-cDNA gigapack III gold clonning kit” (Stratagene) cloning kit were used and the library was prepared according to protocols included in the kits. The size of the prepared cDNA phage library was 7.73 x 105 pfu / ml.
(2) Selection of the Serum cDNA Library [00141] The immunoselection was performed using the dog testis cDNA phage library prepared above. Specifically, an Escherichia coli host (XL1-Blue MRF ') was infected with the phage so that 2210 clones were present on a NZY 90 x 15 mm agarose plate. The cells were cultured at 42 ° C for 3 to 4 hours, in order to cause plaque formation. The plate was covered with a nitrocellulose membrane (Hybond C extra: GE Healthcare Bio-Science) impregnated with IPTG (isopropylβ-D-thiogalactoside) at 37 ° C for 4 hours. The proteins were induced,
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50/82 expressed, and then transferred to the membrane. Subsequently, the membrane was recovered, immersed, and subjected to stirring in TBS (10 mM Tris-HCl, 150 mM NaCl pH 7.5) containing 0.5% skimmed-milk powder at 4 ° C overnight , so that the nonspecific reaction was suppressed. The filter was allowed to react with the dog's serum diluted 500 times at room temperature for 2 to 3 hours.
[00142] As the serum above dogs with cancer, sera collected from dogs with breast cancer were used. The serums were stored at -80 ° C and then subjected to pre-treatment immediately before use. The pretreatment for the serum was carried out by the following method. Specifically, the host Escherichia coli (XL1-blure MRF ') was infected with phage expressed "λ ZAP Express" into which no foreign genes were introduced, and then cultured in a plate with NZY medium at 37 ° C overnight. Subsequently, a 0.2 M NaHCO3 buffer (pH 8.3) containing 0.5 M NaCl was added to the plate, and then the plate was left to stand at 4 ° C for 15 hours. The supernatants were collected as extracts of Escherichia coli / phage. Then, the collected Escherichia coli / phage extracts were passed through an NHS column (GE Healthcare Bio-Science), in order to immobilize the Escherichia coli / phage-derived protein. The serum from a dog with cancer was passed through the column to which the protein was immobilized by reaction, thus removing Escherichia coli and antibodies adsorbed to the phage from the serum. Each fraction of serum that passed through the column was diluted 500 times with TBS containing 0.5% skimmed milk powder, and the resultant was used as an immunoselection material.
[00143] A membrane, to which the treated serum and fusion protein mentioned above were subjected to the blotting technique, was washed 4 times with TBS-T (0.05% Tween 20 / TBS). The membrane was reacted with goat anti-dog IgG antibody (goat anti-dog IgG-h + i conjugated to
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HRP: BETHYL Laboratories), diluted 5,000 times, as a secondary antibody with TBS containing 0.5% skimmed milk powder at room temperature for 1 hour. Detection was carried out by enzymatic color reaction using an NBT / BCIP reaction solution (Roche). Colonies corresponding to the positive color reaction site were collected from NZY 090 x 15 mm agarose plates, and then dissolved in 500 µl SM buffer (100 mM NaCl, 10 mM MgClSO4, 50 mM Tris-HCl , 0.01% gelatin, pH 7.5). Until the unification of positive colonies for the color reaction, secondary selection and tertiary selection were repeated by a method similar to that described above. Thus, 30,940 phage clones that reacted with serum IgG were screened so that 5 positive clones were isolated.
(3) Homology search for the isolated antigen gene [00144] A procedure for converting the phage vectors to plasmid vectors was performed for the 5 positive clones isolated by the method described above for the purpose of subjecting the clones to nucleotide sequence analysis . Specifically, 200 µl of a solution of Escherichia coli hosts (XL1-Blue MRF ') prepared to give an absorbance in OD600 of 1.0; 250 µl of a purified phage solution, and 1 µl of auxiliary phage “ExAssist helper phage” (Stratagene) were mixed and allowed to react at 37 ° C for 15 minutes. Then 3 ml of LB medium was added, the cells were grown at 37 ° C for 2.5 to 3 hours, and then the resultant was immediately placed in a 70 ° C water bath for a 20 minute incubation. . Centrifugation was performed at 4 ° C, 1000 x g for 15 minutes and then the supernatant was collected as a phagemid solution. Subsequently, 200 µl of a solution prepared from the host phagemid Escherichia coli SOLR to give an OD600 absorbance of 1.0; and 10 pL of the purified phage solution was mixed, followed by 15 minutes of reaction at 37 ° C. 50 μ50 pl of the resulting solution was
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52/82 plated on LB agar medium containing ampicillin (in a final concentration of 50 pg / ml) and then grown overnight at 37 ° C. A single transformed SOLR colony was collected and then cultured in LB medium containing ampicillin (in a final concentration of 50 pg / ml) at 37 ° C. After cultivation, plasmid DNA containing an insert of interest was purified using a “QIAGEN plasmid Miniprep Kit” kit (QIAGEN).
[00145] The purified plasmid was subjected to analysis of the entire sequence of the inserted segment by the primer walking method using the T3 primer of SEQ ID NO: 31 and the T7 primer of SEQ ID NO: 32. The sequences of the SEQ ID NOS: 5, 7, 9, 11, and 13 were obtained through sequence analysis. Using the nucleotide sequences of the genes and their amino acid sequences (SEQ ID NOS: 6, 8, 10, 12, and 14), the BLAST homology research program (http: //www.ncbi.nlm .nih.gov / BLAST /) was conducted to search for homology with known genes. As a result, it was revealed that all five genes obtained were genes that encode CAPRIN-1. The sequence identities between the five genes were 100% with respect to the nucleotide sequence and 99% with respect to the amino acid sequence in the regions to be translated into proteins. The sequence identities of these genes with the human homologous coding genes were 94% with respect to the nucleotide sequence and 98% with respect to the amino acid sequence in the regions to be translated into proteins. The nucleotide sequences of human homologues are represented by SEQ ID NOS: 1 and 3, and their amino acid sequences are represented by SEQ ID NOS: 2 and 4. In addition, the sequence identities of the dog genes obtained with the genes coding of cattle counterparts were 94% with respect to the nucleotide sequence and 97% with respect to the amino acid sequence in the regions that are translated into proteins. The nucleotide sequence of the cattle counterpart
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53/82 is represented by SEQ ID NO: 15 and its amino acid sequence is represented by SEQ ID NO: 16. In addition, the sequence identities of the human homolog encoding genes with the cattle homolog encoding genes were 94% with respect to the nucleotide sequence and 97% with respect to the amino acid sequence in the regions that are translated into proteins. In addition, the sequence identities of the dog genes obtained with the coding genes of horse counterparts were 93% with respect to the nucleotide sequence and 97% with respect to the amino acid sequence in the regions that are translated into proteins. The nucleotide sequence of the horse homologue is represented by SEQ ID NO: 17 and its amino acid sequence is represented by SEQ ID NO: 18. In addition, the sequence identities of the human homologous coding genes with the coding genes of horse homologues were 93% with respect to the nucleotide sequence and 97% with respect to the amino acid sequence in the regions that are translated into proteins. In addition, the sequence identities of the dog genes obtained with the mouse coding genes were 87% to 89% with respect to the nucleotide sequence and from 95 to 97% with respect to the amino acid sequence in the regions that are translated in proteins. The nucleotide sequences of the mouse homologues are represented by SEQ ID NOS: 19, 21, 23, 25 and 37; and their amino acid sequences are represented by SEQ ID NOS: 20, 22, 24, 26 and 28. In addition, the sequence identities of the human homolog encoding genes with the mouse homolog encoding genes were 89% to 91% with respect to the nucleotide sequence and 95% to 96% with respect to the amino acid sequence in the regions that are translated into proteins. In addition, the sequence identities of the dog genes obtained with the coding genes of chicken homologs were 82% with respect to the nucleotide sequence and 87% with respect to the
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54/82 amino acid in the regions that are translated into proteins. The nucleotide sequence of the chicken homologue is represented by SEQ ID NO: 29 and its amino acid sequence is represented by SEQ ID NO: 30. In addition, the sequence identities of the genes encoding the human homologue with the coding genes of the chicken homologues were 81% to 82% with respect to the nucleotide sequence; and 86% with respect to the amino acid sequence in the regions that are translated into proteins.
(4) Analysis of gene expression in each tissue [00146] The expression of genes obtained by the method described above was examined in normal tissues of dogs and humans and in different cell lines by the RT-PCR method. The reverse transcription reaction was performed as follows. Specifically, the total RNA was extracted from 50 mg to 100 mg of tissue or 5 to 10 x 10 6 cells of the cell line using a Trizol reagent (Invitrogen) according to the attached protocols. The cDNA was synthesized with the total RNA using a synthesis system for RT-PCR "Superscript First-Strand Synthesis System" (Invitrogen) according to the manufacturer's protocol. PCR was performed as follows using SEQ ID NOS primers: 33 and 34 specific for the obtained genes. Specifically, the reagents and an accompanying buffer were added to 0.25 pL of the sample, prepared by the reverse transcription reaction in a total volume of 25 μL, so that the resultant contained the above primers at 2 μΜ each, 0 dNTPs, 2 mM each, and 0.65U of ExTaq polymerase (Takara Shuzo Co., Ltd.). PCR was performed by repeating a cycle of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds, for 30 cycles, using a Thermal Cycler thermal cycler (BIO RAD). The gene-specific primers above are capable of amplifying the region ranging from nucleotides 206 to 632 in the nucleotide sequence of SEQ ID NO: 5 (dog CAPRIN-1 gene) and the region extending from nucleotides 698 to 1124 in the sequence of nucleotides of
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SEQ ID NO: 1 (human CAPRIN-1 gene). As a control for comparison, GAPDH specific primers of SEQ ID NOS: 35 and 36 were also used simultaneously. As a result, as shown in Fig. 1, a strong expression was observed in the testis among tissues of normal dogs, while expression was observed in the tissues of breast cancer and dog adenocarcinoma. In addition, the observation of the expression of the human homologue from the genes obtained was also performed. As a result, similarly to the case of the dog CAPRIN-1 gene, the expression could be observed only in the testis among normal tissues. However, in the case of cancer cells, the expression has been detected in many types of cancer cell lines, including breast cancer cell lines, brain tumor, leukemia, lung cancer, and esophageal cancer. The expression was seen especially in many of the breast cancer cell lines. These results were confirmed by the results that, with the exception of testis tissue, CAPRIN-1 expression is not observed in normal tissues, whereas CAPRIN-1 was expressed in cancer cells and, specifically, in many cancer cell lines. mama.
[00147] In Fig. 1, reference number 1 on each vertical axis indicates the expression patterns of genes identified above and reference number 2 indicates expression patterns of the GAPDH gene as a control.
(5) Preparation of polyclonal antibody against CAPRIN-1 derived peptide [00148] To obtain an antibody that binds CAPRIN-1, a CAPRIN-1 derived peptide represented by SEQ ID NO: 37 was synthesized. 1 mg of the peptide as an antigen was mixed with an equivalent amount of an incomplete Freund's adjuvant solution (IFA). The mixture was administered subcutaneously to rabbits 4 times every 2 weeks. The blood was then collected and the antiserum containing polyclonal antibodies was
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56/82 obtained. In addition, the antiserum was purified using a G protein transporter (GE Healthcare Bio-Sciences), so that polyclonal antibodies against the CAPRIN-1 derived peptide were obtained. In addition, serum from a rabbit to which the antigen was not administered was purified using a G protein transporter in a similar manner as above, and the resultant was used as a control antibody.
(6) ANALYSIS OF ANTIGEN PROTEIN EXPRESSION ON CANCER CELL [00149] Next, 7 breast cancer cell lines (MDA-MB-157, T47D, MRK-nu-1, MDA-MB-231V, BT20, SK-BR-3 and MDA-MB231T), whose expression of the CAPRIN-1 gene was observed at high levels, were examined for the expression of the CAPRIN-1 protein on cell surfaces. 106 cells from each human breast cancer cell line for gene expression that had been observed previously were centrifuged in a 1.5 ml microcentrifuge tube. 2 μ2 pg (5 pL) of the polyclonal antibodies against the CAPRIN1-derived peptide prepared in item (5) above were added to the tube. After suspension with 95 pL of PBS containing 0.1% fetal bovine serum, the cells were left to rest on ice for 1 hour. After washing with PBS, the resulting product was suspended in PBS containing 5 pL of goat anti-rabbit IgG antibody labeled with FITC (SantaCruz) and 95 pL of 0.1% fetal bovine serum (SBF) and then the The resulting mixture was left to stand on ice for 1 hour. After washing with PBS, the fluorescence intensity was measured using a FACS Calibur (Becton, Dickinson & Company). Meanwhile, procedures similar to the above were performed using the control antibody prepared in item (5) above, instead of polyclonal antibodies against the CAPRIN-1 derived peptide so that a control was prepared. As a result, all cells to which the human anti-CAPRIN-1 antibody was added exhibited fluorescence intensity 30% stronger or more in
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57/82 compared to the control. Specifically, the fluorescence intensity increased by 187% in the case of MDA-MB-231V and 124% in the case of 3-SK-BR. With these results it was revealed that the CAPRIN-1 protein was expressed on the cell membrane surfaces of the human cancer cell lines above. The percentage increase in fluorescence intensity above was expressed as a percentage of the increase in mean fluorescence intensity (MFI level) in each cell type and was calculated using the following formula.
[00150] Percentage increase in mean fluorescence intensity (percentage increase in fluorescence intensity) (%) = ((MFI level in cells that reacted with human anti-CAPRIN-1 antibody) (MFI level of control)) / (MFI level of the control) x 100.
[00151] With a technique similar to the one above, CAPRIN-1 expression was analyzed by 3 kidney cancer cell lines (Caki-1, Caki2, and A498), an ovarian cancer cell line (SKOV3), a lung cancer cell line (QG56), a prostate cancer line (PC3), a cervical cancer cell line (Hela), a fibrosarcoma cell line (HT1080), 2 tumor cell lines brain (T98G and U87MG), two mouse colorectal cancer cell lines (CT26 and colon 26), a mouse breast cancer cell line (4T1), a mouse melanoma cell line (B16), and two strains of mouse neuroblastoma (N1E-115 and Neuro2a). As a result, CAPRIN-1 expression was confirmed in all cell lines. In addition, similar results were obtained in the case of the use of the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody # 1) comprising the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 51 or the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody # 2) comprising the variable region of
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58/82 SEQ ID NO: 47 heavy chain and the light chain variable region of SEQ ID NO: 51, or the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody # 3), comprising the SEQ heavy chain variable region ID NO: 63, and the light chain variable region of SEQ ID NO: 67 (obtained in Example 3).
(7) Immunohistochemical staining (7) -1 CAPRIN-1 expression in normal mouse and dog tissues.
[00152] Mice (Balb / c, females) and dogs (beagles, females) were exsanguinated under anesthesia with ether and anesthesia with ketamine / isoflurane. After laparotomy, each organ (stomach, liver, eyeball, thymus, muscle, bone marrow, uterus, small intestine, esophagus, heart, kidney, salivary gland, large intestine, mammary gland, brain, lung, skin, supra gland kidney, ovary, pancreas, spleen, and bladder) was transferred to a 10 cm plate containing PBS. Each organ was cut in PBS and then subjected to fixation perfusion overnight in 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde (PFA). The infusion solution was discarded, the tissue surface of the organs was washed with PBS, a PBS solution containing 10% sucrose was added to a 50 ml centrifuge tube, each tissue was added to the tube, and then , the tube was shaken using a rotor at 4 ° C for 2 hours. The solution was replaced with a PBS solution containing 20% sucrose, and then left to stand at 4 ° C until the tissue sank. The solution was replaced by a PBS solution containing 30% sucrose and then left to stand at 4 ° C until the tissue sank. The tissue was removed and then necessary portions were excised with a surgical scalpel. Then, an OCT compound (Tissue Tek) was added to the fabric so that it was completely applied to the surface of the fabric, and then the fabric was placed in a cryoform. The cryomold was placed on dry ice for rapid freezing. Then the tissue was cut from 10 pm to 20 pm using a cryostat
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59/82 (Leica). The cuts were dried in air on glass slides, using a hair dryer for 30 minutes, to prepare the cut fabric mounted on a glass slide. Then, each sample was placed in a staining bottle filled with PBS-T (saline solution containing 0.05% Tween 20) and then subjected to PBS-T replacement three times every 5 minutes. The excess water around the cuts was removed with Kimwipes, and then the cuts were circulated using a “DAKOPEN” (DAKO) pen. As blocking solutions, a MOM mouse Ig blocking reagent (Vectastain) and a PBS-T solution containing 10% SBF was placed on the slides on the mouse and dog tissue, respectively, and then left to stand. humid chamber at room temperature for 1 hour. Then, a solution of polyclonal antibodies (reactive with the surfaces of cancer cells, and prepared according to item (5) above), against the peptide derived from CAPRIN-1 (SEQ ID NO: 37), at 10 pg / ml adjusted with a blocking solution, it was placed on the slides and then rested overnight in a humid chamber at 4 ° C. 10-minute washes were performed with PBS-T for 3 times, and then a biotin-labeled anti-IgG MOM antibody (Vectastain) was diluted 250 times with the blocking solution, and then the slides were incubated in room temperature for 1 hour in a humid chamber. After ten (10) minutes of washing with PBS-T for 3 times, an ABC avidin-biotin reagent (Vectastain) was placed on the slides, and then the sample was left to stand in a humid chamber at room temperature for 5 minutes. . After ten (10) minutes of washing with PBS-T for 3 times, a DAB dye solution (10 mg DAB + 30% H2O2 10 pl / 0.05 M Tris-HCl (pH 7.6) was added 50 ml), and then the sample was left to stand in a humid chamber at room temperature for 30 minutes. After washing with distilled water, a hematoxylin reagent (DAKO) was placed on the sample and
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60/82 the sample was left to stand at room temperature for 1 minute and then washed with distilled water. The glass slide was immersed in 70%, 80%, 90%, 95% ethanol solutions, and finally, 100%, for 1 minute each, then the slides were at rest in xylene overnight. The glass slides were removed, sealed with “Glycergel Mounting Medium” (DAKO) mounting medium, and then analyzed. As a result, CAPRIN-1 expression was observed slightly within cells of each tissue of salivary glands, kidney, colon and stomach, but its expression was not observed on cell surfaces. In addition, expression in tissues from other organs was not observed. In addition, similar results were obtained in the case of the use of the anti-CAPRIN1 monoclonal antibody (monoclonal antibody # 1) comprising the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 51, the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody # 2) comprising the heavy chain variable region of SEQ ID NO: 47 and the light chain variable region of SEQ ID NO: 51, or the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody # 3), comprising the heavy chain variable region of SEQ ID NO: 63, and the light chain variable region of SEQ ID NO: 67 (obtained in Example 3).
(7) -2 CAPRIN-1 expression in dog breast cancer tissue.
[00153] Freeze cut slides were prepared by a method similar to that described above using 108 breast cancer specimens frozen from tissue of dogs pathologically diagnosed as having malignant breast cancer, and immunohistochemical staining was performed using the polyclonal antibodies (prepared according to item (5) above) against the peptide derived from CAPRIN-1 (SEQ ID NO: 37). As a result, CAPRIN-1 expression was observed in 100 of the 108 samples (92.5%) and CAPRIN-1 was strongly
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61/82 expressed on the surface of cancer cells with a particularly high degree of atypism. In addition, similar results were obtained when using monoclonal antibody # 1, # 2 or # 3 obtained in the Example
3.
(7) -3 CAPRIN-1 expression in human breast cancer tissue.
[00154] Immunohistochemistry staining was performed using 188 specimens of human breast cancer tissue in a paraffin-embedded human breast cancer tissue array (BIOMAX). After 3 hours of treatment of the array of breast cancer tissue in a matrix at a temperature of 60 ° C, the matrix was placed in a xylene-stained bottle, followed by replacing the xylene three times every 5 minutes. Then, a similar process was carried out with ethanol and PBST instead of xylene. The human breast cancer tissue matrix was placed in a staining bottle with 10 mM citrate buffer (pH 6.0) containing 0.05% Tween 20. After 5 minutes of treatment at 125 ° C, the matrix was left standing at room temperature for 40 minutes or more. The excess water around the cuts was removed with Kimwipes, and the cuts were circulated with a DAKOPEN pen, and an endogenous peroxidase blocker (DAKO) was added dropwise in appropriate amounts. Then the tissue matrix samples were left to stand at room temperature for 5 minutes, the tissue matrix samples were placed in a PBS-T staining bottle, followed by the replacement of the PBS-T three times every 5 minutes. As a blocking solution, a PBS-T solution containing 10% SBF was placed in the matrix, and then the matrix was left to stand in a humid chamber at room temperature for 1 hour. Then, a solution of polyclonal antibodies against the peptide derived from 1-CAPRIN (SEQ ID NO: 37) prepared according to item (5) above, having a concentration of 10 pg / ml adjusted with a solution of PBS-T
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62/82 containing 5% SBF was placed on the slides and the tissue matrix was incubated overnight in a humid chamber at 4 ° C. After ten (10) minutes of washing with PBS-T for 3 times, a peroxidase-labeled conjugated polymer “Peroxidase Labeled Polymer Conjugated (DAKO)” was added dropwise onto the slides in appropriate amounts, and then the tissue arrangement in a matrix it was resting in a humid chamber at room temperature for 30 minutes. After ten (10) minutes after washing with PBS-T for 3 times, a DAB dye solution (DAKO) was placed on the slides and then incubated at room temperature for about 10 minutes. The dye solution was discarded, and was washed for 10 minutes with PBS-T for 3 times, and then washed with distilled water. The matrix tissue arrangement was immersed in 70%, 80%, 90%, 95% ethanol solutions, and finally, 100%, for 1 minute each, then rested overnight in xylene. The glass slides were removed, sealed with “Glycergel Mounting Medium” (DAKO) mounting medium, and then analyzed. As a result, strong CAPRIN-1 expression was observed in 138 (73%) out of a total of 188 breast cancer tissue specimens. In addition, similar results were obtained when monoclonal antibody # 1, # 2 or # 3 obtained in Example 3 was used.
(7) -4 CAPRIN-1 expression in human malignant brain tumor.
[00155] Immunohistochemistry staining was performed according to a method similar to that used in item (7) -3 above with 247 tissue specimens of malignant brain tumors in a tissue array of human malignant brain tumors embedded in paraffin (BIOMAX), using polyclonal antibodies (prepared according to item (5) above) against the peptide derived from CAPRIN-1 (SEQ ID NO: 37). As a result, strong CAPRIN-1 expression was observed in 227 (92%) of a total of 247 specimens of malignant brain tumor tissue. In addition,
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Similar results were obtained when monoclonal antibody # 1, # 2 or # 3 obtained in Example 3 was used.
(7) -5 CAPRIN-1 expression in lymph node with HUMAN BREAST CANCER metastasis.
[00156] Immunohistochemistry staining was performed according to a method similar to that described in item (7) -3 above, with 150 lymph node tissue specimens with breast cancer metastases embedded in a matrix tissue arrangement (micro array) embedded in paraffin from lymph node tissue with human breast cancer metastases (BIOMAX), using polyclonal antibodies against the CAPRIN-1 derived peptide (SEQ ID NO: 37) prepared in item (5) above. As a result, strong CAPRIN-1 expression was observed in 136 of a total of 150 lymph node tissue specimens with breast cancer metastasis (90%). Specifically, it was revealed that CAPRIN-1 was also strongly expressed in cancer tissues that had breast cancer metastases. In addition, similar results were obtained when monoclonal antibody # 1, # 2 or # 3 obtained in Example 3 was used.
(7) -6 CAPRIN-1 expression in different human cancer tissues.
[00157] Immunohistochemistry staining was performed according to a method similar to the previous one, with specimens in different matrix tissue arrangements embedded in human cancer tissue paraffin (BIOMAX), using polyclonal antibodies against the CAPRIN-1 derived peptide. (SEQ ID NO: 37) prepared in item (5) above. As a result, a strong expression of CAPRIN-1 was observed in esophageal cancer, colon cancer, rectal cancer, lung cancer, kidney cancer, bladder cancer and cervical cancer. In addition, similar results were obtained when monoclonal antibody # 1, # 2 or # 3 obtained in Example 3 was used.
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Example 2 Preparation of Human CAPRIN-1 (1) Preparation of recombinant protein.
[00158] Based on the SEQ ID NO: 1 gene obtained in Example 1, a recombinant protein from the human homologous gene was prepared by the following method. A PCR was performed in a total volume of 50 pL, with 1 pL of cDNA, two primers (SEQ ID NOS: 38 and 39, comprising the sequences for cleavage by the restriction enzymes SacI and Xhol) of 0.4 μΜ each, 0 , 2 mM dNTP, and 1.25U PrimeStar HS polymerase (Takara Shuzo Co., Ltd.), prepared by adding the reagents and a follow-up buffer. The expression was confirmed by an RT-PCR method for the cDNA used between the various tissue or cell derived cDNAs prepared in the Example
1. PCR was performed by repeating 98 ° C thermocycles for 10 seconds and 68 ° C for 2.5 minutes for 30 cycles, using a Thermal Cycler thermocycler (BIO RAD). The two primers above are capable of amplifying a coding region of the entire amino acid sequence of SEQ ID NO: 2. After the PCR, the amplified DNA was subjected to 1% agarose gel electrophoresis, then a DNA fragment from about 2.1 kbp was purified using a “QIAquick Gel Extration Kit” kit (QIAGEN).
[00159] The DNA fragment thus purified was linked to a PCR-Blunt cloning vector (Invitrogen). After the transformation of Escherichia coli, with it, the plasmid was collected. It was verified by the sequence of the fragment that the gene thus amplified has the sequence of interest. The plasmid that has a sequence corresponding to the sequence of interest was treated with the restriction enzymes SacI and XhoI and then was purified with a QIAquick gel extraction kit. The gene sequence of interest was introduced into an Escherichia coli expression vector pET30a (Novagen), and treated with the restriction enzymes SacI and XhoI. The protein
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65/82 recombinant fused to the His-tag marker could be produced using the vector. The plasmid was transformed into E. coli for recombinant expression, BL21 (DE3), and then the expression was induced with 1 mM IPTG, so that the protein of interest was expressed in Escherichia coli.
(2) Purification of recombinant protein.
[00160] The recombinant Escherichia coli obtained above that expresses the SEQ ID NO: 1 gene was grown in LB medium containing 30 pg / ml kanamycin at 37 ° C until the absorbance at 600 nm reached about 0.7, was added isopropyl-pD-thiogalactopyranoside at a final concentration of 1 mM, then the cells were cultured at 37 ° C for 4 hours. Subsequently, centrifugation was performed at 4800 rpm for 10 minutes, then the cells were collected. The resulting cell pellet was suspended in phosphate-buffered saline and centrifuged at 4800 rpm for 10 minutes, then the cells were washed.
[00161] The cells were suspended in phosphate-buffered saline and then disrupted using ultrasound on ice. The resulting lysate from ultrasonic Escherichia coli was subjected to centrifugation at 6000 rpm for 20 minutes, and then the resulting supernatant was considered as a soluble fraction and the precipitate was considered as an insoluble fraction.
[00162] The soluble fraction was added to a nickel chelate column adjusted according to a conventional method (carrier: “Chelating Sepharose® Fast Flow” resin (GE Healthcare), 5 ml column capacity, and a balance buffer : 50 mM hydrochloride buffer (pH 8.0)). The non-adsorbed fractions were washed with 50 mM hydrochloride buffer (pH 8.0) in an amount 10 times the column capacity and 20 mM phosphate buffer (pH 8.0) containing 20 mM imidazole. Immediately after washing, 6 beds were eluted with 20 mM phosphate buffer (pH 8.0) containing 100 mM imidazole. THE
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66/82 elution of the protein of interest was confirmed by staining with Coomassie staining in the elution fraction with 20 mM phosphate buffer (pH 8.0) containing 100 mM imidazole and then the elution fraction was added to a strong anion exchange column (carrier: “Q Sepharose® Fast Flow” resin (GE Healthcare), column capacity of 5 ml, and 20 mM phosphate buffer (pH 8.0) as equilibration buffer). An non-adsorbed fraction was washed with 20 mM phosphate buffer (pH 7.0) in an amount 10 times the column capacity and 20 mM phosphate buffer (pH 7.0) containing 200 mM sodium chloride. Immediately after washing, 5 beds were eluted with 20 mM phosphate buffer (pH 7.0) containing 400 mM sodium chloride, thus the purified fraction of the protein having the amino acid sequence represented by SEQ ID NO: 2 was obtained.
[00163] 200 pL of each purified sample obtained by the method described above was distributed in 1 ml of reaction buffer (20 mM Tris-Hcl, 50 mM NaCl, 2 mM CaCl2, pH 7.4), followed by the addition of 2 pL of enterokinase (Novagen). After that, the resultant was left to stand overnight at room temperature so that the His-tag was cleaved, and then purification was performed using an enterokinase cleavage capture kit “Enterokinase Cleavage Capture Kit” (Novagen), according to the manufacturer's protocols. Then, 1.2 ml of purified sample obtained by the method described above were subjected to the replacement of the buffer with physiological phosphate buffer (Nissui Pharmaceutical Co., Ltd.), using NANOSEP 10K OMEGA (PALL) ultrafiltration. Additionally, sterile filtration was performed using a HT Tuffryn Acrodisc 0.22 pm (PALL), then the resultant was used in the next experiment.
Example 3
Preparation of Mouse Monoclonal Antibody Anti-CAPRIN-1 [00164] 100 pg of the antigen protein (human CAPRIN-1) ()
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67/82 represented by SEQ ID NO: 2 prepared in Example 2 was mixed with an equivalent amount of MPL + TDM adjuvant (Sigma), and then this was used as an antigen solution for a mouse. The antigen solution was administered intraperitoneally to 6-week-old Balb / cc mice (Japan SLC Inc.), then administration was performed 7 times each week, and thus, immunization was completed. Each spleen was excised 3 after the final immunization, and placed between two sterilized glass slides and then crushed. The resulting product was washed with PBS (-) (Nissui) and then centrifuged at 1500 rpm for 10 minutes to remove the supernatant. This procedure was repeated 3 times, so that the splenocytes were obtained. The splenocytes thus obtained and the mouse myeloma cells SP2 / 0 (acquired from ATCC) were mixed in a 10: 1 ratio. The PEG solution prepared by mixing 200 pL of RPMI 1640 medium containing 10% PBS heated to 37 ° C and 800 pL of PEG1500 (Boehringer) was added to the mixture, left to stand for 5 minutes for cell fusion, and then subjected to centrifugation at 1700 rpm for 5 minutes. After removing the supernatant, the cells were suspended in 150 ml of RPMI1640 medium containing 15% SBF, supplemented with a HAT solution (Gibco) (2% in equivalents) (selective HAT medium), then the cell suspension was plated on fifteen 96-well plates (Nunc) at 100 pL per well. The cells were cultured for 7 days at 37 ° C under conditions of 5% CO2, so that the hybridomas prepared by the fusion of splenocytes and myeloma cells were obtained.
[00165] Hybridomas were selected using an antibody binding affinity marker produced by hybridomas prepared for the CAPRIN-1 protein. The CAPRIN-1 protein solution (1 pg / ml) prepared in Example 2 was added to a 96-well plate with 100 pL per well and then left to stand at 4 ° C for 18 hours. Each
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68/82 well was washed 3 times with PBS-T, 400 pL of a 0.5% solution of Bovine Serum Albumin (BSA) (Sigma) was added to each well, then the plate was left to stand at room temperature room for 3 hours. The solution was removed and then the wells were washed three times with 400 µl of PBS-T per well. The hybridoma culture supernatant obtained above was added to a volume of 100 pL per well and then the plates were left to stand at room temperature for 2 hours. After washing each well three times with PBS-T, an anti-mouse IgG (H + L) antibody labeled with HRP (Invitrogen) diluted 5000 times with PBS was added to 100 pL per well and the resultant was left in stand at room temperature for 1 hour. After washing the well three times with PBS-T, 100 pL of a TMB substrate solution (Thermo) was added to each well and then the plate was left to stand for 15 to 30 minutes for the staining reaction. After color development, 100 pL of 1N sulfuric acid was added to each well to stop the reaction, then absorbances at 450 nm and 595 nm were measured using an absorption spectrometer. As a result, hybridomas that produce antibodies with high absorbance values were selected.
[00166] The hybridomas so selected were added to a 96-well plate at 0.5 cells per well and then cultured. After 1 week, hybridomas were observed that formed isolated colonies in the wells. These cells in the culture wells were grown for a longer time, then the hybridomas were selected using as a marker the binding affinity of the antibodies produced by the cloned hybridomas with the CAPRIN-1 protein. The CAPRIN-1 protein solution (1 pg / ml) prepared in Example 2 was added to a 96-well plate with 100 pL per well, and then left to stand at 4 ° C for 18 hours. Each well was washed three times with PBS-T, 400 pL of a 0.5% BSA solution was added in
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69/82 each well, then the plate was left to stand at room temperature for 3 hours. The solution was removed and then the wells were washed three times with 400 µl of PBS-T per well. 100 µl of each hybridoma culture supernatant obtained above was added per well, and then the plate was left to stand at room temperature for 2 hours. After washing each well three times with PBS-T, 100 pL of an anti-mouse IgG antibody (H + L) labeled with HRP (Invitrogen) diluted 5,000 times with PBS was added per well and then rested for 1 hour at room temperature. After washing the well three times with PBS-T, 100 pL of a TMB substrate solution (Thermo) was added to each well and then the plate was left to stand for 15 to 30 minutes for the staining reaction. After color development, 100 pL of 1N sulfuric acid was added to each well to stop the reaction, then absorbances at 450 nm and 595 nm were measured using an absorption spectrometer. As a result, 50 hybridoma cell lines producing monoclonal antibodies reactive against the CAPRIN-1 protein were obtained.
[00167] Next, from these monoclonal antibodies, antibodies reactive with the cell surfaces of breast cancer cells expressing CAPRIN-1 were selected. Specifically, 106 cells of the human breast cancer cell line MDA-MB-231V were subjected to centrifugation in a 1.5 ml microcentrifuge tube, and 100 pL of the culture supernatant from each of the above hybridomas was added to the tube. then the tube was left to stand on ice for 1 hour. After washing with PBS, a goat anti-mouse IgG (H + L) labeled with FITC (Invitrogen) diluted 500 times with PBS containing 0.1% SBF was added, then the resulting solution was left to stand on ice for 1 hour. After washing with PBS, the
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70/82 fluorescence intensity was measured using a FACScalibur (Becton, Dickinson & Company). Meanwhile, procedures similar to those mentioned above were performed using the serum of a 6-week-old Balb / c mouse that was not treated with the antibodies and that was diluted 500 times with hybridoma culture medium, so that a control sample was gotten. As a result, three monoclonal antibodies (monoclonal antibodies # 1, # 2 and # 3) were selected that exhibited stronger fluorescence intensity than that of the control, and that reacted with the cell surfaces of breast cancer cells.
Example 4 Characterization of Selected Antibodies (1) Cloning of genes from the variable regions of the mouse anti-CAPRIN-1 monoclonal antibody [00168] The mRNA was extracted from each hybridoma cell line that produces any of the three monoclonal antibodies selected in the Example 3. An RT-PCR method using primers specific for the mouse FR1 derived sequence and mouse FR4 derived sequence was performed for them, and the genes of the heavy chain variable regions (VH) and variable chain regions (VL) of all anti-CAPRIN-1 monoclonal antibodies were obtained. For the determination of the sequence, these genes were cloned into a vector pCR2.1 (Invitrogen).
(2) RT-PCR [00169] mRNA was prepared from 106 cells from each hybridoma cell line using an mRNA micro purification kit (GE HealthCare) RNA purification kit. The mRNA thus obtained was reverse transcribed and then the cDNA was synthesized using a cDNA ribbon synthesis kit "SuperScriptII 1st strand Synthesis Kit" (Invitrogen). These procedures were carried out in accordance with protocols attached to
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71/82 each kit.
[00170] The amplification of the antibody gene was performed by a PCR method using the obtained cDNA. To obtain the VH region gene, a primer (SEQ ID No: 54) specific for the mouse heavy chain FR1 sequence and a primer (SEQ ID No: 55) specific for the mouse heavy chain FR4 sequence were used. In addition, to obtain the VL region gene, a primer (SEQ ID No: 56) specific for the mouse light chain FR1 sequence and a primer (SEQ ID No: 57) specific for the FR4 light chain sequence of mouse were used. These primers were designed with the reference of Jones S.T. and Bending M.M. Bio / technology 9, 88-89 (1991). For PCR, Ex-Taq (Takara Bio Inc.) was used. A cDNA sample was added to 5 μl of 10 x Ex-Taq buffer, 4 μl of dNTP mix (2.5 mM), primers (1.0 μΜ) (2 μL each), and 0.25 μl of ExTaq (5 U / pL), then the total amount of the PCR mixture was adjusted with sterile water to 50 pL. PCR was performed under the following conditions: 2 minutes of treatment at 94 ° C, followed by 30 cycles of 1 minute denaturation at 94 ° C, 30 seconds of annealing at 58 ° C, and 1 minute of extension reaction at 72 ° C ° C.
(3) Cloning [00171] The PCR products thus obtained were subjected to electrophoresis on an agarose gel, and the DNA bands of the VH and VL regions were excised. The DNA fragments were purified using a “QIAquick Gel purification kit” (QIAGEN) purification kit according to the manufacturer's protocol. The purified DNA was cloned into the pCR2.1 vector using the TA cloning kit (Invitrogen). The ligated vector was transformed into DH5a competent cells (TOYOBO), according to a conventional method. 10 clones of each transformant were grown overnight in medium (100 pg / ml ampicillin) at 37 ° C, then the plasmid DNA was purified using
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72/82 a purification kit “Qiaspin Miniprep kit” (QIAGEN).
(4) Determination of the sequence.
[00172] The gene sequences of the VH region and the VL region in each plasmid obtained above were analyzed with an M13 forward primer (SEQ ID NO: 58) and an M13 reverse primer (SEQ ID NO: 59) in a fluorescence sequencer (DNA Sequencer 3130XL; ABI), using a sequencing kit “Big Dye Terminator Cycle Sequencing Kit” Ver3.1 (ABI) according to the manufacturer's protocols. As a result, each gene sequence was determined. The sequences were identical between the 10 clones.
[00173] The gene sequences encoding the monoclonal antibody variable heavy chain regions obtained are presented by SEQ ID NOS: 52, 70, and 68 and their amino acid sequences are presented by SEQ ID NOS: 43, 47, and 51 , and the sequences of genes encoding the monoclonal antibody variable light chain regions obtained are presented by SEQ ID NOS: 53 and 69, and their amino acid sequences are presented by SEQ ID NOS: 51 and 67. Specifically, it was revealed that monoclonal antibody # 1 comprises the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 51; monoclonal antibody # 2 comprises the heavy chain variable region of SEQ ID NO: 47 and the light chain variable region of SEQ ID NO: 51; and monoclonal antibody # 3 comprises the heavy chain variable region of SEQ ID NO: 63 and the light chain variable region of SEQ ID NO: 67.
Example 5 Identification of CAPRIN-1 Epitopes to be Recognized by Anti-CAPRIN-1 Monoclonal Antibodies # 1, # 2 and # 3 [00174] Using anti monoclonal antibodies
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CAPRIN-1 # 1 and # 2 (obtained in Example 3) reactive with the surfaces of cancer cells, regions of CAPRIN-1 epitopes have been identified to be recognized by the antibodies.
[00175] 93 candidate peptides, each consisting of 12 to 16 amino acids in the human CAPRIN-1 protein amino acid sequence, were synthesized, and then each peptide was dissolved in DMSO at a concentration of 1 mg / ml. Each peptide was dissolved in 0.1 M sodium carbonate buffer (pH 9.6) at a concentration of 30 pg / ml, added to a 96-well plate (Nunc, Product No. 436006) at 100 pL per well, then, the plate was left to stand at 4 ° C overnight. The solution was discarded, 10 mM ethanolamine / 0.1 M sodium carbonate buffer (pH 9.6) was added in a volume of 200 pL per well, then the resulting solution was left to stand at room temperature for 1 hour. The solution was then discarded and the plate was washed twice with PBS containing 0.5% Tween-20 (PBST), so that a plate on which each peptide was immobilized was prepared.
[00176] The cell culture supernatant containing the mouse monoclonal antibodies (# 1, # 2 and # 3) obtained in Example 3 was added in a volume of 50 pL per well, then the plate was stirred at room temperature for 1 hour. The solution was removed, followed by three washes with PBST. Then, a secondary antibody solution prepared with an anti-mouse IgG antibody labeled with HRP (Invitrogen) diluted 3000 to 4000 times with PBST was added (50 pL each) to the mouse monoclonal antibodies. The solution was removed, followed by six washes with PBST.
[00177] The TMB substrate solution (Thermo) was added in a volume of 100 pL per well and then left to stand for 15 to 30 minutes for the staining reaction. After color development, 100 pL of
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74/82 1N sulfonic acid was added to each well to stop the reaction, then the absorbances at 450 nm and 595 nm were measured using an absorption spectrometer. As a result, a polypeptide comprising the amino acid sequence of SEQ ID NO: 37 was identified as a partial CAPRIN-1 sequence recognized by all anti-CAPRIN-1 monoclonal antibodies # 1, # 2 and # 3.
[00178] Thus, it was revealed that the polypeptide of SEQ ID NO: 37 contains regions of epitopes for anti-CAPRIN-1 antibodies # 1, # 2 and # 3.
Example 6
CAPRIN-1 Expression on the Surface of Multiple Cancer Cells Using Anti-CAPRIN-1 Antibodies # 1, # 2 and # 3 [00179] Next, 7 breast cancer cell lines (MDA-MB-157, T47D , MRK-nu-1, MDA-MB-231V, BT20, SK-BR-3, and DA-MB231T) in which CAPRIN-1 gene expression was observed, and the remaining three breast cancer cell lines (MDA-MB-231c, MCF-7 and ZR75-1), 5 glioma cell lines (T98G, SNB19, U251, U87MG, and U373), 4 kidney cancer cell lines (Caki-1, Caki-2 , A498 and ACHN), 2 gastric cancer cell lines (MKN28 and MKN45), 5 colorectal cancer cell lines (HT29, LoVo, Caco2, SW480 and HCT116), 3 lung cancer cell lines (A549, QG56 and PC8), 4 leukemia cell lines (AML5, Namalwa, BDCM, RPI1788), a cervical cancer cell line (SW756), a bladder cancer cell line (T24), a cell line from esophageal cancer (KYSE180) and a cell line d and lymphoma (Ramos) were examined for CAPRIN-1 protein expression on the cell surfaces of each cell line using the culture supernatants containing # 1, # 2 and # 3 obtained in Example 3. 106 cells from each cell line cells were centrifuged in a 1.5 ml microcentrifuge tube. Each cell culture supernatant (100
Petition 870200015052, of 01/31/2020, p. 84/181
75/82 pL) containing # 1, # 2 or # 3 was added and then incubated on ice for 1 hour. After washing with PBS, a goat anti-mouse IgG (H + L) labeled with FITC (SouthernBiotech) diluted 500 times with PBS containing 0.1% SBF was added, then the resulting solution was left to stand ice for 1 hour. After washing with PBS, the fluorescence intensity was measured using a FACS Calibur (Becton, Dickinson & Company). The sample subjected to a reaction with only a secondary antibody was used as a negative control. As a result, cells to which antibodies # 1, # 2 and # 3 were added exhibited a stronger fluorescence intensity by 20% or more compared to the negative control. With these results it was revealed that the CAPRIN-1 protein was expressed on the cell membrane surfaces of the human cancer cell lines above. The percentage increase in fluorescence intensity above was expressed as a percentage of the increase in mean fluorescence intensity (MFI level) in each cell type and was calculated using the following formula.
[00180] Percentage increase in mean fluorescence intensity (percentage of increase in fluorescence intensity) (%) = ((MFI level in cells that reacted with human anti-CAPRIN-1 antibody) (MFI level of control)) / (MFI level of the control) x 100.
Example 7
Antitumor Effects (ADCC Activity and CDC Activity) of Anti-CAPRIN-1 Antibodies on Cancer Cells.
[00181] It was evaluated whether anti-CAPRIN-1 antibodies may or may not damage cancer cells that express CAPRIN-1 primarily by measuring ADCC activity. The evaluation was performed using a polyclonal antibody against the human CAPRIN-1 derived peptide (SEQ ID NO: 37) prepared in Example 1. 106 cells of the cancer cell line
Petition 870200015052, of 01/31/2020, p. 85/181
76/82 human breast MDA-MB-157, in which the expression of CAPRIN-1 was confirmed, were collected in a 50 ml centrifuge tube, 100 pCi of chromium-51 was added, and then, incubation was performed at 37 ° C for 2 hours. Then, the resultant was washed three times with RPMI1640 medium containing 10% fetal bovine serum. The cells were added to a 96-well V-bottom plate at a density of 103 cells per well. 1 pg of the polyclonal antibody against the human CAPRIN-1 derived peptide mentioned above was added to the wells, and then lymphocytes (2 x 105 each) separated from the peripheral rabbit blood were added and cultured for 4 hours at 37 ° C under 5% CO2 conditions. After culture, the amount of chromium (Cr) -51 released from the damaged cancer cells in the culture supernatant was measured, so that the ADCC activity of the polyclonal antibodies against the human CAPRIN-1 derived peptide against the cells was calculated. cancerous. As a result, a cytotoxic activity of 17.8% was observed against the MDA-MB-157 strain (see Fig. 2.). On the other hand, when similar procedures were performed using a control antibody (Example 1 (5)) prepared from peripheral blood of a rabbit not immunized with the antigen, and when the antibody was not added, almost no activity was observed (see Fig. 2). Thus, it has been revealed that anti-CAPRIN-1 antibodies can damage cancer cells that express CAPRIN-1.
[00182] In the present case, cytotoxic activity was determined as a cytotoxic activity against a cancer cell line. Specifically, as described above, the result was obtained by mixing an anti-CAPRIN-1 antibody to be used in the present invention, a rabbit lymphocyte, and 103 cells from each cancer cell line that had incorporated chromium-51, culturing the cells for 4 hours, measuring the amount of chromium-51 released into the culture medium, and then calculating
Petition 870200015052, of 01/31/2020, p. 86/181
77/82 cytotoxic activity against the cancer cell line using the following formula *:
* Formula: cytotoxic activity (%) = (the amount of chromium-51 released from cancer cells after the addition of an antiCAPRIN-1 antibody and rabbit lymphocytes) / (amount of chromium-51 released from target cells to which 1N hydrochloric acid was added) x 100.
[00183] Next, mouse monoclonal antibodies anti-CAPRIN-1 # 1, # 2, and # 3 (obtained in Example 3), were evaluated for their cytotoxic activity against cancer cells. Each culture supernatant of antibody producing cells # 1, # 2 or # 3 was purified using a “Hitrap ProteinA Sepharose FF” column (GE Healthcare), subjected to buffer replacement with PBS (-), and then filtered with a 0.22 pm filter (Millipore). The results were used as antibodies to measure activity. 106 cells of the human breast cancer cell line MDA-MB-157 were collected in a 50 ml centrifuge tube, 100 pCi chromium-51 was added, and then incubated at 37 ° C for 2 hours. . Subsequently, the resulting product was washed three times with RPMI 1640 medium containing 10% SBF. The cells were added to a 96-well V-bottom plate at a density of 103 cells per well for use as target cells. The above purified antibodies (1 pg each) were added to the cells. 5x104 mouse spleen cells isolated from the spleen of a 6-week-old BALB / C mouse (Japan SLC Inc.) according to a conventional method, were additionally added and then cultured for 4 hours at 37 ° C under the 5% CO2 conditions. After culture, the amount of chromium-51 released from damaged tumor cells in a culture supernatant was measured, and the cytotoxic activity of each anti-CAPRIN-1 antibody against cancer cells was calculated. As negative control samples, a sample was used
Petition 870200015052, of 01/31/2020, p. 87/181
78/82 prepared by adding PBS instead of antibodies and a sample prepared by adding an isotypic control antibody instead of antibodies. As a result, antibodies # 1, # 2 and # 3 exhibited more than 26% cytotoxic activity against the MDA-MB-157 strain. In contrast, the activity of the negative control sample prepared by adding PBS and the activity in the negative control sample prepared by adding isotypic control antibody were 2.0% and 2.8%, respectively. Likewise, antibodies # 1, # 2 and # 3 were evaluated for their cytotoxic activities against other cancer cells, including U373 and T98G glioma cell lines, lung cancer cell lines A549 and QG56, cell lines renal cancer Caki-1 and ACHN, cervical cancer cell line SW756, bladder cancer cell line T24, esophageal cancer cell line KYSE180, gastric cancer cell line MKN28 and MKN45, cell line colorectal cancer SW480, AML5 leukemia cell line, and a Ramos lymphoma cell line. As a result, antibody # 1 exhibited 11.2% activity against T98G (6.4% in the case of isotypic control), 13.3% activity against U373 (4.3% in the case of isotypic control), 20 , 8% activity against A549 (4.5% in the case of isotypic control), 21.3% activity against QG56 (5.3% in the case of isotypic control), 15.9% activity against Caki-1 (4.5% in the case of isotypic control), 14.7% of activity against ACHN (3.8% in the case of isotypic control), 13.5% of activity against SW756 (5.1% in the case of isotypic control), 11.6 % activity against T24 (3.8% in the case of isotypic control), 16.2% activity against KYSE180 (3.7% in the case of isotypic control), 12.8% activity against MKN28 (4.2% in the case of isotypic control), 13.4% of activity against MKN45 (4.6% in the case of isotypic control), 12.4% of activity against SW480 (6.4% in the case of isotypic control), 10.3 % of activity against AML5 (4.7% in the case of isotypic control), and 7.8% of activity against Ramos (2.6% in the case of control) le isotypic). In addition,
Petition 870200015052, of 01/31/2020, p. 88/181
79/82 antibodies # 2 and # 3 showed similar results. It was demonstrated by the results above that the anti-CAPRIN-1 # 1, # 2 and # 3 antibodies obtained cause damage to cancer cells that express CAPRIN-1 through ADCC activity.
[00184] It was demonstrated by the results above that the mouse monoclonal antibodies anti-CAPRIN-1 # 1, # 2 and # 3 thus obtained cause damage to cancer cells that express CAPRIN-1 through ADCC activity.
[00185] In the present case, cytotoxic activity was determined as a cytotoxic activity against a cancer cell line. Specifically, as described above, the result was obtained by mixing an anti-CAPRIN-1 antibody to be used in the present invention, a mouse splenocyte, and 103 cells from each cancer cell line that had incorporated chromium-51, culturing the cells for 4 hours, measuring the amount of chromium-51 released into the culture medium, and then cytotoxic activity against the cancer cell line was calculated using the following formula *:
* Formula: cytotoxic activity (%) = (the amount of chromium-51 released from cancer cells after the addition of an antiCAPRIN-1 antibody and mouse splenocytes) / (amount of chromium-51 released from target cells to which 1N hydrochloric acid was added) x 100.
[00186] Then, monoclonal antibodies # 1, # 2 and # 3 antiCAPRIN-1 were evaluated for their cytotoxicity activities (CDC activity) against cancer cells. Blood collected from a rabbit was added to an Eppendorf tube and left to stand at room temperature for 60 minutes, then subjected to 5 minutes of centrifugation at 3000 rpm. In this way, the serum for measuring CDC activity was prepared. 105 cells of the human breast cancer cell line MDA-MB-231V were
Petition 870200015052, of 01/31/2020, p. 89/181
80/82 collected in a 50 ml centrifuge tube, 100 pCi of chromium 51 was added, and the incubation was carried out at 37 ° C for 2 hours. The resulting product was washed three times with RPMI 1640 medium containing 10% SBF. Subsequently, the cells were suspended in RPMI medium containing the rabbit serum prepared above (50%), and then the cells were added to a 96-well plate with V bottom at a density of 103 cells per well. 1 pg of each of the # 1 and # 2 mouse monoclonal antibodies was added to the cells, then the cells were cultured for 4 hours at 37 ° C under 5% CO2 conditions. After culture, the amount of chromium51 released from damaged tumor cells in a culture supernatant was measured, and the CDC activity of each antibody against MDA-MB-213V cells was calculated. As a result, both antibodies # 1 and # 2 exhibited more than 21% CDC activity. In addition, no cytotoxic activity was observed in the negative control group to which no antibody was added. Thus, it has been revealed that antibodies # 1 and # 2 can damage tumor cells that express CAPRIN-1 also by CDC activity.
[00187] In the present case, the cytotoxic activity was determined as a cytotoxic activity against a cancer cell line. Specifically, as described above, the result was obtained by mixing an anti-CAPRIN-1 antibody to be used in the present invention, a serum, and 103 cells from each cancer cell line that had incorporated chromium-51, the culture cells for 4 hours, measuring the amount of chromium51 released into the culture medium, and then cytotoxic activity against the cancer cell line was calculated using the following formula *:
* Formula: cytotoxic activity (%) = (the amount of chromium-51 released from cancer cells after adding an antiCAPRIN-1 antibody and serum) / (amount of chromium-51 released from target cells to which 1N hydrochloric acid was added) x 100.
Petition 870200015052, of 01/31/2020, p. 90/181
81/82 [00188] Next, the anti-CAPRIN-1 # 1 and # 2 mouse monoclonal antibodies obtained were evaluated for their in vivo antitumor effects in mice grafted with tumor. The antibodies used in the present were prepared by column purification of the culture supernatant from each cell producing # 1 or # 2 in the same manner as described above.
[00189] The anti-tumor effects of antibodies # 1 and # 2 were examined using tumor-bearing mice in which a mouse-derived cancer cell line expressing CAPRIN-1 was transplanted. 4T1 cells (acquired from ATCC) were transplanted subcutaneously in the dorsal region of 30 Balb / c mice (SLC Japan Inc.) at a density of 5x105 cells per mouse. Tumors were allowed to grow to a size of about 5 mm in diameter. Antibodies # 1 and # 2 were administered intraperitoneally in 20 of the 30 tumor-bearing mice, in an amount of 200 pg (200 pL) / mouse (each antibody was administered to 10 mice). Subsequently, the same amount of antibody was administered intraperitoneally to each tumor-bearing mouse a total of 3 times within 2 days. Tumor sizes were measured every day and the anti-tumor effects were examined by observation. Meanwhile, as a control group, PBS (-) was administered instead of antibodies to the 10 remaining tumor-bearing mice. The tumor size was calculated as a volume using the formula: major axis length x minor axis length x minor axis length x 0.5.
[00190] As a result of observing the anti-tumor effects, it was observed that the tumors almost completely regressed by day 16 after administration of the antibodies in the examination group to which the mouse monoclonal antibodies anti-CAPRIN-1 # 1 and # 2 were
Petition 870200015052, of 01/31/2020, p. 91/181
82/82 administered. On the other hand, in the control group to which PBS was administered (), the tumors had an increase of about 820% on day 12. It was demonstrated by the results that mouse monoclonal antibodies anti-CAPRIN-1 # 1 and # 2 obtained exhibit strong anti-tumor effects in vivo against cancer cells that express CAPRIN-1.
Industrial Applicability [00191] The antibodies of the present invention are useful for treating and / or preventing cancer.
[00192] All publications, patents and patent applications cited in the present are fully incorporated into the present by reference.
Free Text String Listing [00193] SEQ ID NO: 31: T3 primer.
[00194] SEQ ID NO: 32: T7 primer.
[00195] SEQ ID NOS: 33 and 34: primers.
[00196] SEQ ID NOS: 35 and 36: primers for GAPDH.
[00197] SEQ ID NOS: 38 and 39: primers.
Petition 870200015052, of 01/31/2020, p. 92/181

权利要求:
Claims (8)
[1]
Claims
1. MONOCLONAL ANTIBODY, or fragment thereof, characterized by comprising a heavy chain variable region comprising SEQ ID NOS: 40, 41, and 42 and a light chain variable region comprising SEQ ID NOS: 48, 49, and 50; a heavy chain variable region comprising SEQ ID NOS: 44, 45, and 46 and a light chain variable region comprising SEQ ID NOS: 48, 49, and 50; or a heavy chain variable region comprising SEQ ID NOS: 60, 61 and 62 and a light chain variable region comprising SEQ ID NOS: 64, 65, and 66; and have cytotoxic activity against a cancer cell expressing a CAPRIN-1 protein.
[2]
2. PHARMACEUTICAL COMPOSITION, characterized by comprising the antibody or fragment thereof, as defined in the claim
1, as an active ingredient, and a pharmaceutically acceptable carrier.
[3]
PHARMACEUTICAL COMPOSITION, according to claim 2, characterized in that it also comprises an antitumor agent.
[4]
4. PHARMACEUTICAL COMBINATION, characterized by comprising the pharmaceutical composition, as defined in the claim
2, and a pharmaceutical composition comprising an anti-tumor agent.
[5]
5. USE OF AN ANTIBODY, or fragment thereof, as defined in claim 1, characterized by being for the manufacture of a drug to treat and / or prevent cancer.
[6]
6. USE OF A PHARMACEUTICAL COMPOSITION, as defined in any of claims 2 to 4, characterized by being for the manufacture of a drug to treat and / or prevent cancer.
[7]
7. USE OF A PHARMACEUTICAL COMBINATION, as defined in claim 4, characterized by being for the manufacture of a drug to treat and / or prevent cancer.
Petition 870200015052, of 01/31/2020, p. 93/181
2/2
[8]
8. USE, according to any one of claims 5 to
7, characterized by cancer being breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mast cell tumor, kidney cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer or colorectal cancer.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-05-28| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-07-09| B06T| Formal requirements before examination|

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2019-11-05| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|

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2020-03-10| B09A| Decision: intention to grant|

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2020-05-12| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 04/02/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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2020-06-09| B16C| Correction of notification of the grant|Free format text: REF. RPI 2575 DE 12/05/2020 QUANTO AO ENDERECO E A PRIORIDADE UNIONISTA. |

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2020-06-23| B16C| Correction of notification of the grant|Free format text: REF. RPI 2575 DE 12/05/2020 QUANTO A PRIORIDADE UNIONISTA |

优先权:

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申请号 | 申请日 | 专利标题

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JP2010-023453|2010-02-04|

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JP2010023453|2010-02-04|

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JP2010-183116|2010-08-08|

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JP2010183161|2010-08-18|

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PCT/JP2011/052412|WO2011096533A1|2010-02-04|2011-02-04|Pharmaceutical composition for treatment and/or prevention of cancer|

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