Pharmaceutical composition for treating and preventing cancer

Abstract

The present invention relates to a pharmaceutical composition for the treatment and / or prevention of cancer, comprising as active ingredient an antibody or a fragment thereof having immunological reactivity with a CAPRIN-1 protein or a fragment of said protein comprising 7 or more contiguous amino acids.

Description

[0001] This application is a divisional application of Chinese patent application 200980138959.9, the original application being filed on August 5, 2009, with the invention title "Pharmaceutical Composition for Treating and Preventing Cancer". Invention Field

[0002] This invention relates to novel pharmaceutical uses of anti-CAPRIN-1 antibodies or fragments thereof, for example, as therapeutics and / or preventative agents for cancer. Background Technology

[0003] Cancer is a leading cause of death. Currently, cancer treatment primarily involves surgery, which can be combined with radiotherapy and chemotherapy. Despite the development of new surgical methods and the discovery of new anticancer agents in recent years, the outcomes of cancer treatment have not significantly improved, except for a few types of cancer. With the advancements in molecular biology and cancer immunology in recent years, antibodies that specifically react to cancer, cancer antigens recognized by cytotoxic T cells, and genes encoding cancer antigens have been identified, and expectations for antigen-specific immunotherapy have risen (Tsuyoshi AKIYOSHI, "Gan to Kagaku Ryouhou (Cancer and Chemotherapy)," 1997, Vol. 24, pp. 551-519 (Japan), (Cancer and Chemotherapy Publishers Inc., Japan)).

[0004] To mitigate side effects, peptides, polypeptides, or proteins that need to be recognized as target antigens in cancer treatments are not present in almost all normal cells but are specifically found in cancer cells. In 1991, Boon et al. at the Ludwig Institute in Belgium isolated the human melanoma antigen MAGE1, recognized by CD8-positive T cells, using a cDNA expression cloning method with autologous cancer cell lines and cancer-reactive T cells (Bruggen P. et al., Science, 254:1643-1647, 1991). Subsequently, the SEREX method (serological identification of antigens by recombinant expression cloning) was reported, in which gene expression cloning technology can be used to identify tumor antigens that can be recognized by antibodies produced in response to autologous cancer in the body of cancer patients (Proc. Natl. Acad. Sci. USA, 92:11810-11813, 1995; and US Patent No. 5,698,396). Using the SEREX method, some cancer antigens that are not expressed in normal cells but are specifically expressed in cancer cells have been isolated (Int. J. Cancer, 72:965-971 (1997); Cancer Res., 58:1034-1041 (1998); Int. J. Cancer, 29:652-658 (1998); Int. J. Oncol., 14:703-708 (1999); Cancer Res., 56:4766-4772 (1996); and Hum. Mol. Genet 6:33-39, 1997). Furthermore, several clinical trials have been conducted using immune cells that specifically react with cancer antigens (which are some isolated cancer antigens), and using vaccines containing cancer antigens, etc., to induce cancer-specific immunotherapy.

[0005] Meanwhile, in recent years, various antibody drugs targeting antigenic proteins on cancer cells have emerged for cancer treatment. These drugs, used as cancer-specific therapeutic agents, have shown some efficacy and have therefore attracted attention. However, most target antigen proteins are also expressed in normal cells. Administration of antibodies can damage not only cancer cells but also normal cells that have expressed the target antigens, causing side effects (or adverse reactions), which is a problem. Therefore, it is anticipated that if cancer antigens specifically expressed on the surface of cancer cells can be identified, and antibodies targeting these antigens can be used as drugs, treatment with fewer side effects will be possible.

[0006] Cytoplasmic and proliferation-associated protein 1 (CAPRIN-1) is an intracellular protein expressed when quiescent normal cells are activated or undergo cell division. CAPRIN-1 is also known to be involved in the formation of intracellular stress granules from RNA in cells and is involved in the regulation of mRNA transport and translation. CAPRIN-1 has different names, such as GPI-anchored membrane protein 1 and membrane component surface marker 1 protein (M11S1), which suggests that this protein is considered a membrane protein. These different names stem from reports that the CAPRIN-1 gene sequence originally contained a GPI-binding region and that CAPRIN-1 is a membrane protein expressed in colorectal cancer cells (J. Biol. Chem., 270:20717-20723, 1995). Later reports indicated that the CAPRIN-1 gene sequence described in the report was incorrect. Specifically, a single nucleotide deletion in the CAPRIN-1 gene sequence currently registered in GenBank et al. caused a frameshift, resulting in the deletion of 80 amino acids from the carboxyl terminus. The resulting artificial product (74 amino acids) was the GPI-binding region mentioned in the aforementioned report. Furthermore, an error existed on the 5' side of this sequence, resulting in the deletion of 53 amino acids from the amino terminus (J. Immunol., 172:2389-2400, 2004). In addition, it has been reported that the protein encoded by the CAPRIN-1 gene sequence currently registered in GenBank et al. is not a cell membrane protein (J. Immunol., 172:2389-2400, 2004).

[0007] Furthermore, based on the report in J. Biol. Chem., 270:20717-20723, 1995 that CAPRIN-1 is a cell membrane protein, US 2008 / 0075722 and WO 2005 / 100998 disclose that CAPRIN-1, named M11S1, can be used as a target for cancer therapeutic antibody drugs and is classified as a cell membrane protein; however, the examples do not include a description of applying antibodies against this protein to treat cancer. However, as reported in J. Immunol., 172:2389-2400, 2004, since the application of US 2008 / 0075722 until now, it has been generally believed that CAPRIN-1 is not expressed on the cell surface, and therefore, it is clear that the disclosures of US 2008 / 0075722 and WO 2005 / 100998, based solely on the incorrect information that CAPRIN-1 is a cell membrane protein, should not be construed as common knowledge to those skilled in the art. Invention Overview

[0008] The problem that the invention aims to solve

[0009] One objective of this invention is to identify cancer antigen proteins specifically expressed on the surface of cancer cells, and to provide the use of antibodies targeting such proteins as cancer therapeutics and / or preventative agents.

[0010] Problem-solving methods

[0011] Through in-depth research, using the SEREX method and employing a cDNA library derived from testicular tissue and serum from dogs with breast cancer, the inventors have obtained cDNA encoding a protein that binds to antibodies present in the serum of tumor-bearing organisms. Using the obtained canine gene and homologous human, bovine, equine, mouse, and chicken genes, the CAPRIN-1 protein with the amino acid sequence shown in SEQ ID NO:2-30 (i.e., SEQ ID NO:2-30 with even numbers) and antibodies against the CAPRIN-1 protein have been prepared. Furthermore, the inventors have discovered that CAPRIN-1 is specifically expressed in breast cancer, brain tumors, leukemia, lymphoma, lung cancer, esophageal cancer, colorectal cancer, gastric cancer, and kidney cancer cells, and that a portion of the CAPRIN-1 protein is specifically expressed on the surface of these tumor cells. Moreover, the present invention has found that antibodies against the portion of CAPRIN-1 expressed on the surface of cancer cells can damage (impair) cancer cells expressing CAPRIN-1. These findings led to the completion of this invention.

[0012] Therefore, the present invention has the features described below.

[0013] The present invention provides a pharmaceutical composition for treating and / or preventing cancer, comprising, as an active ingredient, an antibody or a fragment thereof having immunoreactivity with CAPRIN-1 protein, or with a CAPRIN-1 protein fragment comprising seven or more consecutive amino acids, wherein the CAPRIN-1 protein has an amino acid sequence shown in any one of SEQ ID NO: 2-30 with even-numbered numbers, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence of any one of SEQ ID NO: 2-30 with even-numbered numbers.

[0014] In one embodiment of the invention, the cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, esophageal cancer, colorectal cancer, stomach cancer, and kidney cancer.

[0015] In another embodiment of the invention, the antibody is a monoclonal antibody or a polyclonal antibody.

[0016] In another embodiment of the present invention, the antibody is a human antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, or a bispecific antibody.

[0017] In another embodiment of the invention, the antibody is an antibody having immunological reactivity with a polypeptide or a fragment thereof, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO:37 or SEQ ID NO:136, or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence.

[0018] In another embodiment of the invention, in a pharmaceutical composition for treating and / or preventing cancer comprising an antibody as an active ingredient, the antibody is any one of the following antibodies (a) to (k) and has an immunological reactivity with CAPRIN-1 protein:

[0019] (a) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:40, 41 and 42, and a light chain variable region comprising the sequences shown in SEQ ID NO:44, 45 and 46;

[0020] (b) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:40, 41 and 42, and a light chain variable region comprising the sequences shown in SEQ ID NO:50, 51 and 52;

[0021] (c) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:40, 41 and 42, and a light chain variable region comprising the sequences shown in SEQ ID NO:55, 56 and 57;

[0022] (d) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:40, 41 and 42, and a light chain variable region comprising the sequences shown in SEQ ID NO:60, 61 and 62;

[0023] (e) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:40, 41 and 42, and a light chain variable region comprising the sequences shown in SEQ ID NO:65, 66 and 67;

[0024] (f) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:70, 71 and 72, and a light chain variable region comprising the sequences shown in SEQ ID NO:74, 75 and 76;

[0025] (g) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:80, 81 and 82, and a light chain variable region comprising the sequences shown in SEQ ID NO:84, 85 and 86;

[0026] (h) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:90, 91 and 92, and a light chain variable region comprising the sequences shown in SEQ ID NO:94, 95 and 96;

[0027] (i) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:100, 101 and 102, and a light chain variable region comprising the sequences shown in SEQ ID NO:104, 105 and 106;

[0028] (j) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:110, 111 and 112, and a light chain variable region comprising the sequences shown in SEQ ID NO:114, 115 and 116;

[0029] (k) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:120, 121 and 122, and a light chain variable region comprising the sequences shown in SEQ ID NO:124, 125 and 126.

[0030] Invention Effects

[0031] The anti-CAPRIN-1 antibody used in this invention damages (or destroys) cancer cells. Therefore, such anti-CAPRIN-1 antibodies can be used to treat or prevent cancer. Brief description of the attached diagram

[0032] Figure 1 The expression patterns of genes encoding the CAPRIN-1 protein are shown in normal tissues and tumor cell lines. In this figure, reference numeral 1 represents the expression pattern of each gene encoding the CAPRIN-1 protein, and reference numeral 2 represents the expression pattern of the GAPDH gene.

[0033] Figure 2 The cytotoxic activity of anti-CAPRIN-1 antibody (or anti-CAPRIN-1 antibody) against the CAPRIN-1 gene-expressing breast cancer cell line (T47D) is shown in the figure. In this figure, reference numeral 3 shows the activity after the addition of anti-CAPRIN-1 antibody, reference numeral 4 shows the activity after the addition of a control antibody, and reference numeral 5 shows the activity in the absence of any antibody.

[0034] Figure 3The cytotoxic activity of the anti-CAPRIN-1 antibody (or anti-CAPRIN-1 antibody) against the CAPRIN-1 gene-expressing breast cancer cell line (MDA-MB-157) is shown. In this figure, reference number 6 shows the activity after the addition of the anti-CAPRIN-1 antibody, reference number 7 shows the activity after the addition of the control antibody, and reference number 8 shows the activity in the absence of any antibody.

[0035] Figure 4 Cytotoxicity against the MDA-MB-157 breast cancer cell line expressing the CAPRIN-1 gene was demonstrated, wherein the cytotoxicity was demonstrated by anti-CAPRIN-1 monoclonal antibodies (i.e., monoclonal antibodies #1 to #11) that react with the surface of cancer cells. Specifically, this figure shows the activity levels after adding anti-CAPRIN-1 monoclonal antibody #1 (reference number 9), anti-CAPRIN-1 monoclonal antibody #2 (reference number 10), anti-CAPRIN-1 monoclonal antibody #3 (reference number 11), anti-CAPRIN-1 monoclonal antibody #4 (reference number 12), anti-CAPRIN-1 monoclonal antibody #5 (reference number 13), anti-CAPRIN-1 monoclonal antibody #6 (reference number 14), anti-CAPRIN-1 monoclonal antibody #7 (reference number 15), anti-CAPRIN-1 monoclonal antibody #8 (reference number 16), anti-CAPRIN-1 monoclonal antibody #9 (reference number 17), anti-CAPRIN-1 monoclonal antibody #10 (reference number 18), and anti-CAPRIN-1 monoclonal antibody #11 (reference number 19); the activity level after adding a monoclonal antibody that reacts with the CAPRIN-1 protein itself but not with the surface of cancer cells (reference number 20); and the activity level after adding PBS instead of each antibody (reference number 21).

[0036] Figures 5a to 5cThe antitumor activity of anti-CAPRIN-1 monoclonal antibodies (i.e., monoclonal antibodies #1 to #11) reacting with the surface of cancer cells in Balb / c mice transplanted with the CAPRIN-1-expressing mouse cancer CT26 cell line is shown. These figures show the administration of anti-CAPRIN-1 monoclonal antibody #1 (reference number 22), anti-CAPRIN-1 monoclonal antibody #2 (reference number 23), anti-CAPRIN-1 monoclonal antibody #3 (reference number 24), anti-CAPRIN-1 monoclonal antibody #4 (reference number 25), anti-CAPRIN-1 monoclonal antibody #5 (reference number 26), anti-CAPRIN-1 monoclonal antibody #6 (reference number 27), anti-CAPRIN-1 monoclonal antibody #7 (reference number 28), and anti-C... Tumor size in mice after administration of APRIN-1 monoclonal antibody (reference number 29), #9 anti-CAPRIN-1 monoclonal antibody (reference number 30), #10 anti-CAPRIN-1 monoclonal antibody (reference number 31), and #11 anti-CAPRIN-1 monoclonal antibody (reference number 32), tumor size in mice after administration of monoclonal antibodies that react with CAPRIN-1 protein itself but not with the surface of cancer cells (reference number 33), and tumor size in mice after administration of PBS in place of each antibody (reference number 34).

[0037] Figures 6a to 6c The antitumor activity of anti-CAPRIN-1 monoclonal antibodies (i.e., monoclonal antibodies #1 to #11) reacting with the surface of cancer cells in Balb / c mice transplanted with a mouse cancer N1E cell line expressing CAPRIN-1 is shown. These figures show the administration of anti-CAPRIN-1 monoclonal antibody #1 (reference number 35), anti-CAPRIN-1 monoclonal antibody #2 (reference number 36), anti-CAPRIN-1 monoclonal antibody #3 (reference number 37), anti-CAPRIN-1 monoclonal antibody #4 (reference number 38), anti-CAPRIN-1 monoclonal antibody #5 (reference number 39), anti-CAPRIN-1 monoclonal antibody #6 (reference number 40), anti-CAPRIN-1 monoclonal antibody #7 (reference number 41), and anti-C... Tumor size in mice after administration of APRIN-1 monoclonal antibody (reference number 42), #9 anti-CAPRIN-1 monoclonal antibody (reference number 43), #10 anti-CAPRIN-1 monoclonal antibody (reference number 44), and #11 anti-CAPRIN-1 monoclonal antibody (reference number 45), tumor size in mice after administration of monoclonal antibodies that react with CAPRIN-1 protein itself but not with the surface of cancer cells (reference number 46), and tumor size in mice after administration of PBS in place of each antibody (reference number 47).

[0038] Implementation schemes of the present invention

[0039] As described below, the antitumor activity of antibodies against any of the even-numbered peptides in SEQ ID NO: 2-30 used in this invention can be evaluated by examining tumor growth inhibition in tumor-bearing animals in vivo or by examining in vitro whether they exhibit immune cell-mediated or complement-mediated cytotoxic activity against tumor cells expressing the following peptides.

[0040] In addition, the polynucleotide sequences encoding proteins consisting of the amino acid sequences shown in SEQ ID NO:2 to 30 (i.e., SEQ ID NO:2, 4, 6...28 and 30) are shown in SEQ ID NO:1 to 29 (i.e., SEQ ID NO:1, 3, 5...27 and 29) with odd numbers.

[0041] The amino acid sequences shown in SEQ ID NO: 6, 8, 10, 12, and 14 of the sequence listing disclosed in this invention are the amino acid sequences of the CPRIN-1 protein, which are isolated by the SEREX method using a cDNA library derived from canine testicular tissue and serum from dogs with mammary cancer, as a polypeptide that can bind to antibodies specifically present in the serum of tumor-bearing dogs; the amino acid sequences shown in SEQ ID NO: 2 and 4 are the amino acid sequences of the CPRIN-1 protein isolated as a human homolog of the canine polypeptide; the amino acid sequence shown in SEQ ID NO: 16 is the amino acid sequence of the CPRIN-1 protein isolated as a bovine homolog of the canine polypeptide; the amino acid sequence shown in SEQ ID NO: 18 is the amino acid sequence of the CPRIN-1 protein isolated as an equine homolog of the canine polypeptide; the amino acid sequences shown in SEQ ID NO: 20 to 28 (even numbers) are the amino acid sequences of the CPRIN-1 protein isolated as a murine homolog of the canine polypeptide; and SEQ ID The amino acid sequence shown in NO:30 is the amino acid sequence of the CPRIN-1 protein isolated as a chicken homolog of the canine polypeptide (see Example 1 described below). CAPRIN-1 is known to be expressed when normal cells in a quiescent phase are activated or undergo cell division.

[0042] It is known that CAPRIN-1 is not expressed on the cell surface. However, as a result of examinations related to this invention, it has now been revealed that certain portions of CAPRIN-1 are expressed on the surface of various cancer cells. According to the invention, it is preferred to use an antibody that binds to the portion of the CAPRIN-1 protein expressed on the surface of cancer cells. Examples of peptides in the CAPRIN-1 protein expressed on the surface of cancer cells include polypeptides consisting of seven or more consecutive amino acids in the region of amino acid residue No. (or amino acid (aa)) 50-98, or amino acid residue No. (aa): 233-305, of any of the even-numbered amino acid sequences shown in SEQ ID NO:2 to 30 (excluding SEQ ID NO:6 and 18). Specific examples include amino acid sequences shown in SEQ ID NO:37 or 136 (preferably, the region of amino acid sequence shown in SEQ ID NO:137 or 138 in the amino acid sequence shown in SEQ ID NO:136), or amino acid sequences having 80% or more, preferably 85% or more, more preferably 90% or more, and more preferably 95% or more sequence identity with said amino acid sequence. The antibodies of the present invention include all antibodies that can bind to the above-mentioned peptides and have anti-tumor activity.

[0043] The anti-CAPRIN-1 antibodies used in this invention as described above can be of any type, as long as they exhibit antitumor activity. Examples include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies (scFV), and fragments thereof, such as Fab and F(ab')2. These antibodies and fragments thereof can be prepared by methods known in the art. In this invention, antibodies capable of specifically binding to the CAPRIN-1 protein are desired. Such antibodies are preferably monoclonal antibodies; however, polyclonal antibodies may also be used as long as homologous antibodies can be stably produced. Furthermore, if the subject is human, human antibodies or humanized antibodies are desired to avoid or suppress immune rejection.

[0044] The term "specifically binds to CAPRIN-1 protein" as used in this article refers to the target antibody specifically binding to the CAPRIN-1 protein and essentially not binding to other proteins.

[0045] As described below, the antitumor activity of the antibodies used in this invention can be evaluated by examining tumor growth inhibition in tumor-bearing animals in vivo, or by examining in vitro whether they exhibit immune cell-mediated or complement-mediated cytotoxic activity against tumor cells expressing the polypeptide.

[0046] Furthermore, the subjects for cancer treatment and / or prevention requiring the present invention are mammals, such as humans, pets, livestock, or racing animals. Humans are the preferred subjects.

[0047] The preparation of antigens, antibodies, and pharmaceutical compositions related to this invention will be explained below.

[0048] Preparation of antigens used to prepare antibodies

[0049] The proteins or fragments thereof used as sensitizing antigens to obtain anti-CAPRIN-1 antibodies in this invention are not limited to their source, such as animals, e.g., humans, dogs, cattle, horses, mice, rats, and chickens. However, such antibodies or fragments thereof are preferably selected based on their compatibility with parental cells used for cell fusion. Proteins derived from mammals are generally preferred, and proteins derived from humans are particularly preferred. For example, if CAPRIN-1 is human CAPRIN-1, human CAPRIN-1 protein, its partial peptides, or cells capable of expressing human CAPRIN-1 can be used.

[0050] For example, the nucleotide and amino acid sequences of human CAPRIN-1 and its homologs can be obtained by logging into GenBank (NCBI, USA) and applying the BLAST or FASTA algorithm (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997).

[0051] According to the present invention, when the nucleotide sequence (SEQ ID NO: 1 or 3) or amino acid sequence (SEQ ID NO: 2 or 4) of human CAPRIN-1 is used as the base sequence, the target is each nucleic acid or protein composed of a sequence having 70%-100%, preferably 80%-100%, more preferably 90%-100%, and more preferably 95%-100% (e.g., 97%-100%, 98%-100%, 99%-100%, or 99.5%-100%) sequence identity with the ORF or mature portion of the base nucleotide sequence or amino acid sequence. As used herein, the term "% sequence identity" refers to the percentage (%) of the number of identical amino acids (or nucleotides) to the total number of amino acids (or nucleotides) when cleavage is introduced or not introduced to align two sequences to achieve maximum similarity.

[0052] The fragment length of the CAPRIN-1 protein is from the length of the epitope (or antigenic determinant) amino acid of the smallest unit of the antigen recognized by the antibody to less than the full length of the protein. The epitope refers to a polypeptide fragment that is antigenic or immunogenic in mammals, preferably humans. The smallest unit of this polypeptide fragment consists of about 7 to 12 amino acids, for example, 8 to 11 amino acids. Specific examples are the amino acid sequences shown in SEQ ID NO:37, SEQ ID NO:137, or SEQ ID NO:138, or amino acid sequences having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with said amino acid sequences.

[0053] Peptides containing the aforementioned human CAPRIN-1 protein and some of its peptides can be synthesized using chemical synthesis methods, such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (tert-butyloxycarbonyl method) (Japanese Biochemical Society, Biochemistry Experiment Tutorial (Seikagaku Jikken Kouza) 1, Protein Chemistry IV, Chemical Modification and Peptide Synthesis, Kagaku-dojin Publishing Company, Inc., Japan, 1981). Alternatively, the peptides can be synthesized using conventional techniques using various commercially available peptide synthesizers. Furthermore, polynucleotides encoding the aforementioned polypeptides can be prepared using known genetic engineering methods (Sambrook et al., Molecular Cloning, 2nd ed., Current Protocols in Molecular Biology (1989), ColdSpring Harbor Laboratory Press; Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., A Compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons et al.), each polynucleotide is integrated into an expression vector, and the vector is introduced into a host cell, thereby allowing the host cell to produce the polypeptide, thus obtaining the target polypeptide. The desired polypeptide can be obtained through such methods.

[0054] Polynucleotides encoding the aforementioned polypeptides can be readily prepared using known genetic engineering techniques or conventional techniques using commercially available nucleic acid synthesizers. For example, DNA containing the nucleotide sequence shown in SEQ ID NO:1 can be prepared by PCR using a human chromosomal DNA or cDNA library as a template and a primer pair designed to amplify the nucleotide sequence shown in SEQ ID NO:1. PCR conditions can be appropriately determined. For example, such conditions may include 30 cycles of the following reaction steps (as one cycle): 94°C for 30 seconds (denaturation); 55°C for 30 seconds to 1 minute (annealing); and at 72°C for 2 minutes (extension), using a thermostable DNA polymerase (e.g., Taq polymerase) and a Mg2+-containing... 2+ The PCR buffer was then used, followed by 30 cycles and incubated at 72°C for 7 minutes. However, the present invention is not limited to the exemplary PCR conditions described above. PCR techniques and conditions are described, for example, 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).

[0055] Additionally, appropriate probes and primers can be prepared based on the nucleotide and amino acid sequences shown in SEQ ID NO:1 to 30 of the sequence listing described herein, and human cDNA libraries can be screened using such probes and primers, thereby isolating the target DNA. Such cDNA libraries are preferably prepared from cells, organs, or tissues expressing any of the proteins shown in SEQ ID NO:2 to 30. Examples of cells or tissues include those from the testes and cells or tissues from cancers or tumors such as leukemia, breast cancer, lymphoma, brain tumors, lung cancer, and colorectal cancer. The operations described above, such as the preparation of probes or primers, the construction of cDNA libraries, the screening of cDNA libraries, and the cloning of the target gene, are known to those skilled in the art, and can be performed, for example, according to the methods described in Sambrook et al., Molecular Cloning, 2nd edition, Current Protocols in Molecular Biology, (1989), and Ausubel et al. (ibid.). DNA encoding the human CAPRIN-1 protein and its partial peptides can be obtained from the DNA thus obtained.

[0056] The host cells described above can be any cells, as long as they express the aforementioned polypeptides. Examples of prokaryotic host cells include, but are not limited to, *Escherichia coli*. Examples of eukaryotic host cells include, but are not limited to, mammalian cells such as monkey kidney cells (COS 1), Chinese hamster ovary cells (CHO), human embryonic kidney cell line (HEK293), and fetal mouse skin cell line (NIH3T3); yeast cells such as budding yeast cells and dividing yeast cells; silkworm cells and Xenopus oocytes.

[0057] When using prokaryotic cells as host cells, expression vectors with origins of replication, promoters, ribosome binding sites, multiple cloning sites, terminators, drug resistance genes, and auxotrophic complement genes that can replicate in prokaryotic cells can be used. Examples of *E. coli* expression vectors include pUC vectors, pBluescriptII, pET expression systems, and pGEX expression systems. DNA encoding the aforementioned polypeptide can be integrated into such expression vectors, prokaryotic host cells can be transformed using these vectors, and the resulting transformed cells can then be cultured. Therefore, the polypeptide encoded by this DNA can be expressed in prokaryotic host cells. In this case, the polypeptide can also be expressed as a fusion protein with another protein.

[0058] When using eukaryotic cells as host cells, eukaryotic expression vectors with promoters, splice regions, and polyadenylate addition sites are used. Examples of such expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vectors, pRS, pcDNA3, and pYES2. DNA encoding the aforementioned polypeptide is integrated into such expression vectors using a method similar to that described above. Eukaryotic host cells are then transformed with this vector, and the resulting transformed cells are subsequently cultured. This allows the expression of the polypeptide encoded by the DNA within the eukaryotic host cells. When pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, etc., are used as expression vectors, the aforementioned polypeptides can be expressed as fusion proteins with tags such as His tags (e.g., (His)6 to (His) tags). 10 ), FLAG tag, myc tag, HA tag or GFP.

[0059] To introduce expression vectors into host cells, known methods can be used, such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection method, viral infection, lipid transfection method, or binding to cell membrane permeable peptides.

[0060] Target peptides can be isolated and purified from host cells by combining known separation techniques. Examples of such known techniques include, but are not limited to, treatment with denaturing agents such as urea or surfactants, ultrasonic disruption, enzymatic digestion, salting out, solvent fractionation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric point focusing electrophoresis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reversed-phase chromatography.

[0061] Antibody Structure

[0062] Generally, antibodies are heteropolyglycoproteins, each containing at least two heavy chains and two light chains. Furthermore, antibodies other than IgM are heterotetrameric glycoproteins (approximately 150 kDa), each containing two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to the heavy chain by a single covalent disulfide bond. However, the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has intrachain disulfide bonds. Each heavy chain has a variable domain (VH region) at one end, with several constant regions sequentially linked to it. Each light chain has a variable domain (VL region) at one end and a single constant region at the opposite end. The constant region of the light chain is aligned with the first constant region of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Specific regions of the antibody variable domain, called the "complementarity-determining region (CDR)," exhibit specific variability, thereby conferring the antibody's binding specificity. The relatively conserved portion of the variable region is called the "framework region (FR)." The complete heavy or light chain variable domain contains four receptor regions (FRs), which are linked together by three chain decanters (CDRs). These CDRs are named "CDRH1", "CDRH2", and "CDRH3" in order from the N-terminus of the heavy chain. Similarly, for the light chain, they are named "CDRL1", "CDRL2", and "CDRL3". CDRH3 plays the most important role in antibody-antigen binding specificity. Furthermore, the CDRs in each chain maintain their proximity to each other through the FR regions, and they, together with the CDRs of the corresponding chains, form antibody-antigen binding sites. Constant regions do not directly affect antigen-antibody binding. However, they exhibit various effector effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis via binding to the Fcγ receptor, half-life / clearance rate via the neonatal Fc receptor (FcRn), and complement-dependent cytotoxicity (CDC) via C1q in the complement cascade.

[0063] Antibody Preparation

[0064] The term "anti-CAPRIN-1 antibody" as used in this invention refers to an antibody that has an immunological reactivity with the full-length CAPRIN-1 protein or a fragment thereof.

[0065] As used herein, the term "immunologic reactivity" refers to the property of an antibody to bind to the CAPRIN-1 antigen in vivo. The function of damaging tumors (e.g., death, suppression, or regression) can be exerted as a result of such binding. In particular, any type of antibody can be used in this invention, as long as it can bind to the CAPRIN-1 protein to damage tumors or cancers such as leukemia, lymphoma, breast cancer, brain tumors, lung cancer, esophageal cancer, gastric cancer, kidney cancer, or colorectal cancer.

[0066] Examples of such antibodies include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, and antibody fragments (e.g., Fab and F(ab')2). Furthermore, examples of any immunoglobulin type of such antibodies include IgG, IgE, IgM, IgA, IgD, and IgY, and examples of any immunoglobulin subclass include IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0067] In addition to glycosylation, antibodies can also be modified by acetylation, formylation, amidation, phosphorylation, or polyethylene glycol (PEG) modification.

[0068] Examples of antibody preparation are described below.

[0069] When the target antibody is a monoclonal antibody, mice were immunized with CAPRIN-1-expressing breast cancer cell lines (SK-BR-3), and spleens were subsequently extracted from these mice. Cells were isolated from each spleen and then fused with mouse myeloma cells. Clones capable of producing antibodies with inhibitory effects on cancer cell growth were screened from the resulting fusion cells (hybridomas). Hybridomas producing monoclonal antibodies with inhibitory effects on cancer cell growth were isolated and cultured. The target antibody could then be purified from the culture supernatant using conventional affinity purification methods.

[0070] In addition, hybridomas that produce monoclonal antibodies can be prepared, for example, by the following method. First, animals are immunized with a sensitized antigen using known methods. In general methods, immunization is performed by intraperitoneal or subcutaneous injection of the sensitized antigen into the mammal. Specifically, the sensitized antigen is diluted or suspended in PBS (phosphate-buffered saline), physiological saline, etc., to a suitable yield. If necessary, a suitable amount of conventional adjuvant (e.g., Freund's complete adjuvant) is mixed in. After emulsification, the product is administered to the mammal several times every 4 to 21 days. Furthermore, a sufficient carrier can be used with the sensitized antigen for immunization.

[0071] As described above, after immunizing a mammal and determining that serum antibody levels have risen to the desired level, immune cells are collected from the mammal and fused. A particularly preferred example of immune cells is the spleen cell.

[0072] Mammalian myeloma cells were used as the relevant parental cells for fusion with the aforementioned immune cells. For such myeloma cells, the following known cell lines were preferred: 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 (de St. 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).

[0073] Cell fusion of immune cells and the aforementioned myeloma cells can be performed using known methods, such as those of Kohler and Milstein (Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46).

[0074] More specifically, the aforementioned cell fusion can be performed, for example, in a culture solution containing conventional nutrients in the presence of a cell fusion promoter. Examples of usable fusion promoters include polyethylene glycol (PEG) and Sendai virus (HVJ: Japanese hemagglutination virus). If desired, adjuvants such as dimethyl sulfoxide can be further added to improve fusion efficiency.

[0075] The ratio of immune cells to myeloma cells used can be arbitrarily determined. For example, the ratio of immune cells to myeloma cells is preferably from 1:1 to 10:1. Examples of culture media suitable for the above-mentioned cell fusion include RPMI 1640 and MEM media, which are suitable for the growth of the above-mentioned myeloma cell lines, as well as other conventional culture media for this type of cell culture. In addition, serum substitutes, such as fetal bovine serum (FCS), can be used in conjunction with these media.

[0076] To perform cell fusion, the aforementioned immune cells and myeloma cells are thoroughly mixed in a predetermined amount in culture medium. A PEG solution (e.g., average molecular weight: approximately 1000 to 6000), previously heated to approximately 37°C, is added to the mixture at a typical concentration of 30% to 60% (w / v), followed by mixing. This results in the formation of the target hybridoma. Subsequently, the steps of successively adding suitable culture medium and removing the supernatant by centrifugation are repeated to remove cell fusion agents unsuitable for hybridoma growth.

[0077] The resulting hybridomas are cultured in a standard selection medium such as HAT medium (containing hypoxanthine, aminopterin, and thymidine) for selection. Culture in HAT medium is continued for a sufficient period (typically several days to several weeks) to kill cells other than the target hybridoma (non-fusion cells). Next, hybridomas producing the target antibody are screened using standard limiting dilution methods, yielding monoclonal antibodies.

[0078] In addition, hybridomas that produce human antibodies with desired activity (e.g., cell growth inhibitory activity) can be obtained by means of immunizing non-human animals with antigens. Human lymphocytes (e.g., human lymphocytes infected with EBV) are sensitized in vitro with proteins, cells expressing the proteins, or their lysate products, and the sensitized lymphocytes are fused with perpetually dividing human myeloma cells (e.g., U266) (accession number TIB196).

[0079] The monoclonal antibody-producing hybridomas generated above can be passaged in standard culture medium. Furthermore, they can be stored in liquid nitrogen for a period of time.

[0080] Specifically, immunization is performed using the desired antigen or cells expressing the desired antigen as sensitizing antigens according to conventional immunization methods. The resulting immune cells are fused with known parental cells using conventional cell fusion methods. Then, cells (hybridomas) that produce monoclonal antibodies are screened using conventional screening methods. Therefore, antibody preparation can be performed.

[0081] Other examples of antibodies that can be used in this invention include polyclonal antibodies. For example, polyclonal antibodies can be obtained using the methods described below.

[0082] Serum was obtained by immunizing small animals such as mice, mice producing human antibodies, or rabbits with naturally occurring CAPRIN-1 protein, recombinant CAPRIN-1 protein expressed in microorganisms such as E. coli as a protein fused with GST, or a partial peptide thereof. The serum was purified by ammonium sulfate precipitation, protein A / protein G column chromatography, DEAE ion exchange chromatography, affinity column chromatography (using a column conjugated with CAPRIN-1 protein or a synthetic peptide), or similar techniques used to prepare polyclonal antibodies. In the examples described below, a rabbit polyclonal antibody was prepared and its antitumor effect was confirmed. This antibody targets a partial peptide (having the sequence shown in SEQ ID NO:37) of the structural domain of the amino acid sequence of the CAPRIN-1 protein expressed on the surface of cancer cells.

[0083] Known human antibody-producing mice used in this article include, for example, KM mice (Kirin Pharma / Medarex) or XenoMouse (Amgen) (e.g., WO02 / 43478 and WO02 / 092812). Complete human polyclonal antibodies can be obtained from the blood of such mice when they are immunized with CAPRIN-1 protein or fragments thereof. Alternatively, human monoclonal antibodies can be prepared by fusing spleen cells collected from immunized mice with myeloma cells.

[0084] Antigens can be prepared using methods such as those employing animal cells (Japanese Patent Publication No. 2007-530068) or those employing baculoviruses (e.g., WO98 / 46777). If the antigen has low immunogenicity, it is bound to an immunogenic macromolecule (such as albumin). The antigen can then be used for immunization.

[0085] Alternatively, recombinant antibodies can be used, which are prepared by cloning an antibody gene from a hybridoma, integrating the clone into a suitable vector, introducing the vector into a host, and applying gene recombination technology. (See, for example, Carl, AKBorrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, published by MACMILLANPUBLISHERS LTD, UK, 1990). Specifically, the variable region (V region) cDNA of the antibody is synthesized from hybridoma mRNA using reverse transcriptase. After obtaining the DNA encoding the V region of the target antibody, this DNA is ligated to the DNA encoding the desired constant region (C region) of the antibody. The resulting product is integrated into an expression vector. Alternatively, the DNA encoding the V region of the antibody can be integrated into an expression vector containing the antibody C region DNA. This DNA is integrated into the expression vector so that it is expressed under the control of expression control regions such as enhancers or promoters. Subsequently, host cells are transformed with this expression vector, thereby enabling antibody expression.

[0086] The anti-CAPRIN-1 antibody of the present invention is preferably a monoclonal antibody. However, it can be a polyclonal antibody, a genetically modified antibody (such as a chimeric antibody and a humanized antibody), etc.

[0087] Monoclonal antibodies include human monoclonal antibodies and non-human animal monoclonal antibodies (e.g., mouse monoclonal antibodies, rat monoclonal antibodies, rabbit monoclonal antibodies, and chicken monoclonal antibodies). Monoclonal antibodies can be obtained by culturing hybridomas, wherein the hybridoma is obtained by fusing myeloma cells with spleen cells from non-human mammals (e.g., mice or mice producing human antibodies) immunized with the CAPRIN-1 protein. In the examples described below, mouse monoclonal antibodies were generated and their antitumor activity was demonstrated. These monoclonal antibodies comprise a heavy chain variable (VH) region having the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:73, SEQ ID NO:83, SEQ ID NO:93, SEQ ID NO:103, SEQ ID NO:113, or SEQ ID NO:123, and a light chain variable (VL) region having the amino acid sequences shown in SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:68, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO:97, SEQ ID NO:107, SEQ ID NO:117, or SEQ ID NO:127. Here, the VH region comprises: CDR1 represented by the amino acid sequence of SEQ ID NO:40, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, SEQ ID NO:100, SEQ ID NO:110 or SEQ ID NO:120; CDR2 represented by the amino acid sequence of SEQ ID NO:41, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, SEQ ID NO:101, SEQ ID NO:111 or SEQ ID NO:121; and CDR3 represented by the amino acid sequence of SEQ ID NO:42, SEQ ID NO:72, SEQ ID NO:82, SEQ ID NO:92, SEQ ID NO:102, SEQ ID NO:112 or SEQ ID NO:122.The VL region comprises: CDR1 represented by the amino acid sequences of SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:55, SEQ ID NO:60, SEQ ID NO:65, SEQ ID NO:74, SEQ ID NO:84, SEQ ID NO:94, SEQ ID NO:104, SEQ ID NO:114, or SEQ ID NO:124; CDR2 represented by the amino acid sequences of SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:61, SEQ ID NO:66, SEQ ID NO:75, SEQ ID NO:85, SEQ ID NO:95, SEQ ID NO:105, SEQ ID NO:115, or SEQ ID NO:125; and CDR2 represented by the amino acid sequences of SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:86, SEQ ID NO:96, SEQ ID NO:124, ...124, SEQ ID NO:124, SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO: CDR3 is represented by the amino acid sequences of NO:106, SEQ ID NO:116, or SEQ ID NO:126.

[0088] Chimeric antibodies are antibodies produced by combining sequences from different animals. An example is an antibody composed of the variable regions of the heavy and light chains of a mouse antibody and the constant regions of the heavy and light chains of a human antibody. Such chimeric antibodies can be prepared using known methods. For instance, they can be produced by integrating the product into an expression vector by linking DNA encoding the V region of an antibody to DNA encoding the C region of a human antibody, and then introducing this vector into host cells used for antibody preparation.

[0089] Polyclonal antibodies include those obtained by immunizing animals (e.g., mice) that produce human antibodies with CAPRIN-1 antibodies.

[0090] Humanized antibodies are modified antibodies, sometimes referred to as "reconstructed human antibodies." It is known to construct humanized antibodies by transplanting the CDR (Copyright Determinant Region) of an antibody from an immunized animal into the complementarity-determining region (CDR) of a human antibody. This general gene recombination technique is also well-known.

[0091] Specifically, DNA sequences designed to allow the linking of mouse antibody CDRs to human antibody framework regions (FRs) are synthesized via PCR, wherein the PCR uses several oligonucleotides prepared in a manner that have overlapping portions at one end. Humanized antibodies are obtained by linking the DNA obtained above to DNA encoding the constant region of a human antibody, integrating the product into an expression vector, and introducing the vector into host cells for antibody production (see EP-A-239400 and WO96 / 02576). The human antibody FRs linked to each other via CDRs are selected based on the assumption that the complementarity-determining regions (CDGs) form good antigen-binding sites. If desired, amino acids in the framework region of the antibody variable region can be replaced in a manner that allows the CDGs in the reconstructed human antibody to form suitable antigen-binding sites (Sato K. et al., Cancer Research 1993, 53:851-856). Furthermore, the framework region can be replaced with a framework region from a different human antibody (see WO99 / 51743).

[0092] The complementarity-determining regions (CDRs) of human antibodies, which are linked together by CDRs, are selected based on the following assumptions to form good antigen-binding sites. If necessary, the amino acids in the CDRs of the antibody variable region can be replaced in such a way that the CDRs in the reconstructed human antibody form suitable antigen-binding sites (Sato K. et al., Cancer Research 1993, 53:851-856).

[0093] Once chimeric or humanized antibodies are generated, amino acids in the variable region (e.g., FR) or constant region can be replaced, for example, with different amino acids.

[0094] In this document, the amino acid substitutions referred to are, for example, substitutions of less than 15, less than 10, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, or no more than 2 amino acids, preferably 1 to 5 amino acids, and more preferably 1 or 2 amino acids. The substituted antibody should be functionally equivalent to the unsubstituted antibody. The substitutions are preferably conservative amino acid substitutions, i.e., substitutions between amino acids that have similar properties in terms of charge, side chain, polarity, aromaticity, etc. For example, amino acids with similar properties can be classified into the following types: basic amino acids (arginine, lysine, and histidine); acidic amino acids (aspartic acid and glutamic acid); non-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).

[0095] One example of antibody modification is an antibody linked to a molecule such as polyethylene glycol (PEG). Regarding the antibody modifications of this invention, the substance binding to the antibody is not limited. Such antibody modifications can be obtained by chemically modifying antibodies. Methods for such modifications have been established in the relevant art.

[0096] As used herein, the expression "functionally equivalent" refers to a situation where the target antibody has similar biological or biochemical activity to the antibody of the present invention. In particular, such antibodies have the function of damaging tumors and do not substantially induce rejection when administered to humans. Examples of such activity are cell growth inhibitory activity or binding activity.

[0097] Known methods for preparing peptides that are functionally equivalent to a given peptide, as understood by those skilled in the art, include methods for introducing mutations into the peptide. For example, those skilled in the art can appropriately introduce mutations into the antibodies of the present invention by 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, 488-492; or Kunkel (1988) Methods Enzymol. 85, 2763-2766) or similar methods. This allows for the preparation of antibodies functionally equivalent to those of the present invention.

[0098] The aforementioned antibodies capable of recognizing the CAPRIN-1 protein epitope recognized by the anti-CAPRIN-1 antibody can be obtained by methods known to those skilled in the art. For example, they can be obtained by methods including: identifying the CAPRIN-1 protein epitope recognized by the anti-CAPRIN-1 antibody using conventional methods (e.g., epitope mapping), and using a polypeptide having the amino acid sequence contained in that epitope as an immunogen to generate an antibody; or methods including identifying the epitope of the antibody generated by conventional methods, and screening for antibodies having the same epitope as the anti-CAPRIN-1 antibody. Hereinafter, "epitaph" refers to a polypeptide fragment that is antigenic or immunogenic in mammals, particularly humans. Its smallest unit consists of about 7 to 12 amino acids, preferably 8 to 11 amino acids.

[0099] The affinity constant Ka(k) of the antibody of this invention on / k off Preferably at least 10 7 M -1 At least 10 8 M -1 At least 5×10 8 M -1 At least 10 9 M -1 At least 5×10 9 M -1 At least 10 10 M -1 At least 5×10 10 M -1 At least 10 11 M -1 At least 5×10 11 M -1 At least 10 12 M -1 or at least 10 13 M -1 .

[0100] The antibody of the present invention can be conjugated with an antitumor agent. The binding between the antibody and the antitumor agent can be achieved by a spacer having a group that reacts with amino, carboxyl, hydroxyl, thiol, etc. (e.g., iminosuccinate group, formyl group, 2-pyridyl dithio group, maleimide group, alkoxycarbonyl group, or hydroxyl group).

[0101] Examples of antitumor agents include the following, which are well-known in the references: paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, indomethacin, piperosulfan, benzodopa, carboquinone, metoprolol, uredopa, hexamethylmelamine, triethylene melamine, triethylenephosphamide, triethylenethiophosphamide, trimethylolmelamine, bullatacin, bullatacinone, camptothecin, lichenin, callystatin, cryptophycin 1, cryptophycin 8. Dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, naphthalenemustine, cholophosphamide, estradiol, ifosfamide, nitrogen mustard, oxyhydrochloride nitrogen mustard, phenylalanine nitrogen mustard, neonitrogen mustard, cholesterol-p-phenylacetic acid nitrogen mustard, pinesol benzenemustine, chlorotetracycline, uracil nitrogen mustard, nitrosourea nitrogen mustard, pyrimidine nitrosourea, flotinib, cyclohexylnitrosourea, pyrimidine nitrosourea, renosulfuron, calicheamicin, dynemicin, chlorphosphatate, esperamicin, aclarubicin, actinomycin, authramycin, diazoacetylserine, bleomycin, actinomycin C, carabicin Erythromycin, Carcinobacterium, Chromomycin, Actinomycin, Detorbicin, 6-Diazono-5-oxo-L-leucine, Doxorubicin, Epirubicin, Isorubicin, Demethoxybenzamine, Ephedrine, Mitomycin C, Mycophenolic acid, Nogamycin, Oligomycin, Pelomycin, Potfiromycin, Purulin, Triamcinolone Acetonide, Rodoxine, Streptomycin, Streptozotocin, Tuberculin, Ubenimex, Netostat D, Zorubicin, Folic Acid, Pteranoside, Trimethoprim, Fludarabine, 6-Mercaptopurine, Thiomipril, 2-Aminopurine-6-Mitrol, Cyclocytidine, Azacitidine, 6-Zazouridine, Carmoflu, Cytarabine, Dideoxyuridine, Deoxyfluorouridine, Enoxabine, 5-Fluorodeoxyuridine, androgens such as dimethyltestosterone, Methylandrostenone propionate, Cyclothrostenol, Meandrosten, Testrolide, Aminophenepiperidone, Mitotan, Tralostan, Frolinicacid, acetolactone, aldehyde phosphoramidone, aminovaleric acid, enuramicin, acridine, bestrabucil, pyrazinamide, edatrazine, defofamine, decarbonyl colchicine, iminoquinone amino ester, elfornithine, elifonitrile, epoxy tubulin, ethoxylated pyrazinamide, mushroom polysaccharide, chlordamine, maytansine, ansamitocine, propamidone, mitoxantrone, mopidanmol, nitraerine, pentostatin, methamidophos, pirarubicin, loxoantrone, podophylloidic acid, 2-ethylhydrazine, methylbenzylhydrazine, propylimine, rhizomycin, schizotypalin, germanium spiroamine, sigmazoic acid, Triaminoquinone, Baculosporin A, Anguidine, Ethyl carbamate, Deacetylvinamide, Dazometazidine, Mannitol Mustard, Dibromomannitol, Dibromoeusine, Piperobromide, Gacyclidine, Taxene, Chlorbutazone Mustard, Gemcitabine, 6-Thioguanine, Mercaptopurine, Cisplatin, Oxyceplatin, Carboplatin, Vincristine, Etoposide, Ifosfamide, Mitoxantrone, Vincristine, Vinorelbine, Norfloxacin, Teniposide, Edatraxa, Donomycin, Aminopterin, Xeloda, Ibandronate, Irinotecan, Topoisomerase inhibitors, Difluoromethylornithine (DMFO), Retinoic acid, Capecitabine, and their pharmaceutically acceptable salts or derivatives.

[0102] Alternatively, a radioactive isotope may be linked to the antibody of the present invention, such as those known in the references. 211 At、 131 I, 125 I, 90 Y、 186 Re、 188 Re、 153 Sm、 212 Bi、 32 P, 175 Lu or 176 Lu et al. expressed hope that such radioactive isotopes would be effective in treating or diagnosing tumors.

[0103] The antibodies of the present invention are antibodies that are immunoreactive with CAPRIN-1, or antibodies that specifically recognize CAPRIN-1. Such antibodies should have a structure that allows the test animal administered the antibody to completely or almost completely avoid rejection. If the test animal is human, examples of the above-mentioned antibodies include human antibodies, humanized antibodies, chimeric antibodies (e.g., human-mouse chimeric antibodies), single-chain antibodies, and bispecific antibodies. Such antibodies are: recombinant antibodies having variable regions of heavy and light chains derived from human antibodies; recombinant antibodies having variable regions of heavy and light chains (each consisting of a complementarity-determining region (CDR1, CDR2, and CDR3) derived from non-human animal antibodies and a framework region derived from human antibodies; or recombinant antibodies having variable regions of heavy and light chains derived from non-human animal antibodies and constant regions of heavy and light chains derived from human antibodies. The first two types of antibodies are preferred.

[0104] The recombinant antibody described above can be generated by the method described below. DNA encoding an anti-human CAPRIN-1 monoclonal antibody (e.g., human monoclonal antibody, mouse monoclonal antibody, rat monoclonal antibody, rabbit monoclonal antibody, or chicken monoclonal antibody) is cloned from antibody-producing cells such as hybridomas. Using the obtained clone as a template, DNA encoding the light chain variable region and heavy chain variable region of the antibody is generated by methods such as RT-PCR. The sequences of the light chain variable region and heavy chain variable region, or the sequences of CDR1, CDR2, and CDR3, are then determined using the Kabat EU coding system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institute of Health, Bethesda, Md. (1991)).

[0105] Furthermore, DNA encoding the variable region or the CDR can be generated using gene recombination technology (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) or a DNA synthesizer. Here, the hybridomas that produce human monoclonal antibodies described above can be generated by immunizing an animal (e.g., a mouse) with human CAPRIN-1 and fusing spleen cells from the spleen of that animal with myeloma cells. In addition to the above, if necessary, DNA encoding the variable and constant regions of the light or heavy chain from which the human antibody originates can be generated using gene recombination technology or a DNA synthesizer.

[0106] In the case of humanized antibodies, DNA is produced in which the CDR coding sequence in the DNA encoding the variable region of the light or heavy chain from the human antibody source has been replaced by the corresponding CDR coding sequence from an antibody derived from a non-human animal (e.g., mouse, rat, or chicken). The DNA obtained as described above is then ligated with DNA encoding the constant region of the light or heavy chain from the human antibody source. This produces DNA encoding the humanized antibody.

[0107] In the case of chimeric antibodies, DNA encoding the variable region of the heavy or light chain of an antibody derived from a non-human animal (e.g., mouse, rat, or chicken) is linked to DNA encoding the constant region of the light or heavy chain of a human antibody. This produces DNA encoding the chimeric antibody.

[0108] A single-chain antibody is an antibody in which the heavy chain variable region and the light chain variable region are linearly linked together by a linker. DNA encoding a single-chain antibody can be generated by binding DNA encoding the heavy chain variable region, DNA encoding the linker, and DNA encoding the light chain variable region. Here, the heavy chain variable region and the light chain variable region are those derived from human antibodies, or those derived from human antibodies in which only the CDR has been replaced by the CDR of an antibody derived from a non-human animal (e.g., mouse, rat, or chicken). Furthermore, the linker consists of 12 to 19 amino acids. An example of this is (G4S)3, which consists of 15 amino acids (GBKim et al., Protein Engineering Design and Selection 2007, 20(9):425-432).

[0109] A diabody is an antibody that specifically binds to two different epitopes, wherein, for example, DNA encoding heavy chain variable region A, DNA encoding light chain variable region B, DNA encoding heavy chain variable region B, and DNA encoding light chain variable region A are linked together in such a sequence (provided that the DNA encoding light chain variable region B and the DNA encoding heavy chain variable region B are linked together by DNA encoding the aforementioned linker). This produces DNA encoding a bispecific antibody. Here, both the heavy chain variable region and the light chain variable region are derived from human antibodies, or from human antibodies where only the CDR has been replaced by an antibody CDR derived from a non-human animal (e.g., mouse, rat, or chicken).

[0110] The recombinant DNA generated as described above is integrated into one or more suitable vectors. Each such vector is introduced into a host cell (e.g., mammalian cell, yeast cell, or insect cell) for (co)expression. This produces a recombinant antibody (PJ Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997; WILEY, P. Shepherd and C. Dean., Monoclonal Antibodies., 2000; OXFORD UNIVERSITY PRESS; JWGoding, Monoclonal Antibodies: Principles and Practice., 1993; ACADEMIC PRESS).

[0111] Examples of antibodies of the present invention produced by the above method include the following antibodies (a) to (k).

[0112] (a) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:40, 41 and 42, and a light chain variable region comprising the sequences shown in SEQ ID NO:44, 45 and 46 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO:43 and the light chain variable region of SEQ ID NO:47).

[0113] (b) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:40, 41 and 42, and a light chain variable region comprising the sequences shown in SEQ ID NO:50, 51 and 52 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO:43 and the light chain variable region of SEQ ID NO:53).

[0114] (c) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:40, 41 and 42, and a light chain variable region comprising the sequences shown in SEQ ID NO:55, 56 and 57 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO:43 and the light chain variable region of SEQ ID NO:58).

[0115] (d) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:40, 41 and 42, and a light chain variable region comprising the sequences shown in SEQ ID NO:60, 61 and 62 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO:43 and the light chain variable region of SEQ ID NO:63).

[0116] (e) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:40, 41 and 42, and a light chain variable region comprising the sequences shown in SEQ ID NO:65, 66 and 67 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO:43 and the light chain variable region of SEQ ID NO:68).

[0117] (f) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:70, 71 and 72, and a light chain variable region comprising the sequences shown in SEQ ID NO:74, 75 and 76 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO:73 and the light chain variable region of SEQ ID NO:77).

[0118] (g) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO:80, 81 and 82, and a light chain variable region comprising the sequences shown in SEQ ID NO:84, 85 and 86 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO:83 and the light chain variable region of SEQ ID NO:87).

[0119] (h) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO: 90, 91 and 92, and a light chain variable region comprising the sequences shown in SEQ ID NO: 94, 95 and 96 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO: 93 and the light chain variable region of SEQ ID NO: 97).

[0120] (i) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO: 100, 101 and 102, and a light chain variable region comprising the sequences shown in SEQ ID NO: 104, 105 and 106 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO: 103 and the light chain variable region of SEQ ID NO: 107).

[0121] (j) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO: 110, 111 and 112, and a light chain variable region comprising the sequences shown in SEQ ID NO: 114, 115 and 116 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO: 113 and the light chain variable region of SEQ ID NO: 117).

[0122] (k) An antibody comprising a heavy chain variable region comprising the sequences shown in SEQ ID NO: 120, 121 and 122, and a light chain variable region comprising the sequences shown in SEQ ID NO: 124, 125 and 126 (and preferably an antibody comprising the heavy chain variable region of SEQ ID NO: 123 and the light chain variable region of SEQ ID NO: 127).

[0123] Here, the amino acid sequences shown in SEQ ID NO:40, 41 and 42, SEQ ID NO:70, 71 and 72, SEQ ID NO:80, 81 and 82, SEQ ID NO:90, 91 and 92, SEQ ID NO:100, 101 and 102, SEQ ID NO:110, 111 and 112, or SEQ ID NO:120, 121 and 122 correspond to CDR1, CDR2 and CDR3 of the variable region of the mouse antibody heavy chain, respectively. Furthermore, the amino acid sequences shown in SEQ ID NO:44, 45 and 46, SEQ ID NO:50, 51 and 52, SEQ ID NO:55, 56 and 57, SEQ ID NO:60, 61 and 62, SEQ ID NO:65, 66 and 67, SEQ ID NO:74, 75 and 76, SEQ ID NO:84, 85 and 86, SEQ ID NO:94, 95 and 96, SEQ ID NO:104, 105 and 106, SEQ ID NO:114, 115 and 116, or SEQ ID NO:124, 125 and 126 correspond to CDR1, CDR2 and CDR3 of the variable region of the mouse antibody light chain, respectively.

[0124] Furthermore, the humanized antibodies, chimeric antibodies, single-chain antibodies, or bispecific antibodies of the present invention are, for example, the following antibodies (i) or (ii) (examples of antibody (a) are described below).

[0125] (i) An antibody comprising: a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:40, 41 and 42 and the amino acid sequence of the human antibody-derived framework region; and a light chain variable region comprising the amino acid sequences of SEQ ID NO:44, 45 and 46 and the amino acid sequence of the human antibody-derived framework region (and preferably an antibody comprising the amino acid sequence of SEQ ID NO:43 in the heavy chain variable region and the amino acid sequence of SEQ ID NO:47 in the light chain variable region).

[0126] (ii) An antibody comprising: a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:40, 41 and 42 and the amino acid sequence of the human antibody-derived framework region; a heavy chain constant region comprising the amino acid sequence of the human antibody-derived source; a light chain variable region comprising the amino acid sequences of SEQ ID NO:44, 45 and 46 and the amino acid sequence of the human antibody-derived framework region; and a light chain constant region comprising the amino acid sequence of the human antibody-derived source (and preferably comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:43; a heavy chain constant region comprising the amino acid sequence of the human antibody-derived source; a light chain variable region comprising the amino acid sequence of SEQ ID NO:47; and a light chain constant region comprising the amino acid sequence of the human antibody-derived source).

[0127] In addition, sequences of the constant and variable regions of the heavy and light chains of human antibodies can be obtained, for example, from NCBI (USA: GenBank, UniGene, etc.). For example, the following sequences can be used as reference sequences for the corresponding regions: the sequence with accession number J00228 for the human IgG1 heavy chain constant region; the sequence with accession number J00230 for the human IgG2 heavy chain constant region; the sequence with accession number X03604 for the human IgG3 heavy chain constant region; the sequence with accession number K01316 for the human IgG4 heavy chain constant region; the sequence with accession numbers V00557, X64135, or X64133 for the human light chain κ constant region; and the sequence with accession numbers X64132 or X64134 for the human light chain λ constant region.

[0128] The antibodies mentioned above preferably have cytotoxic activity, thereby exhibiting antitumor activity.

[0129] Furthermore, the specific sequences of the antibody heavy and light chain variable regions and CDRs described above are merely illustrative. The invention is obviously not limited to these specific sequences. Hybridomas capable of producing different human antibodies or non-human animal antibodies (e.g., mouse antibodies) against human CAPRIN-1 are generated. Monoclonal antibodies produced from these hybridomas are collected. Then, using immunobinding activity and cytotoxic activity with respect to human CAPRIN-1 as indicators, it is determined whether the obtained antibody is the target antibody. The target hybridoma producing the monoclonal antibody is thus identified. Subsequently, as described above, DNA encoding the heavy and light chain variable regions of the target antibody is generated from the hybridoma for sequence determination. This DNA is used to produce different antibodies.

[0130] Furthermore, the antibodies described above in this invention can be any of the antibodies (i) to (iv) described above having one or more (preferably 1 or 2) amino acid substitutions, deletions, or additions (especially in the framework region sequence and / or constant region sequence), as long as they have the specific property of specifically recognizing CAPRIN-1. Here, the term "several amino acids" means 2 to 5, preferably 2 or 3 amino acids.

[0131] Furthermore, according to the present invention, DNA encoding the above-described antibody of the present invention, DNA encoding the heavy chain or light chain of the antibody, or DNA encoding the variable region of the heavy chain or light chain of the antibody are also provided. For example, in the case of antibody (a), examples of such DNA include: heavy chain variable region encoding DNA comprising a nucleotide sequence encoding the amino acid sequences of SEQ ID NO:40, 41, and 42; and light chain variable region encoding DNA comprising a nucleotide sequence encoding the amino acid sequences of SEQ ID NO:44, 45, and 46.

[0132] The complementarity-determining region (CDR) encoded by the DNA sequence described above is the region that determines antibody specificity. Therefore, the sequences encoding other regions of the antibody (i.e., constant regions and framework regions) can be sequences from different antibodies. Here, different antibodies include antibodies from non-human organisms. However, considering the reduction of side effects, human-derived antibodies are preferred. That is, in the above case, the DNA sequence encoding the heavy and light chain framework regions and the constant region preferably contains a nucleotide sequence encoding the relevant amino acid sequence from a human antibody.

[0133] Furthermore, different examples of DNA encoding the antibodies of the present invention (such as antibody (a)) include: DNA encoding the heavy chain variable region comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:43, and DNA wherein the region encoding the light chain variable region comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:47. Here, an example of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:43 is the nucleotide sequence of SEQ ID NO:48. Furthermore, an example of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:47 is the nucleotide sequence of SEQ ID NO:49. Additionally, the DNA encoding the heavy chain and light chain constant regions described above preferably comprises a nucleotide sequence encoding the corresponding human antibody-derived amino acid sequence.

[0134] The DNA of the present invention can be obtained, for example, by the methods described above or the following methods. First, total RNA is prepared from the hybridoma of the antibody of the present invention using a commercially available RNA extraction kit. Then, cDNA is synthesized by reverse transcriptase using random primers, etc. Next, the cDNA encoding the antibody is amplified by PCR using oligonucleotides having conserved sequences in the variable regions of the known mouse antibody heavy and light chain genes as primers. The sequence encoding the constant region can be obtained by amplifying known sequences using PCR. The nucleotide sequence of the DNA can be determined by general methods, wherein such methods include, for example, integration into a plasmid or phage for sequence determination.

[0135] We believe that the anti-CAPRIN-1 antibody used in this invention exhibits antitumor activity against CAPRIN-1-expressing cancer cells through the cytotoxic mechanism described below.

[0136] The cytotoxicity includes effector cell-mediated antibody-dependent cytotoxicity (ADCC) against CAPRIN-1-expressing cells and complement-dependent cytotoxicity (CDC) against CAPRIN-1-expressing cells.

[0137] Therefore, the activity of the anti-CAPRIN-1 antibody used in this invention can be evaluated by determining ADCC or CDC activity against cancer cells expressing CAPRIN-1 in vitro and then in vivo, as specifically described in the following examples.

[0138] The anti-CAPRIN-1 antibody used in this invention binds to the CAPRIN-1 protein on cancer cells and exhibits an anti-tumor effect based on the aforementioned activity. Therefore, it is believed that such antibodies can be used for cancer treatment or prevention. In particular, according to the present invention, a pharmaceutical composition for treating and / or preventing cancer is provided, comprising an anti-CAPRIN-1 antibody as an active ingredient. When the anti-CAPRIN-1 antibody is used for the purpose of administering antibodies to humans (antibody therapy), it is preferable to use it in the form of a human antibody or a humanized antibody to reduce immunogenicity.

[0139] Furthermore, the higher the binding affinity between anti-CAPRIN-1 antibodies and the CAPRIN-1 protein on the surface of cancer cells, the stronger the anti-CAPRIN-1 antibody can exhibit anti-tumor activity. Therefore, if anti-CAPRIN-1 antibodies with high binding affinity to the CAPRIN-1 protein can be obtained, stronger anti-tumor activity can be expected. Thus, it becomes possible to use such antibodies as pharmaceutical compositions for tumor treatment and / or prevention. As mentioned above, for high binding affinity, the affinity constant Ka(k) on / k off Preferably at least 10 7 M -1 At least 10 8 M -1 At least 5×10 8 M -1 At least 10 9 M -1 At least 5×10 9 M -1 At least 10 10 M -1 At least 5×10 10 M -1 At least 10 11 M -1 At least 5×10 11 M -1 At least 10 12 M -1 or at least 10 13 M -1 .

[0140] <Binding with cells expressing antigens>

[0141] The ability of an antibody to bind to CAPRIN-1 can be determined by using binding assays such as ELISA, Western blotting, immunofluorescence, or flow cytometry as described in the examples.

[0142] Immunohistochemical staining

[0143] Frozen tissue sections fixed with paraformaldehyde or acetone, or paraformaldehyde-fixed paraffin-embedded tissue sections, can be used to identify antibodies against CAPRIN-1 based on a reactivity test with CAPRIN-1 using immunohistochemical methods known to those skilled in the art. Such sections are prepared from tissues obtained during surgery from patients or from animals carrying xenograft tissue, wherein the animals are inoculated with native or transformed cell lines expressing CAPRIN-1.

[0144] For immunohistochemical staining, antibodies that react with CAPRIN-1 can be stained using various methods. For example, the antibody can be visualized by reacting with goat anti-mouse antibodies or goat anti-rabbit antibodies conjugated with horseradish peroxidase.

[0145] <Pharmaceutical Composition>

[0146] The target of the pharmaceutical composition for treating and / or preventing cancer of the present invention is not particularly limited, as long as the target is a cancer (cell) expressing the CAPRIN-1 gene.

[0147] The terms “tumor” and “cancer” used in this article both refer to malignant growths and are therefore used interchangeably.

[0148] In this invention, cancers that can be targeted are genes expressing polypeptides encoding an amino acid sequence comprising any one of the even-numbered SEQ ID NO: 2 to 30, or a partial sequence consisting of seven or more consecutive amino acids of said amino acid sequence. Preferred examples include breast cancer, brain tumors, leukemia, lung cancer, lymphoma, mast cell tumor, esophageal cancer, and colorectal cancer.

[0149] Examples of these specific tumors include, but are not limited to, breast cancer, complex breast cancer, mixed malignant tumors of the breast, intraductal papillary carcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, glioma (i.e., neuroepithelial tumor), ependymoma, neurocellular tumor, embryonal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, digestive lymphoma, small cell to medium cell lymphoma, cecal cancer, ascending colon cancer, descending colon cancer, transverse colon cancer, sigmoid colon cancer, and rectal cancer.

[0150] Furthermore, the test animals of this invention are mammals. Examples include mammals such as primates, pets, livestock, and racing animals. Humans, dogs, and cats are particularly preferred.

[0151] When the antibodies used in this invention are used as pharmaceutical compositions, they can be formulated using methods known to those skilled in the art. For example, they can be used parenterally as a parenteral injection: a sterile solution comprising water or a pharmaceutically acceptable non-aqueous solution; or a suspension. For example, in one possible scenario, a pharmaceutically acceptable carrier or medium, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, or binder, can be used in combination by mixing in a unit dosage form required for a generally acceptable pharmaceutical composition. The amount of the active ingredient in the formulation is determined to achieve a suitable dose within the indicated range.

[0152] Sterile compositions for injection can be prepared using a carrier such as distilled water for injection according to general formulation methods.

[0153] Examples of aqueous solutions for injection include physiological saline and isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Such solutions can be used with suitable solubilizing agents. Examples of such solubilizing agents include alcohols, such as ethanol and polyols, propylene glycol, polyethylene glycol, and nonionic surfactants, such as polysorbate 80. (TM) And HCO-60.

[0154] Examples of oily fluids include sesame oil and soybean oil. Such oily fluids can be used in combination with solubilizers such as benzyl benzoate or benzyl alcohol. Furthermore, they can be mixed with buffers (such as phosphate buffer solutions, acetate buffer solutions), analgesics (such as procaine hydrochloride), stabilizers (such as benzyl alcohol, phenol), or antioxidants. Generally, the prepared injectable solution is introduced into a suitable ampoule.

[0155] The above-described pharmaceutical composition is administered orally or parenterally. Parenterally administration is preferred. Specific examples of dosage forms include injectable formulations, nasal formulations, pulmonary formulations, and dermal formulations. For example, injectable formulations can be administered systemically or locally via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0156] Furthermore, the method of administration can be appropriately determined based on the patient's age, weight, sex, and symptoms. A single dose of a pharmaceutical composition containing an antibody or a polynucleotide encoding an antibody can be selected, for example, in the range of 0.0001 mg to 1000 mg / kg body weight. Alternatively, a dose can be selected, for example, in the range of 0.001 to 100,000 mg per patient; however, it is not necessarily limited to this. Dosage and method of administration vary depending on the patient's age, weight, sex, and symptoms. However, those skilled in the art can appropriately select the dosage and method.

[0157] <Polypeptides and DNA>

[0158] According to the present invention, the following polypeptides and DNA of the above-mentioned antibodies (a) to (k) are also provided.

[0159] (i) a polypeptide comprising the amino acid sequences of SEQ ID NO:43, SEQ ID NO:73, SEQ ID NO:83, SEQ ID NO:93, SEQ ID NO:103, SEQ ID NO:113 and SEQ ID NO:123, and the DNA encoding the polypeptide.

[0160] (ii) A polypeptide comprising the amino acid sequences of SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:68, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO:97, SEQ ID NO:107, SEQ ID NO:117 and SEQ ID NO:127, and the DNA encoding the polypeptide.

[0161] (iii) DNA containing the nucleotide sequences of SEQ ID NO:48, SEQ ID NO:78, SEQ ID NO:88, SEQ ID NO:98, SEQ ID NO:108, SEQ ID NO:118 and SEQ ID NO:128.

[0162] (iv) DNA containing the nucleotide sequences of SEQ ID NO:49, SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:79, SEQ ID NO:89, SEQ ID NO:99, SEQ ID NO:109, SEQ ID NO:119 and SEQ ID NO:129.

[0163] (v) A heavy chain CDR polypeptide comprising the amino acid sequences selected from SEQ ID NO:40, 41 and 42; the amino acid sequences selected from SEQ ID NO:70, 71 and 72; the amino acid sequences selected from SEQ ID NO:80, 81 and 82; the amino acid sequences selected from SEQ ID NO:90, 91 and 92; the amino acid sequences selected from SEQ ID NO:100, 101 and 102; the amino acid sequences selected from SEQ ID NO:110, 111 and 112; and the amino acid sequences selected from SEQ ID NO:120, 121 and 122, and the DNA encoding the polypeptide.

[0164] (vi) A light chain CDR polypeptide comprising the amino acid sequences selected from SEQ ID NO:44, 45, and 46; SEQ ID NO:50, 51, and 52; SEQ ID NO:55, 56, and 57; SEQ ID NO:60, 61, and 62; SEQ ID NO:65, 66, and 67; SEQ ID NO:74, 75, and 76; SEQ ID NO:84, 85, and 86; SEQ ID NO:94, 95, and 96; SEQ ID NO:104, 105, and 106; SEQ ID NO:114, 115, and 116; and SEQ ID NO:124, 125, and 126; and DNA encoding the polypeptide.

[0165] These polypeptides and DNA can be produced using the aforementioned gene recombination technology. Example

[0166] The invention will be described in more detail below with reference to the following embodiments, but the scope of the invention is not limited thereto.

[0167] Example 1: Identification of novel cancer antigen proteins using the SEREX method

[0168] (1) Construction of cDNA library

[0169] Total RNA was extracted from testicular tissue of healthy dogs using the guanidine-phenol-chloroform method, and polyA RNA was purified using the Oligotex-dT30 mRNA purification kit (Takara Shuzo Co., Ltd.) according to the included instructions.

[0170] A canine testis cDNA phage library was synthesized using the mRNA (5 μg) obtained in this manner. The cDNA phage library was constructed according to the instructions included in each kit, using a cDNA synthesis kit, a ZAP-cDNA synthesis kit, and a ZAP-cDNA Gigapack III Gold Cloning kit (STRATAGENE). The size of the resulting cDNA phage library was 7.73 × 10⁻⁶. 5 pfu / ml.

[0171] (2) Using serum to screen cDNA libraries

[0172] Immunoscreening was performed using the canine testis cDNA phage library constructed above. Specifically, host *E. coli* (XL1-Blue MRF') was infected with phages on NZY agarose plates (Φ90 x 15 mm), yielding 2210 clones. *E. coli* cells were cultured at 42°C for 3–4 hours to form plaques. The plates were covered with a nitrocellulose membrane (Hybond C Extra: GE Healthcare Bio-Science) impregnated with IPTG (isopropyl-β-D-thiogalactopyranoside) at 37°C for 4 hours to induce and express the protein, which was then transferred to the membrane. Subsequently, the membrane was recovered and immersed in TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.5% powdered skim milk and shaken overnight at 4°C to inhibit nonspecific reactions. The membrane was then reacted with 500-fold diluted serum from infected dogs at room temperature for 2–3 hours.

[0173] For the serum from the aforementioned affected dogs, serum collected from dogs with mammary cancer was used. This serum was stored at -80°C and pretreated before use. The method for pretreating the serum samples was as follows: Specifically, host *Escherichia coli* (XL1-BLue MRF') was infected with a phage expressing λZAP without the inserted foreign gene, and subsequently cultured overnight at 37°C on NZY agar plates. Then, a buffer containing 0.5M NaCl (0.2M NaHCO3, pH 8.3) was added to the plates, and the plates were incubated at 4°C for 15 hours. The supernatant was then collected as the *E. coli* / phage extract. The collected *E. coli* / phage extract was then passed through an NHS column (GE Healthcare Bio-Science), thereby immobilizing the proteins derived from *E. coli* / phages onto the column. The serum from the affected dogs was passed through the protein-immobilized column to react with it, and antibodies adsorbed onto *E. coli* and phages were removed from the serum. The serum fractions that had flowed through the column were diluted 500-fold with TBS containing 0.5% powdered skim milk. The resulting product was used as an immunoassay material.

[0174] The membrane, already stained with the treated serum and the aforementioned fusion protein, was washed four times with TBS-T (0.05% Tween 20 / TBS). Then, goat anti-canine IgG (goat anti-canine IgG-h+I HRP conjugated (BETHYL Laboratories)) diluted 5000-fold with TBS containing 0.5% powdered skim milk as a secondary antibody was incubated with the membrane at room temperature for 1 hour. Detection was performed using an enzymatic colorimetric reaction with NBT / BCIP reaction solution (Roche). Colonies corresponding to positive colorimetric sites were collected from NZY agarose plates (Φ90 x 15 mm) and lysed in 500 μl of SM buffer (100 mM NaCl, 10 mM MgClSO4, 50 mM Tris-HCl, 0.01% gelatin, pH 7.5). The second and third screenings were repeated using a method similar to that described above, thereby screening 30,940 phage clones that reacted with serum IgG until only one colony was identified as positive for the chromogenic reaction. Five positive clones were thus isolated.

[0175] (3) Homology search of isolated antigen genes

[0176] To perform nucleotide sequence analysis on the five positive clones isolated using the above method, the phage vector was converted into a plasmid vector. Specifically, 200 μl of plasmid vector with an absorbance OD of [missing value] was prepared. 600 The solution contained 1.0 μL of host *Escherichia coli* (XL1-Blue MRF'). This was mixed with 250 μL of purified phage solution, followed by 1 μL of ExAssist helper phage (STRATAGENE), and incubated at 37 °C for 15 min. 3 mL of LB medium was added, and the mixture was incubated at 37 °C for 2.5–3 h. Immediately after incubation, the solution temperature was maintained at 70 °C for 20 min using a water bath, followed by centrifugation at 4 °C and 1000 × g for 15 min. The supernatant was then collected as the phage particle solution. Subsequently, 200 μL of absorbance OD was prepared. 600 A 1.0 μL solution of host *Escherichia coli* (SOLR) containing phage particles was prepared. This solution was mixed with 10 μL of purified phage solution and incubated at 37 °C for 15 min. 50 μL of the solution was inoculated onto LB agar medium containing ampicillin (final concentration: 50 μg / ml) and incubated overnight at 37 °C. Transformed SOLR single colonies were collected and incubated at 37 °C on LB medium containing ampicillin (final concentration: 50 μg / ml). Plasmid DNA containing the target insert was purified using the QIAGEN Plasmid Mini-Preparation Kit.

[0177] The purified plasmid was analyzed for its full-length insert sequence using primer walking (SEQ ID NO:31 T3 primer and SEQ ID NO:32 T7 primer). Sequence analysis yielded gene sequences of SEQ ID NO:5, 7, 9, 11, and 13. A BLAST homology search (http: / / www.ncbi.nlm.nih.gov / BLAST / ) was performed using the nucleotide and corresponding amino acid sequences of these genes (SEQ ID NO:6, 8, 10, 12, and 14). As a result of this homology search using known genes, all five obtained genes encoded CAPRIN-1. The sequence identity among these five genes was 100% nucleotide and 99% amino acid, respectively, for the region to be translated into protein. Furthermore, the sequence identity of these genes with genes encoding human homologs was 94% nucleotide and 98% amino acid, respectively, for the region to be translated into protein. The nucleotide sequences of the human homolog are shown in SEQ ID NO:1 and 3, and its amino acid sequences are shown in SEQ ID NO:2 and 4. Furthermore, regarding the region to be translated into protein, the obtained canine gene exhibits 94% nucleotide sequence identity and 97% amino acid sequence identity with the gene encoding the bovine homolog. The nucleotide sequence of the bovine homolog is shown in SEQ ID NO:15, and its amino acid sequence is shown in SEQ ID NO:16. Furthermore, regarding the region to be translated into protein, the sequence identity between the gene encoding the human homolog and the gene encoding the bovine homolog is 94% nucleotide sequence identity and 93% to 97% amino acid sequence identity. Furthermore, regarding the region to be translated into protein, the obtained canine gene exhibits 93% nucleotide sequence identity and 97% amino acid sequence identity with the gene encoding the equine homolog. The nucleotide sequence of the equine homolog is shown in SEQ ID NO:17, and its amino acid sequence is shown in SEQ ID NO:18. Furthermore, regarding the regions to be translated into proteins, the sequence identity between the gene encoding the human homolog and the gene encoding the equine homolog is 93% nucleotide sequence identity and 96% amino acid sequence identity. Additionally, regarding the regions to be translated into proteins, the sequence identity between the obtained canine gene and the gene encoding the mouse homolog is 87% to 89% nucleotide sequence identity and 95% to 97% amino acid sequence identity. The nucleotide sequences of the mouse homolog are shown in SEQ ID NO: 19, 21, 23, 25, and 27, and their amino acid sequences are shown in SEQ ID NO: 20, 22, 24, 26, and 28.Furthermore, regarding the regions to be translated into proteins, the sequence identity between the gene encoding the human homolog and the gene encoding the mouse homolog is 89% to 91% nucleotide sequence identity and 95% to 96% amino acid sequence identity. Furthermore, regarding the regions to be translated into proteins, the sequence identity between the obtained canine gene and the gene encoding the chicken homolog is 82% nucleotide sequence identity and 87% amino acid sequence identity. The nucleotide sequence of the chicken homolog is shown in SEQ ID NO:29, and its amino acid sequence is shown in SEQ ID NO:30. Furthermore, regarding the regions to be translated into proteins, the sequence identity between the gene encoding the human homolog and the gene encoding the chicken homolog is 81% to 82% nucleotide sequence identity and 86% amino acid sequence identity.

[0178] (4) Gene expression analysis in each tissue

[0179] The expression of genes obtained in normal canine and human tissues and various cell lines using the above method was examined by RT-PCR. Reverse transcription was performed as follows: Specifically, TRIZOL reagent (Invitrogen) was used, according to the included protocol, from each tissue (50 mg to 100 mg) and each cell line (5 to 10 x 10⁻⁶ mg). 6 Total RNA was extracted from cells. cDNA was synthesized from the extracted total RNA using the Superscript first-strand synthesis system (Invitrogen) for RT-PCR according to the protocol contained therein. PCR was performed using primers specific to the obtained genes (SEQ ID NO: 33 and 34) as follows. Specifically, a reaction solution was prepared to a total volume of 25 μl by adding the following reagents (0.25 μl of sample prepared by reverse transcription, the above primers (2 μM each), dNTPs (0.2 mM each), and 0.65 U ExTaq polymerase (Takara Shuzo Co., Ltd.)) and the accompanying buffer, and a Thermal Cycler (BIO RAD) for the following 30 cycles: 94 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds. In addition, the aforementioned gene-specific primers were used to amplify the region between nucleotides 206 and 632 of the nucleotide sequence of SEQ ID NO:5 (canine CAPRIN-1 gene) and the region between nucleotides 698 and 1124 of the nucleotide sequence of SEQ ID NO:1 (human CAPRIN-1 gene). As a comparative control, GAPDH-specific primers (SEQ ID NO:35 and 36) were also used. The results are as follows: Figure 1The study showed strong expression in the testicular tissue of healthy dogs, as well as in canine mammary and adenocarcinoma tissues. Furthermore, expression of the human homolog of the obtained gene was confirmed. As a result, similar to the case of the canine CAPRIN-1 gene, its expression was only confirmed in the testes in normal tissues. However, in cancer cell lines, expression was detected in many types of cancer cell lines, such as breast cancer, brain tumors, leukemia, lung cancer, and esophageal cancer cell lines. Its expression was particularly confirmed in many breast cancer cell lines. Based on these results, it was confirmed that CAPRIN-1 expression is not observed in normal tissues other than the testes; however, CAPRIN-1 is expressed in many cancer cell lines, especially in breast cancer cell lines.

[0180] In addition, Figure 1 In the diagram, reference number 1 on the vertical axis shows the expression pattern of each gene identified above, and reference number 2 on the same axis shows the expression pattern of the GAPDH gene used as a comparison control.

[0181] (5) Preparation of polyclonal antibodies against CAPRIN-1-derived peptides

[0182] To obtain an antibody binding to CAPRIN-1, a CAPRIN-1-derived peptide (Arg-Asn-Leu-Glu-Lys-Lys-Gly-Lys-Leu-Asp-Asp-Tyr-Gln (SEQ ID NO:37)) was synthesized. The peptide (1 mg) was mixed as the antigen with an equal volume of incomplete Freund's adjuvant (IFA) solution. This mixture was administered subcutaneously to rabbits four times every two weeks. Blood was then collected to obtain antiserum containing polyclonal antibodies. Furthermore, this antiserum was purified using protein G support (GE Healthcare Bio-Sciences) to obtain polyclonal antibodies against the CAPRIN-1-derived peptide. Additionally, antibodies obtained by purifying the serum of rabbits that had not been administered the antigen using protein G support in the same manner were selected as control antibodies.

[0183] (6) Analysis of antigen protein expression on cancer cells

[0184] Next, we examined whether CAPRIN-1 protein was expressed on the cell surface of seven breast cancer cell lines (MDA-MB-157, T47D, MRK-nu-1, MDA-MB-231V, BT20, SK-BR-3, and MDA-MB-231T), in which CAPRIN-1 gene expression was strongly confirmed. For each human breast cancer cell line (10) in which gene expression had been confirmed as described above... 6Centrifuge cells in 1.5 ml microcentrifuge tubes. Add the polyclonal antibody (2 μg) (5 μl) of the anti-CAPRIN-1 derived peptide prepared in (5) above. Resuspend the product in PBS containing 0.1% fetal bovine serum (95 μl) and incubate on ice for 1 hour. After washing with PBS, resuspend the product in PBS containing FITC-labeled goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, Inc.) (5 μl) and 0.1% fetal bovine serum (FBS) (95 μl) and incubate on ice for 1 hour. After washing with PBS, measure the fluorescence intensity using FACSCalibur (Becton, Dickinson and Company). Simultaneously, use the control antibody prepared in (5) instead of the polyclonal antibody of the anti-CAPRIN-1 derived peptide to perform similar steps to prepare a control. As a result, it was found that the fluorescence intensity in all cells supplemented with the anti-human CAPRIN-1 antibody was at least 30% stronger than that in the control cells. Specifically, the following enhancements in fluorescence intensity were confirmed: MDA-MB-157: 184%, T47D: 221%, MRK-nu-1: 115%, MDA-MB-231V: 82%, BT20: 32%, SK-BR-3: 279%, and MDA-MB-231T: 80%. Based on these results, CAPRIN-1 protein expression on the cell surface of the aforementioned human cancer cell lines was confirmed. Furthermore, the rate of increase in fluorescence intensity was expressed as the rate of increase in mean fluorescence intensity (MFI value) within the cells, calculated using the following equation.

[0185] The rate of increase in average fluorescence intensity (%) = ((MFI value of cells reacting with anti-human CAPRIN-1 antibody) - (MFI value of control)) / (MFI value of control) × 100

[0186] (7) Immunohistochemical staining

[0187] (7)-1: CAPRIN-1 expression in normal mouse and dog tissues

[0188] Mice (Balb / c, female) and dogs (Beagles, female) were bled under ether anesthesia and ketamine / isoflurane anesthesia. Following laparotomy, organs (stomach, liver, eyeball, thymus, muscle, bone marrow, uterus, small intestine, esophagus, heart, kidney, salivary glands, large intestine (colon), mammary gland, brain, lung, skin, adrenal gland, ovary, pancreas, spleen, and bladder) were transferred separately to 10-cm petri dishes containing PBS. Each organ was dissected in PBS and fixed overnight by refluxing with 0.1M phosphate-buffered saline (pH 7.4) containing 4% paraformaldehyde (PFA). The refluxing solution was discarded, and the tissue surface of each organ was rinsed with PBS. A PBS solution containing 10% sucrose was added to 50-ml microcentrifuge tubes. Each tissue was then placed in its respective tube and shaken at 4°C for 2 hours using a rotor. Each solution was replaced with PBS containing 20% ​​sucrose, and the tubes were then allowed to stand at 4°C until sedimentation. Replace each solution with PBS containing 30% sucrose and allow it to stand at 4°C until the tissue settles. Remove each tissue and cut the desired area with a scalpel. Then, apply OCT compound (Tissue Tek) to each tissue surface and spread it out, subsequently placing the tissue on a cryomold. Place the cryomold on dry ice for rapid freezing. Use a cryostat (LEICA) to cut the tissue into sections 10 μm to 20 μm thick, and air-dry the tissue sections on the slides with a hairdryer for 30 minutes to prepare slides with the tissue sections on them. Then, place each slide in a staining bottle filled with PBS-T (physiological saline containing 0.05% Tween 20), changing the PBS-T every 5 minutes for 3 times. Wipe away excess moisture around each sample with Kimwipes paper towels and delineate the sections with DAKOPEN (DAKO). MOM mouse Ig blocking reagent (VECTASTAIN) was applied to mouse tissue as a blocking solution, and PBS-T solution containing 10% fetal bovine serum was applied to dog tissue as a blocking solution. These products were incubated in a humidified chamber at room temperature for 1 hour. Subsequently, a solution containing a monoclonal antibody against CAPRIN-1 (monoclonal antibody #6) was prepared, wherein the antibody was prepared in Example 4 and contained the heavy chain variable region of SEQ ID NO:73 and the light chain variable region of SEQ ID NO:77, and reacted with the surface of cancer cells. The antibody was adjusted to a concentration of 10 μg / ml in the blocking solution. This solution was applied to each slide and then incubated overnight at 4°C in a humidified chamber. After washing three times with PBS-T for 10 minutes each time, MOM biotin-labeled anti-IgG antibody (VECTASTAIN) diluted 250-fold with the blocking solution was applied to each slide, followed by incubation in a humidified chamber at room temperature for 1 hour.After washing three times with PBS-T for 10 minutes each time, apply avidin-biotin ABC reagent (VECTASTAIN) to the slide and then incubate at room temperature for 5 minutes in a humidified chamber. After washing three times with PBS-T for 10 minutes each time, apply DAB staining solution (10 mg DAB + 10 μl 30% H2O2 / 0.05 M Tris-HCl, pH 7.6, 50 ml) to the slide and then incubate at room temperature for 30 minutes in a humidified chamber. Rinse the slide with distilled water and then apply hematoxylin reagent (DAKO). After incubating the slide at room temperature for 1 minute in a humidified chamber, rinse with distilled water. Immerse the slide sequentially in 70%, 80%, 90%, 95%, and 100% ethanol solutions for 1 minute each time, and then incubate overnight in xylene. The slides were removed, mounted with Glycergel mounting medium (DAKO), and then observed. As a result, weak CAPRIN-1 expression was observed intracellularly in cells from all salivary gland, kidney, colon, and stomach tissues; however, no CAPRIN-1 expression was observed on the cell surface. Furthermore, no expression was observed at all in tissues from other organs. Similar results were obtained when using an anti-CAPRIN-1 monoclonal antibody (monoclonal antibody #9) containing the heavy chain variable region of SEQ ID NO:103 and the light chain variable region of SEQ ID NO:107.

[0189] (7)-2: CAPRIN-1 expression in canine mammary cancer tissue

[0190] Using 108 frozen canine mammary gland tissue samples from dogs diagnosed with malignant mammary gland disease by pathological diagnosis, frozen sections were prepared onto glass slides using a method similar to that described above, and immunohistochemical staining was performed using monoclonal antibody #6 prepared in Example 4. As a result, CAPRIN-1 expression was confirmed in 100 (92.5%) of the 108 samples. CAPRIN-1 is particularly strongly expressed on the surface of highly atypically differentiated cancer cells. Furthermore, similar results were obtained when using monoclonal antibody #9 generated in Example 4.

[0191] (7)-3: CAPRIN-1 expression in human breast cancer tissue

[0192] Immunohistochemical staining was performed on 188 breast cancer tissue samples using a paraffin-embedded human breast cancer tissue array (BIOMAX). After treatment at 60°C for 3 hours, the human breast cancer tissue array was added to staining bottles filled with xylene, which was then replaced every 5 minutes for 3 times. Subsequently, a similar procedure was repeated using ethanol and PBS-T instead of xylene. The human breast cancer tissue array was added to staining bottles filled with 10 mM citrate buffer (pH 6.0) containing 0.05% Tween 20, treated at 125°C for 5 minutes, and allowed to stand at room temperature for 40 minutes or longer. Excess moisture was wiped away around each sample using Kimwipes paper towels, each section was circled with DAKOPEN, and a sufficient amount of peroxidase inhibitor (DAKO) was added dropwise. After allowing the results to stand at room temperature for 5 minutes, they were then added to staining bottles filled with PBS-T. The PBS-T was replaced every 5 minutes for 3 times. A PBS-T solution containing 10% FBS was applied to the array as a blocking solution, and the array was placed in a humidifier at room temperature for 1 hour. Subsequently, a solution containing monoclonal antibody #6 prepared in Example 4, which reacts with the surface of cancer cells, was prepared and adjusted to a concentration of 10 μg / ml using a PBS-T solution containing 5% FBS. This solution was applied, and the slide was then incubated overnight at 4°C in a humidifier. After washing three times with PBS-T for 10 minutes each time, an appropriate amount of Peroxidase Labelled Polymer Conjugated (DAKO) was added dropwise, and the slide was then incubated in a humidifier at room temperature for 30 minutes. After washing three times with PBS-T for 10 minutes each time, DAB staining solution (DAKO) was applied, and the slide was incubated at room temperature for approximately 10 minutes. The DAB staining solution was discarded, and the slide was then washed three times with PBS-T for 10 minutes each time. The slides were rinsed with distilled water and then sequentially immersed in 70%, 80%, 90%, 95%, and 100% ethanol solutions for 1 minute each, followed by overnight incubation in xylene. The slides were then removed, mounted with Glycergel mounting medium (DAKO), and observed. As a result, strong CAPRIN-1 expression was observed in 138 (73%) of all 188 breast cancer tissue samples. Furthermore, similar results were obtained when using the monoclonal antibody #9 prepared in Example 4.

[0193] (7)-4: CAPRIN-1 expression in human malignant brain tumors

[0194] Immunohistochemical staining was performed on 247 malignant brain tumor tissue samples from a paraffin-embedded human malignant brain tumor tissue array (BIOMAX) using monoclonal antibody #6 prepared in Example 4, in a manner similar to that described above (7)-3. As a result, strong CAPRIN-1 expression was observed in 227 (92%) of the 247 malignant brain tumor tissue samples. Furthermore, similar results were obtained when using monoclonal antibody #9 prepared in Example 4.

[0195] (7)-5: CAPRIN-1 expression in lymph nodes of human breast cancer metastases

[0196] Immunohistochemical staining was performed on 150 human breast cancer metastatic lymph node tissue samples from a paraffin-embedded human breast cancer metastatic lymph node tissue array (BIOMAX) using monoclonal antibody #6 prepared in Example 4, in a manner similar to that described above (7)-3. As a result, strong CAPRIN-1 expression was observed in 136 (90%) of the 150 human breast cancer metastatic lymph node tissue samples. In particular, it was revealed that CAPRIN-1 is also strongly expressed in cancer tissue metastasized from breast cancer. Furthermore, similar results were obtained when using monoclonal antibody #9 prepared in Example 4.

[0197] Example 2: Antitumor effect of anti-CAPRIN-1 antibody on cancer cells (ADCC activity)

[0198] Next, we examined whether the anti-CAPRIN-1 antibody could damage tumor cells expressing CAPRIN-1. The evaluation was performed using the polyclonal antibody against the human CAPRIN-1-derived peptide prepared in Example 1. Two human breast cancer cell lines (T47D and MDA-MB-157) confirmed to express CAPRIN-1 (10 cells each) were used for the evaluation. 6 Cells were collected into 50 ml centrifuge tubes. Chromium 51 (100 μCi) was added, followed by incubation at 37°C for 2 hours. Afterwards, the cells were washed three times with RPMI 1640 medium containing 10% fetal bovine serum, and then added to the wells of a 96-well V-bottom culture plate (10 cells per well). 3 Add the above-mentioned polyclonal antibody against human CAPRIN-1 derived peptide (1 μg per well) to each well. Then, add lymphocytes isolated from rabbit peripheral blood (2 × 10⁶ cells per well). 5Cells were cultured at 37°C and 5% CO2 for 4 hours. After culture, the level of chromium (Cr)51 released from damaged tumor cells in each culture supernatant was determined. The ADCC activity of the polyclonal antibody against the human CAPRIN-1 derived peptide was then calculated against cancer cells. The results confirmed ADCC activity against T47D (15.4%) and MDA-MB-157 (17.3%) (see [link to relevant documentation]). Figure 2 and 3 Meanwhile, when the same method was performed using a control antibody prepared from peripheral blood of rabbits that had never been immunized with the antigen (Example 1(5)), or when no antibody was added, essentially no activity was observed (see Example 1(5)). Figure 2 and 3 Therefore, this reveals that ADCC activity induced by the use of anti-CAPRIN-1 antibody can damage tumor cells expressing CAPRIN-1.

[0199] Furthermore, regarding cytotoxic activity, the anti-CAPRIN-1 antibody, mouse lymphocytes, and 10 chromium-51-doped [unclear] used in this invention were [unclear]. 3 Cells from a leukemia cell line were mixed together and cultured for 4 hours. The level of chromium 51 released into the culture medium was then determined. Cytotoxic activity against the leukemia cell line was then calculated using the following equation*.

[0200] *Equation: Cytotoxic activity (%) = [(Chromium 51 level released from T47D or MDA-MB-157 mouse lymphoma cells supplemented with anti-CAPRIN-1 antibody and MDA-MB-157) / (Chromium 51 level released from target cells supplemented with 1N hydrochloric acid)] × 100

[0201] Example 3: Preparation of a novel human cancer antigen protein

[0202] (1) Preparation of recombinant proteins

[0203] Recombinant proteins of human homologous genes were prepared based on the gene obtained in Example 1, using the following method. PCR was performed using a Thermal Cycler (BIO RAD) and a reaction solution adjusted to a total volume of 50 μl by adding reagents and the accompanying buffer (1 μl cDNA (derived from tissue / cell-derived cDNA prepared in Example 1, and whose expression was observed by RT-PCR), two types of primers containing SacI and XhoI restriction enzyme site sequences (0.4 μM each, SEQ ID NO:38 and 39), 0.2 mM dNTPs, and 1.25 U PrimeSTAR HS polymerase (Takara Shuzo)). The PCR was performed for 30 cycles at 98°C for 10 seconds and 68°C for 2.5 minutes. The region encoding the full-length amino acid sequence of SEQ ID NO:2 was amplified using the aforementioned two primers. After PCR, the amplified DNA was electrophoresed on a 1% agarose gel and a DNA fragment of approximately 2.1 kbp was purified using the QIAquick Gel Extraction Kit (QIAGEN).

[0204] The purified DNA fragment was ligated into the pCR-Blunt cloning vector (Invitrogen). The resulting product was transformed into *E. coli*, and the plasmid was recovered. Sequencing confirmed that the amplified gene fragment matched the target sequence. The plasmid matching the target sequence was treated with SacI and XhoI restriction enzymes, and the product was then purified using a QIAquick gel extraction kit. The target gene sequence was then inserted into the *E. coli* pET30b expression vector (Novagen) treated with SacI and XhoI restriction enzymes. This vector can produce a recombinant protein fused with a His tag. The plasmid was transformed into *E. coli* BL21(DE3) for expression, and expression was induced with 1 mM IPTG, thereby expressing the target protein in *E. coli*.

[0205] (2) Purification of recombinant proteins

[0206] Each of the recombinant *E. coli* strains expressing SEQ ID NO:1 obtained above was cultured in LB medium containing 30 μg / ml kanamycin at 37°C until the absorbance at 600 nm reached approximately 0.7. Isopropyl-β-D-1-thiogalactopyranoside was then added to a final concentration of 1 mM, followed by incubation at 37°C for 4 hours. Cells were then collected by centrifugation at 4800 rpm for 10 minutes. The cell pellet was resuspended in phosphate-buffered saline and centrifuged at 4800 rpm for 10 minutes to wash the cells.

[0207] Cells were suspended in phosphate-buffered saline and then sonicated on ice. The lysate of the sonicated E. coli was centrifuged at 6000 rpm for 20 minutes. The resulting supernatant was used as the soluble fraction, and the resulting precipitate was used as the insoluble fraction.

[0208] Soluble fractions were added to a nickel chelate column prepared according to standard techniques (carrier: Chelateing Sepharose Fast Flow (GE Health Care); column volume: 5 ml; 50 mM hydrochloric acid buffer (pH 8.0) as equilibration buffer). Unadsorbed fractions were washed away with approximately 10 column volumes of 50 mM hydrochloric acid buffer (pH 8.0) and 20 mM phosphate buffer (pH 8.0) containing 20 mM imidazole. Immediately after washing, six beds were eluted with 20 mM phosphate buffer (pH 8.0) containing 100 mM imidazole. The eluted fraction in 20 mM phosphate buffer (pH 8.0) containing 100 mM imidazole (the elution of the target protein was confirmed by Coomassie staining) was added to a strong anion exchange column (carrier: Q Sepharose Fast Flow (GE Health Care); column volume: 5 ml; 20 mM phosphate buffer (pH 8.0) as equilibration buffer). Unadsorbed fractions were washed away with 10 column volumes of 20 mM phosphate buffer (pH 7.0) and 20 mM phosphate buffer (pH 7.0) containing 200 mM sodium chloride. Immediately after washing, five beds were eluted with 20 mM phosphate buffer (pH 7.0) containing 400 mM sodium chloride. This yielded purified fractions of proteins with the amino acid sequences shown in SEQ ID NO:2.

[0209] 200 μl of each purified preparation obtained by the above method was dispersed into 1 ml of reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2, pH 7.4), and then 2 μl of enterokinase (Novagen) was added. The preparations were allowed to react overnight at room temperature to remove the His tag, and then purified using an enterokinase cleavage and capture kit (Novagen) according to the accompanying instructions. Subsequently, 1.2 ml of each purified preparation obtained by the above method was replaced with physiological phosphate buffer (Nissui Pharmaceutical Co., Ltd.) using NANOSEP 10K OMEGA (PALL) ultrafiltration. Aseptic filtration was performed through 0.22 μm HT Tuffryn Acrodisc (PALL), and the resulting product was used in the following experiments.

[0210] Example 4: Preparation of anti-CAPRIN-1 monoclonal antibody

[0211] The antigen protein (human CAPRIN-1) (100 μg) shown in SEQ ID NO:2 prepared in Example 3 was mixed with an equal volume of MPL+TDM adjuvant (Sigma). This mixture was used as the antigen solution for each mouse. The antigen solution was administered intraperitoneally to 6-week-old Balb / c mice (Japan SLC Inc.), and the solution was administered 3 or 24 times at weekly intervals to complete the immunization. Three days after the final immunization, the spleen was removed and then ground between two sterile glass slides. Each product was washed with PBS(-) (Nissui) and then centrifuged at 1500 rpm for 10 minutes, and the procedure of removing the supernatant was repeated 3 times. Thus, spleen cells were obtained. The spleen cells obtained therefrom were mixed with mouse myeloma cells SP2 / 0 (purchased from ATCC) at a ratio of 10:1. PEG solution was added to the cells, wherein the PEG solution was prepared by mixing 200 μl of RPMI 1640 medium containing 10% FBS heated at 37°C with 800 μl of PEG1500 (Boehringer). The solution was allowed to stand for 5 minutes to allow cell fusion. The resulting mixture was centrifuged at 1700 rpm for 5 minutes to remove the supernatant. The cells were resuspended in 150 ml of RPMI 1640 medium (HAT selective medium) containing 15% FBS and supplemented with 2% equivalent HAT solution (Gibco), and seeded at 100 μl per well in 15 96-well plates (Nunc). The cells were cultured at 37°C and 5% CO2 for 7 days to obtain hybridomas resulting from the fusion of spleen cells and myeloma cells.

[0212] Hybridomas were selected based on the binding affinity of antibodies generated from these hybridomas to the CAPRIN-1 protein. The CAPRIN-1 protein solution (1 μg / ml) prepared in Example 3 was added to 100 μl per well of a 96-well plate, and the plate was incubated at 4°C for 18 hours. Each well was washed three times with PBS-T, and 400 μl of 0.5% bovine serum albumin (BSA) solution (Sigma) was added to each well, followed by incubation at room temperature for 3 hours. The solution was removed, and each well was washed three times with 400 μl of PBS-T. 100 μl of the culture supernatant from each hybridoma obtained above was added to each well, and the plate was incubated at room temperature for 2 hours. Each well was washed three times with PBS-T, and 100 μl of HRP-labeled anti-mouse IgG (H+L) antibody (Invitrogen) diluted 5000-fold with PBS was added to each well, followed by incubation at room temperature for 1 hour. Each well was washed three times with PBS-T. 100 μl of TMB substrate solution (Thermo) was added to each well, and the mixture was allowed to stand for 15 to 30 minutes to allow for color development. After color development, 100 μl of 1N sulfuric acid was added to each well to terminate the reaction. The absorbance at 450 nm and 595 nm was measured using a spectrophotometer. As a result, multiple hybridomas with high absorbance values ​​that produced antibodies were selected.

[0213] The selected hybridomas were added to 96-well plates at a rate of 0.5 hybridoma cells per well and cultured. After 1 week, single colony hybridomas were observed to form in the wells. The cells in these wells were further cultured. Hybridomas were selected using the binding affinity of an antibody (derived from the cloned hybridoma) to the CAPRIN-1 protein as an indicator. The CAPRIN-1 protein solution (1 μg / ml) prepared in Example 3 was added to 96-well plates at a rate of 100 μl per well and the plates were incubated at 4°C for 18 hours. Each well was washed three times with PBS-T, and 400 μl of 0.5% BSA solution was added to each well, and the plates were then incubated at room temperature for 3 hours. The solution was removed, and each well was washed three times with 400 μl of PBS-T. 100 μl of the culture supernatant obtained above for each hybridoma was added to each well, and the plates were incubated at room temperature for 2 hours. Each well was washed three times with PBS-T. 100 μl of HRP-labeled anti-mouse IgG (H+L) antibody (Invitrogen) diluted 5000-fold with PBS was added to each well, and the mixture was incubated at room temperature for 1 hour. Each well was then washed three times with PBS-T. 100 μl of TMB substrate solution (Thermo) was added to each well, and the mixture was incubated for 15 to 30 minutes to allow for color development. After color development, 100 μl of 1N sulfuric acid was added to each well to terminate the reaction. The absorbance at 450 nm and 595 nm was measured using a spectrophotometer. As a result, 150 hybridoma cell lines were obtained that produced monoclonal antibodies exhibiting reactivity to CAPRIN-1 protein.

[0214] Next, monoclonal antibodies that showed reactivity with the surface of breast cancer cells expressing CAPRIN-1 were selected from these monoclonal antibodies. Specifically, they were centrifuged in 1.5 ml microcentrifuge tubes for 10 minutes. 6 MDA-MB-231V human breast cancer cell line was used. Each hybridoma supernatant prepared above (100 μl) was added and incubated on ice for 1 hour. After washing with PBS, FITC-labeled goat anti-mouse IgG antibody (Invitrogen) diluted 500-fold with PBS containing 0.1% FBS was added and incubated on ice for 1 hour. After washing with PBS, fluorescence intensity was measured using a FACSCalibur (Becton, Dickinson & Company). A control was prepared by performing a similar procedure using untreated 6-week-old Balb / c mouse serum diluted 500-fold with hybridoma medium instead of the antibody. As a result, 11 monoclonal antibodies (#1 to #11) with stronger fluorescence intensity than the control were selected; that is, monoclonal antibodies that react with the surface of breast cancer cells were selected.

[0215] Example 5: Characterization of the selected antibody

[0216] (1) Cloning of the variable region gene of the anti-CAPRIN-1 monoclonal antibody

[0217] mRNA was extracted from the hybridoma cell lines that produced the 11 monoclonal antibodies selected in Example 4. Using primers specific to sequences derived from mouse FR1 and mouse FR4, the heavy chain variable (VH) region gene and light chain variable (VL) region gene of each anti-CAPRIN-1 monoclonal antibody were obtained by RT-PCR. For sequencing, these genes were cloned into the pCR2.1 vector (Invitrogen).

[0218] (1)-1RT-PCR

[0219] mRNA micropurification kit (GE Healthcare) was used to purify mRNA from each hybridoma cell line (10 6 mRNA was prepared from individual cells. Each obtained mRNA was reverse transcribed using the SuperScript II First-Strand Synthesis Kit (Invitrogen) for cDNA synthesis. The procedure described above was performed according to the protocol provided with the kit.

[0220] Each obtained cDNA was used for antibody gene amplification via PCR.

[0221] To obtain the VH region gene, primers specific to the mouse heavy chain FR1 sequence (SEQ ID NO: 130) and primers specific to the mouse heavy chain FR4 sequence (SEQ ID NO: 131) were used. Similarly, to obtain the VL region gene, primers specific to the mouse light chain FR1 sequence (SEQ ID NO: 132) and primers specific to the mouse light chain FR4 sequence (SEQ ID NO: 133) were used. These primers were designed according to Jones, ST and Bending, MMBio / Technology 9, 88-89 (1991). For PCR, Ex-taq (Takara Bio Inc.) was used. Each cDNA sample was mixed with 10x EX Taq buffer (5 μl), dNTP mixture (2.5 mM) (4 μl), primers (1.0 μM) (2 μl each), and Ex Taq (5 U / μl) (0.25 μl). The total volume was adjusted to 50 μl with sterile water. PCR was performed under the following conditions: after treatment at 94°C for 2 minutes, no more than 30 cycles were performed: denaturation at 94°C for 1 minute, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute.

[0222] (1)-2 clones

[0223] Each PCR product obtained above was subjected to agarose gel electrophoresis, followed by excision of the VH and VL DNA bands. The DNA was purified using the QIAquick Gel Purification Kit (QIAGEN) according to the kit's instructions. Each purified DNA was cloned into the pCR2.1 vector using the TA Cloning Kit (Invitrogen). Each DNA-ligated vector was transformed into DH5α competent cells (TOYOBO) using standard methods. Each transformant (10 clones) was cultured overnight at 37°C in medium (100 μg / ml ampicillin). The obtained plasmid DNA was purified using the Qiaspin Mini-Preparation Kit (QIAGEN).

[0224] (1)-3 sequencing

[0225] Using a fluorescence sequencer (ABI; DNA sequencer 3130XL) and the BigDye terminator Ver. 3.1 cycle sequencing kit (ABI), gene sequence analysis was performed on the VH and VL regions of each plasmid obtained above, following the kit instructions, using the M13 forward primer (SEQ ID NO: 134) and the M13 reverse primer (SEQ ID NO: 135). The results confirmed that each gene sequence was identical across the 10 clones.

[0226] The amino acid sequences of the variable regions of the heavy chains of the obtained monoclonal antibodies are shown in SEQ ID NO:43, SEQ ID NO:73, SEQ ID NO:83, SEQ ID NO:93, SEQ ID NO:103, SEQ ID NO:113 and SEQ ID NO:123; the amino acid sequences of the variable regions of the light chains of the obtained monoclonal antibodies are shown in SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:68, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO:97, SEQ ID NO:107, SEQ ID NO:117 and SEQ ID NO:127.

[0227] Specifically, monoclonal antibody #1 comprises the heavy chain variable region of SEQ ID NO:43 and the light chain variable region of SEQ ID NO:47. Monoclonal antibody #2 comprises the heavy chain variable region of SEQ ID NO:43 and the light chain variable region of SEQ ID NO:53. Monoclonal antibody #3 comprises the heavy chain variable region of SEQ ID NO:43 and the light chain variable region of SEQ ID NO:58. Monoclonal antibody #4 comprises the heavy chain variable region of SEQ ID NO:43 and the light chain variable region of SEQ ID NO:63. Monoclonal antibody #5 comprises the heavy chain variable region of SEQ ID NO:43 and the light chain variable region of SEQ ID NO:68. Monoclonal antibody #6 comprises the heavy chain variable region of SEQ ID NO:73 and the light chain variable region of SEQ ID NO:77. Monoclonal antibody #7 comprises the heavy chain variable region of SEQ ID NO:83 and the light chain variable region of SEQ ID NO:87. Monoclonal antibody #8 contains the heavy chain variable region of SEQ ID NO:93 and the light chain variable region of SEQ ID NO:97. Monoclonal antibody #9 contains the heavy chain variable region of SEQ ID NO:103 and the light chain variable region of SEQ ID NO:107. Monoclonal antibody #10 contains the heavy chain variable region of SEQ ID NO:113 and the light chain variable region of SEQ ID NO:117. Monoclonal antibody #11 contains the heavy chain variable region of SEQ ID NO:123 and the light chain variable region of SEQ ID NO:127.

[0228] (2) Application of the obtained monoclonal antibody CAPRIN-1 expression on the cell surface of different cells

[0229] Next, we examined whether CAPRIN-1 protein was expressed on the cell surface of seven breast cancer cell lines confirmed to express the CAPRIN-1 gene (MDA-MB-157, T47D, MRK-nu-1, MDA-MB-231V, BT20, SK-BR-3, and DA-MB-231T), three other breast cancer cell lines (MDA-MB-231C, MCF-7, and ZR75-1), six glioma cell lines (T98G, SNB19, U251, and U87G), three renal cell carcinoma cell lines (Caki-1, Caki2, and A498), one gastric cancer cell line (MKN45), one colorectal cancer cell line (Caco2), three lung cancer cell lines (A549, QG56, and PC8), and three leukemia cell lines (Namalwa, BDCM, and RPI1788). Each cell line was centrifuged in 1.5 ml microcentrifuge tubes (10... 6(cells). Hybridoma supernatants containing anti-CAPRIN-1 monoclonal antibodies #1 to #10, prepared in Example 4 and reacting with the cancer cell surface (100 μl of each), were added and incubated on ice for 1 hour. After washing with PBS, each product was suspended in FITC-labeled goat anti-mouse IgG antibody (Invitrogen Corporation) diluted 500-fold with PBS containing 0.1% FBS, and then incubated on ice for 1 hour. After washing with PBS, fluorescence intensity was measured using a FACSCalibur (Becton, Dickinson and Company). Simultaneously, control antibodies prepared in (5) above were used as a control instead of hybridoma supernatants containing anti-CAPRIN-1 monoclonal antibodies #1 to #11, and the same procedure was performed to obtain a control. The results showed that all cells with added monoclonal antibodies #1 to #11 exhibited at least 30% stronger fluorescence intensity than the control cells. Specifically, for example, when using monoclonal antibody #9, the following enhancements in fluorescence intensity were observed: MDA-MB-157: 211%; T47D: 145%; MRK-nu-1: 123%; MDA-MB-231V: 251%; BT20: 168%; and MDA-MB-231T: 94%. Based on this, CAPRIN-1 protein expression on the cell surface of the aforementioned human cancer cell lines was confirmed. Furthermore, the enhancement rate of fluorescence intensity was expressed as the enhancement rate of the mean fluorescence intensity (MFI value) in the cells. This was calculated using the following equation.

[0230] The average fluorescence intensity enhancement rate (%) = ((MFI value of cells reacting with anti-human CAPRIN-1 antibody) - (MFI value of control)) / (MFI value of control) × 100

[0231] (3) Antitumor effect of anti-CAPRIN-1 antibody on cancer cells (ADCC activity)

[0232] The selected anti-CAPRIN-1 monoclonal antibodies #1 to #11 were evaluated based on their cytotoxic activity (ADCC activity) against cancer cells. Hybridomas producing monoclonal antibodies #1 to #11 were cultured in hybridoma SFM medium (Invitrogen). Each supernatant was purified using Hitrap protein A agarose FF (GE Healthcare), followed by replacement with PBS(-) and purification through a 0.22-μm filter (Millipore). Each yield was used as an antibody for activity determination. Human breast cancer MDA-MB-157 cell line (10 6Cells were collected into 50 ml centrifuge tubes. Chromium 51 (100 μCi) was added, followed by incubation at 37°C for 2 hours. Afterwards, the cells were washed three times with RPMI 1640 medium containing 10% FBS. Cells were then added to the wells of a 96-well V-bottom culture plate (10 cells per well). 3 (100 cells). This prepares the target cells. The purified antibody (1 μg per well) is then added. Next, mouse lymphocytes isolated from mouse spleens (2 × 10⁶ cells per well) are added. 5 (cells), and then cultured at 37°C and 5% CO2 for 4 hours. After culture, the level of chromium (Cr)51 released from the damaged tumor cells in each culture supernatant was determined. Then, the ADCC activity of each polyclonal antibody against the human CAPRIN-1 derived peptide against the cancer cells was calculated. As a result, all monoclonal antibodies #1 to #11 showed ADCC activity (20% or more) against MDA-MB-157. Specifically, for example, the following cytotoxic activities were obtained: #1: 22.1%; #2: 29.1%; #6: 30.2%; and #9: 32.4% (see Figure 4 Meanwhile, when the monoclonal antibody prepared in Example 4, which reacts with the CAPRIN-1 protein itself but not with the surface of cancer cells, was used in a similar manner, no cytotoxic activity was confirmed (see Example 4). Figure 4 The results above demonstrate that the obtained anti-CAPRIN-1 monoclonal antibodies (#1 to #11) impair CAPRIN-1-expressing cancer cells by exhibiting ADCC activity.

[0233] (4) Antitumor effect of anti-CAPRIN-1 antibody on cancer cells (CDC activity)

[0234] The selected anti-CAPRIN-1 monoclonal antibodies #1 to #11 were evaluated based on their cytotoxic activity (CDC activity) against cancer cells. Blood collected from rabbits via blood sampling was added to Eppendorf tubes, incubated at room temperature for 60 minutes, and then centrifuged at 3000 rpm for 5 minutes. Serum for CDC activity determination was thus prepared. Human breast cancer MDA-MB-231V cell line (10... 5 Cells were collected into 50 ml centrifuge tubes. Chromium 51 (100 μCi) was added, followed by incubation at 37°C for 2 hours. Afterwards, the cells were washed three times with RPMI medium containing 10% FBS, and then resuspended in RPMI medium containing 50% rabbit serum prepared above. Cells were then added to the wells of a 96-well V-bottom culture plate (10 cells per well). 3(cells). Antibodies #1 to #11 obtained above (3) were added to wells (1 μg per well) and then cultured at 37°C and 5% CO2 for 4 hours. After culture, the level of chromium (Cr)51 released from the damaged tumor cells in each culture supernatant was determined. Then, the CDC activity against MDA-MB-231V exhibited by the anti-CAPRIN-1 monoclonal antibody in each hybridoma supernatant was calculated. As a result, all monoclonal antibodies #1 to #11 exhibited CDC activity (30% or more). Meanwhile, when the same method was performed using the monoclonal antibody prepared in Example 4 that reacts with the CAPRIN-1 protein itself but not with the surface of cancer cells, no cytotoxic activity was confirmed (see Figure 4 Therefore, it was revealed that anti-CAPRIN-1 monoclonal antibodies (#1 to #11) can also impair CAPRIN-1 expression in cancer cells by demonstrating CDC activity.

[0235] Example 6: In vivo antitumor effect of anti-CAPRIN-1 monoclonal antibody on mice

[0236] Next, the in vivo antitumor effects of the obtained anti-CAPRIN-1 monoclonal antibodies #1 to #11 on tumor-bearing mice were evaluated. The antibodies used in this example were obtained by column purification of the supernatant from each hybridoma using the method described above.

[0237] The antitumor efficacy of anti-CAPRIN-1 monoclonal antibodies #1 to #11 was examined in tumor-bearing mice, in which mouse-derived cancer cell lines expressing CAPRIN-1 were transplanted into the tumor-bearing mice. CT26 cells (purchased from ATCC) were subcutaneously transplanted into the backs of 70 Balb / c mice (Japan SLC, Inc.) (10 cells per mouse). 6Each tumor was allowed to grow until its diameter became approximately 7 mm. Anti-CAPRIN-1 monoclonal antibodies #1 to #11 and a class of monoclonal antibodies prepared in Example 4 (reacting to the CAPRIN-1 protein itself but not to the surface of cancer cells) were administered intraperitoneally to the tumor-bearing mice (60 of 70 mice) at a dose of 300 μg (300 μl) per mouse. Subsequently, each antibody was administered intraperitoneally to the relevant tumor-bearing mice three times at the same dose over a total of 2 days. Tumor size was measured daily to observe the anti-tumor effect. The remaining 10 tumor-bearing mice were given PBS(-) instead of the antibodies. This group of mice was designed as a control group. As an observation of the anti-tumor effect, in the test groups administered anti-CAPRIN-1 monoclonal antibodies #1 to #11, the tumor regression was such that the tumor volume (100%) at the start of antibody administration decreased to 50% on day 4, to approximately 10% on day 6, and to a few percent on day 8. Substantial tumor regression occurred between days 11 and 14 (see Figure 5). In contrast, in the control group, tumor volume increased to 260%, 350%, 550%, and 800% of the original volume on days 4, 6, 8, and 11, respectively (see Figure 5). Furthermore, in mice treated with monoclonal antibodies (which react with the CAPRIN-1 protein itself but not with the cancer cell surface), no antitumor effect was observed, and tumor growth occurred as in the control group. These results indicate that the obtained anti-CAPRIN-1 monoclonal antibodies #1 to #11 exhibit a strong in vivo antitumor effect against CAPRIN-1-expressing cancer cells. Additionally, tumor volume was calculated using the formula: long diameter × short diameter × short diameter × 0.5.

[0238] Furthermore, anti-CAPRIN-1 monoclonal antibodies #1 through #11 were administered to tumor-bearing mice (Balb / c) in the same manner as described above, to which mouse N1E cancer cells (purchased from ATCC) were transplanted. This resulted in complete tumor regression by day 15 post-antibody administration. In contrast, in the control group, tumor volume increased to approximately 950% of its original volume (see Figure 6).

[0239] Example 7: Identification of peptides in CAPRIN-1 protein, wherein the peptides bind to anti-CAPRIN-1 antibodies that react with the surface of cancer cells.

[0240] By applying anti-CAPRIN-1 monoclonal antibodies #1 to #11 (obtained above) that react with the surface of cancer cells, partial sequences of the CAPRIN-1 protein recognized by these monoclonal antibodies were identified.

[0241] First, a solution was prepared by dissolving recombinant CAPRIN-1 protein in PBS to a concentration of 1 μg / μl. DTT (Fluka) was added to 100 μl of this solution to a final concentration of 10 mM, followed by reaction at 95 °C for 5 min, thereby reducing the disulfide bonds within the CAPRIN-1 protein. Next, iodoacetamide (Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 20 mM, and the thiol groups were alkylated at 37 °C for 30 min under light-shielded conditions. 50 μg of each anti-CAPRIN-1 monoclonal antibody #1 to #11 was added to 40 μg of the thus obtained reduced alkylated CAPRIN-1 protein. The mixture volume was adjusted to 1 mL of 20 mM phosphate buffer (pH 7.0), and each mixture was then stirred and reacted overnight at 4 °C.

[0242] Then, trypsin (Promega) was added to a final concentration of 0.2 μg. After reacting at 37°C for 1 h, 2 h, 4 h, and 12 h, the product was mixed with protein A-glass beads (GE) in 1 mM calcium carbonate and NP-40 buffer (20 mM phosphate buffer (pH 7.4), 5 mM EDTA, 150 mM NaCl, and 1% NP-40) and reacted for 30 min. The protein A-glass beads were previously blocked with PBS containing 1% BSA (Sigma) and then washed with PBS.

[0243] Each reaction mixture was washed with 25 mM ammonium carbonate buffer (pH 8.0), and then the antigen-antibody complex was eluted with 100 μl of 0.1% formic acid. The eluents were analyzed by LC-MS using a Q-TOF Premier (Waters-MicroMass) according to the instrument's instructions.

[0244] As a result, the peptide of SEQ ID NO:136 was identified as a partial sequence of CAPRIN-1, which was recognized by all anti-CAPRIN-1 monoclonal antibodies #1 to #11. Furthermore, the peptide of SEQ ID NO:137 was identified as a partial sequence of the aforementioned peptide of SEQ ID NO:136, and was recognized by monoclonal antibodies #2 to #5, #6 to #8, and #10. It was also revealed that monoclonal antibodies #2 to #5 recognize the peptide of SEQ ID NO:138, which is a partial sequence peptide of the peptide of SEQ ID NO:137.

[0245] Industrial applicability

[0246] The antibodies of this invention can be used to treat and / or prevent cancer.

[0247] This specification includes all or part of the disclosure of the specification and / or drawings of Japanese Patent Application No. 2009-087285, to which this application claims priority. Furthermore, all publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0248] Sequence List Independent Text

[0249] Primers: SEQ ID NO: 31 to 39 and 130 to 135

Claims

1. An antibody or a fragment thereof that specifically binds to CAPRIN-1 protein expressed on the surface of cancer cells, wherein said antibody or fragment thereof is any one of the following (A) to (K): (A) The antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 40, 41 and 42, respectively, and the light chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 44, 45 and 46, respectively, and has immunological reactivity with CAPRIN-1 protein; (B) The antibody or its fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 40, 41 and 42, respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 50, 51 and 52, respectively, and has immunological reactivity with CAPRIN-1 protein; (C) The antibody or its fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 40, 41 and 42, respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 55, 56 and 57, respectively, and has immunological reactivity with CAPRIN-1 protein; (D) The antibody or its fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 40, 41 and 42, respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 60, 61 and 62, respectively, and has immunological reactivity with CAPRIN-1 protein; (E) The antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 40, 41 and 42, respectively, and the light chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 65, 66 and 67, respectively, and has immunological reactivity with CAPRIN-1 protein; (F) The antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 70, 71 and 72, respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 74, 75 and 76, respectively, and has immunoreactivity with CAPRIN-1 protein; (G) The antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 80, 81 and 82, respectively, and the light chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 84, 85 and 86, respectively, and has immunoreactivity with CAPRIN-1 protein; (H) The antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 90, 91 and 92, respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 94, 95 and 96, respectively, and has immunoreactivity with CAPRIN-1 protein; (I) The antibody or its fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 100, 101 and 102, respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 104, 105 and 106, respectively, and has immunological reactivity with CAPRIN-1 protein; (J) The antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO: 110, 111, and 112, respectively, and the light chain variable region comprises CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO: 114, 115, and 116, respectively, and has immunological reactivity with CAPRIN-1 protein; and (K) The antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 120, 121 and 122, respectively, and the light chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 124, 125 and 126, respectively, and has immunoreactivity with CAPRIN-1 protein.

2. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof is a human antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, or a bispecific antibody.

3. A pharmaceutical composition comprising, as an active ingredient, an antibody or fragment thereof that specifically binds to CAPRIN-1 protein expressed on the surface of cancer cells, as described in claim 1.

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