Pharmaceutical composition for the treatment and / or prevention of cancer expressing caprin-1

Abstract

Pharmaceutical compositions, antibodies and uses of an antibody or a fragment thereof. The present invention relates to a pharmaceutical composition for the treatment and / or prevention of cancer, comprising, as an active ingredient, an antibody or a fragment thereof that has an immunological reactivity to a caprin-i protein or a fragment thereof comprising 7 or more consecutive amino acids.

Description

[0001] The present invention relates to an innovative medical use of antibodies to CAPRIN-1 or fragments thereof as, for example, therapeutic and / or preventive agents for cancer. Background of the Invention

[0002] Cancer is the leading cause of death. Currently, cancer treatment is primarily surgical therapy, which may be combined with radiotherapy or chemotherapy. Despite the development of new surgical methods and the discovery of new anticancer agents in recent years, treatment outcomes for this disease are not significantly improved at present, except for some cancers. Through recent progress in molecular biology and cancer immunology, antibodies that are specifically reactive to cancers, cancer antigens recognized by cytotoxic T cells, as well as the genes encoding cancer antigens have been identified, and expectations for specific immunotherapies targeting cancer antigens have been raised (Tsuyoshi AKIYOSHI, Gan To Kagaku-Ryoho (Cancer and Chemotherapy), 1997, vol. 24, pp. 551-519 (Jp) (Cancer and Chemotherapy Publishers, Inc., Japan)).

[0003] In cancer treatment methods, in order to reduce side effects, it is desirable that peptides, polypeptides, or proteins recognized as cancer antigens be absent in almost all normal cells, but specifically present in cancerous cells. In 1991, Boon et al., from the Ludwig Institute in Belgium, isolated the human melanoma MAGE 1 antigen recognized by CD8-positive T cells by the cDNA expression cloning method using a Petition 870260065851, dated 03 / 07 / 2026, page 12 / 105 2 / 83 autologous cancer cell line 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, whereby tumor antigens recognized by antibodies produced through the response to an autologous cancer in the body of a cancer patient can be identified using the gene expression cloning technique (Proc. Natl. Acad. Sci. USA, 92:11810-11813 (1995); and patent document no. US 5,698,396). Using the SEREX method, some cancer antigens, which are not substantially expressed in normal cells but are specifically expressed in cancerous cells, were 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: 3339, 1997).Additionally, clinical trials of cell therapies using immunocytes that specifically react with cancer antigens, which are some of the isolated cancer antigens, and cancer-specific immunotherapies using vaccines comprising cancer antigens or similar antigens have been conducted.

[0004] In the meantime, in recent years, a variety of antibody-based drugs for cancer treatment that target antigen proteins in cancer cells have begun to emerge. Such drugs, used as cancer-specific therapeutic agents, exhibit drug efficacy up to a certain point and, in this way, they have gained attention. However, most target antigen proteins are also expressed in normal cells. As a result of antibody administration, not only cancer cells but also normal cells in which a target antigen has been expressed can be damaged, thus causing a problematic side effect (or adverse effect). Petition 870260065851, dated 03 / 07 / 2026, page 13 / 105 3 / 83 Consequently, it is expected that if it becomes possible to identify the cancer antigens that are specifically expressed on the surface of a cancer cell, and to use antibodies targeting those antigens as drugs, then antibody-based drug treatments with fewer side effects could be realized.

[0005] Cytoplasmic proliferation-associated protein 1 (CAPRIN-1) is an intracellular protein that is expressed when normal resting cells are activated or undergo cell division. CAPRIN-1 is also known to be involved in the regulation of mRNA transport and translation through the formation of cytoplasmic stress granules containing RNA in a cell. CAPRIN-1 has different names, such as GPI-anchored membrane protein 1 and surface marker membrane component protein 1 (M11S1), if this protein is known as a membrane protein. These different names are derived from the publication (J. Biol. Chem., 270: 20717-20723, 1995) that the CAPRIN-1 gene sequence originally has a GPI-binding region and that CAPRIN-1 is a membrane protein expressed in colon cancer cells.It was subsequently reported that the CAPRIN-1 gene sequence described in the aforementioned publication was incorrect; that is, a frame-shift alteration occurred by deleting a single nucleotide from the CAPRIN-1 gene sequence currently registered in GenBank or similar databases, such that 80 amino acids were deleted from the C-terminal region and the resulting artifact (74 amino acids) was the GPI-binding portion of the publication; and another error was also present at the 5' end of the gene sequence, thus resulting in the deletion of 53 amino acids from the N-terminal region (J. Immunol., 172: 2389-2400, 2004). Additionally, it has been reported that the protein encoded by the CAPRIN-1 gene sequence currently registered in GenBank or similar databases is not a cell membrane protein (J. Immunol., 172: 2389, Petition 870260065851, dated 03 / 07 / 2026, p. 14 / 105). 4 / 83 2400, 2004).

[0006] Furthermore, based on the report in J. Biol. Chem., 270: 20717-20723, 1995, CAPRIN-1 is a cell membrane protein. Patent documents US2008 / 0075722 and WO2005 / 100998 reveal that CAPRIN-1 under the name M11S1 can be used for cancer therapy as a target for antibody-based drugs for cancer therapy and as one of the cell membrane proteins; however, the Examples do not contain a description of cancer therapy using an antibody against the protein. However, as reported in J. Immunol., 172:2389-2400, 2004, it was believed that, from the filing of document US2008 / 0075722 until the present time, CAPRIN-1 was not expressed on the surface of a cell and, therefore, it is obvious that the contents of documents US2008 / 0075722 and WO2005 / 100998, based solely on this incorrect information that CAPRIN-1 is a cell membrane protein, should not be interpreted as common technical knowledge of persons skilled in the art. Brief Description of the Invention Problem to be Solved by the Invention

[0007] An object of the present invention is to identify cancer antigen proteins specifically expressed on the surface of cancer cells and to provide a use of antibodies that target such proteins as therapeutic and / or preventive (or prophylactic) agents for cancer. Ways to Solve the Problem

[0008] As a result of intensive studies, the present inventors have now obtained cDNA encoding a protein that binds to an antibody present in the serum of a tumor-containing organism through the SEREX method using cDNA libraries derived from testicular tissue and sera from dogs with breast cancer. With the use of canine genes Petition 870260065851, dated 03 / 07 / 2026, page 15 / 105 5 / 83 obtained and from homologous genes of human, bovine, equine, mouse and chicken, the CAPRIN-1 proteins having amino acid sequences shown in even numbers of SEQ ID NOS: 2 to 30 (i.e., SEQ ID NOS with even numbers: 2 to 30) and antibodies against the CAPRIN-1 proteins have now been prepared. Furthermore, the present inventors have concluded that CAPRIN-1 is specifically expressed in breast cancer, brain tumor, leukemia, lymphoma, lung cancer, esophageal cancer, colon cancer, gastric cancer and kidney cancer cells, and that those portions of the CAPRIN-1 proteins are specifically expressed on the surface of such cancerous cells. Additionally, the present inventors have now concluded that antibodies against the portions of CAPRIN-1 expressed on cancerous cell surfaces can damage (or impair) cancerous cells expressing CAPRIN-1. These findings lead to the conclusion of the present invention.

[0009] Therefore, the present invention has characteristics as described below.

[0010] The present invention provides a pharmaceutical composition for the treatment and / or prevention of cancer, comprising, as an active ingredient, an antibody or a fragment thereof that has an immunological reactivity to a CAPRIN-1 protein, which has an amino acid sequence shown in any of the even-numbered sequences of SEQ ID Nos: 2 to 30, or an amino acid sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, and more preferably 95% or more of sequence identity with the amino acid sequence of any of the even-numbered sequences of SEQ ID Nos: 2 to 30, or with a fragment of the CAPRIN-1 protein comprising 7 or more consecutive amino acids.

[0011] In one embodiment of the present invention, cancer is Petition 870260065851, dated 03 / 07 / 2026, p. 16 / 105 6 / 83 breast cancer, brain tumor, leukemia, lymphoma, lung cancer, esophageal cancer, colon cancer, gastric (or stomach) cancer, or kidney cancer.

[0012] In another embodiment of the present invention, the antibody is a monoclonal or polyclonal antibody.

[0013] 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.

[0014] In another embodiment of the present invention, the antibody is an antibody that has an immunological reactivity to a polypeptide that has the amino acid sequence shown in SEQ ID NO: 37 or SEQ ID NO: 136, or an amino acid sequence that has 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, or with a fragment of the polypeptide.

[0015] In another embodiment of the present invention, in the pharmaceutical composition for the treatment and / or prevention of cancer comprising the antibody as an active ingredient, the above antibody is one of the antibodies (a) to (k) described below and has an immunological reactivity with a CAPRIN-1 protein. (a) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41 and 42, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 44, 45 and 46; (b) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41 and 42, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 50, 51 and 52; (c) An antibody comprising a variable chain region Petition 870260065851, dated 03 / 07 / 2026, page 17 / 105 7 / 83 heavy comprising the sequences shown in SEQ ID NOs: 40, 41 and 42, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 55, 56 and 57; (d) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41 and 42, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 60, 61 and 62; (e) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41 and 42, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 65, 66 and 67; (f) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 70, 71 and 72, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 74, 75 and 76; (g) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 80, 81 and 82, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 84, 85 and 86; (h) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 90, 91 and 92, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 94, 95 and 96; (i) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 100, 101 and 102, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 104, 105 and 106; (j) An antibody comprising a variable chain region Petition 870260065851, dated 03 / 07 / 2026, page 18 / 105 8 / 83 heavy comprising the sequences shown in SEQ ID NOs: 110, 111 and 112, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 114, 115 and 116; (k) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 120, 121 and 122, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 124, 125 and 126. Effects of the Invention

[0016] The antibodies against CAPRIN-1 used in the present invention damage (or impair) cancer cells. Therefore, such antibodies against CAPRIN-1 are useful for the treatment or prevention of cancers. Brief Description of the Figures

[0017] Figure 1 shows the expression patterns of genes encoding CAPRIN-1 proteins in normal tissues and tumor cell lines. In this figure, reference #1 shows the expression pattern of each gene encoding CAPRIN-1, and reference #2 shows the expression pattern of the GAPDH gene.

[0018] Figure 2 shows the cytotoxic activity of an antibody to CAPRIN-1 (or anti-CAPRIN-1 antibody) against the breast cancer cell line expressing the CAPRIN-1 gene (T47D). In this figure, reference #3 shows the activity after the addition of the anti-CAPRIN-1 antibody, reference #4 shows the activity after the addition of a control antibody, and reference #5 shows the activity in the absence of any antibody.

[0019] Figure 3 shows the cytotoxic activity of an antibody to CAPRIN-1 (or anti-CAPRIN-1 antibody) against the breast cancer cell line expressing the CAPRIN-1 gene (MDA-MB-157). In this figure, reference number 6 shows the activity after the addition of the anti-CAPRIN-1 antibody. Petition 870260065851, dated 03 / 07 / 2026, page 19 / 105 9 / 83 CAPRIN-1, reference #7 shows the activity after the addition of control antibody and reference #8 shows the activity in the absence of any antibody.

[0020] Figure 4 shows cytotoxicity against the MDA-MB-157 breast cancer cell line expressing CAPRIN-1, where cytotoxicity is exhibited by monoclonal antibodies to CAPRIN-1 (i.e., monoclonal antibodies no. 1 and no. 11), which are reactive with the surface of the cancer cell.Specifically, this figure shows the activity levels after the addition of monoclonal antibody #1 to CAPRIN-1 (reference #9), monoclonal antibody #2 to CAPRIN-1 (reference #10), monoclonal antibody #3 to CAPRIN-1 (reference #11), monoclonal antibody #4 to CAPRIN-1 (reference #12), monoclonal antibody #5 to CAPRIN-1 (reference #13), monoclonal antibody #6 to CAPRIN-1 (reference #14), monoclonal antibody #7 to CAPRIN-1 (reference #15), monoclonal antibody #8 to CAPRIN-1 (reference #16), monoclonal antibody #9 to CAPRIN-1 (reference #17), monoclonal antibody #10 to CAPRIN-1 (reference #18), and antibody monoclonal antibody no. 11 to CAPRIN-1 (reference no. 19), the level of activity after the addition of a monoclonal antibody reactive with the CAPRIN-1 protein itself, but not with the surface of the cancer cell (reference no. 20), and the level of activity after the addition of PBS instead of each antibody (reference no. 21).

[0021] Figures 5a to 5c show the antitumor effect of monoclonal antibodies to CAPRIN-1 (i.e., monoclonal antibodies no. 1 and no. 11) reactive with the surface of a cancer cell in Balb / c mice in which mouse carcinoma of the CT26 cell line expressing CAPRIN-1 was transplanted. These figures show the mouse tumor sizes after administration of monoclonal antibody no. 1 to CAPRIN-1 (reference no. 22), and monoclonal antibody no. 2 to CAPRIN-1. Petition 870260065851, dated 03 / 07 / 2026, page 20 / 105 10 / 83 (reference no. 23), of monoclonal antibody no. 3 in CAPRIN-1 (reference no. 24), of monoclonal antibody no. 4 in CAPRIN-1 (reference no. 25), of monoclonal antibody no. 5 in CAPRIN-1 (reference no. 26), of monoclonal antibody no. 6 in CAPRIN-1 (reference no. 27), of monoclonal antibody no. 7 in CAPRIN-1 (reference no. 28), of monoclonal antibody no. 8 in CAPRIN-1 (reference no. 29), of monoclonal antibody no. 9 in CAPRIN-1 (reference no. 30), of monoclonal antibody no. 10 in CAPRIN-1 (reference no. 31), and of monoclonal antibody no. 11 in CAPRIN-1 (reference no. 32), of mouse tumor size after administration of a monoclonal antibody reactive with the self-protein. CAPRIN-1, but not with the surface of the cancerous cell (reference no. 33), and the size of a mouse tumor after administration of PBS instead of each antibody (reference no. 34).

[0022] Figures 6a to 6c show the antitumor effect of monoclonal antibodies to CAPRIN-1 (i.e., monoclonal antibodies n°1 an°11) reactive with the surface of a cancer cell, in Balb / c mice into which the N1E mouse carcinoma cell line expressing CAPRIN-1 was transplanted.These figures show the mouse tumor sizes after administration of monoclonal antibody #1 to CAPRIN-1 (reference #35), monoclonal antibody #2 to CAPRIN-1 (reference #36), monoclonal antibody #3 to CAPRIN-1 (reference #37), monoclonal antibody #4 to CAPRIN-1 (reference #38), monoclonal antibody #5 to CAPRIN-1 (reference #39), monoclonal antibody #6 to CAPRIN-1 (reference #40), monoclonal antibody #7 against CAPRIN-1 (reference #41), monoclonal antibody #8 against CAPRIN-1 (reference #42), monoclonal antibody #9 against CAPRIN-1 (reference #43), monoclonal antibody #10 to CAPRIN-1 (reference #44), and of Monoclonal antibody no. 11 against CAPRIN-1 (reference no. 45), mouse tumor size after administration of a monoclonal antibody reactive with a. Petition 870260065851, dated 03 / 07 / 2026, p. 21 / 105 11 / 83 own protein CAPRIN-1, but not with the surface of the cancer cell (reference no. 46), and the size of mouse tumors after administration of PBS instead of each antibody (reference no. 47). Detailed Description of the Invention

[0023] As described below, the antitumor activity of antibodies to the polypeptide shown in any of the SEQ ID NOs with even numbers: 2 to 30 (i.e., SEQ ID NOs: 2, 4, 6...28 and 30) used in the present invention can be evaluated by examining in vivo the inhibition of tumor growth in an animal with a tumor, or by examining in vitro to determine whether complement- or immunocyte-mediated cytotoxic activity against tumor cells expressing the polypeptide is exhibited or not.

[0024] Furthermore, the nucleotide sequences of polynucleotides that encode proteins consist of the amino acid sequences shown in the SEQ ID NOs with even numbers: 2 to 30 are shown in the SEQ ID NOs with odd numbers: 1 to 29 (i.e., SEQ ID NOs: 1, 3, 5...27 and 29), respectively.

[0025] The amino acid sequences shown in SEQ ID NOs: 6, 8, 10, 12 and 14 in the Sequence Listing disclosed according to the present invention are the amino acid sequences of the CPRIN-1 proteins, which were isolated using the SEREX method with cDNA libraries derived from canine testicular tissue and sera from dogs with breast cancer, as polypeptides capable of binding to specific antibodies that exist in the sera of dogs with tumors; the amino acid sequences shown in SEQ ID NOs: 2 and 4 are the amino acid sequences of the CPRIN-1 proteins isolated as human homologs of said canine polypeptides; the amino acid sequence shown in SEQ ID NO: 16 is the amino acid sequence of the protein isolated as a bovine homolog of said polypeptide. Petition 870260065851, dated 03 / 07 / 2026, p. 22 / 105 12 / 83 canine; the amino acid sequence shown in SEQ ID NO: 18 is the amino acid sequence of the protein isolated as an equine homolog of said canine polypeptide; the amino acid sequences shown in SEQ ID NOs (with even numbers): 20 to 28 are the amino acid sequences of the CPRIN-1 proteins isolated as mouse homologs of said canine polypeptides; and the amino acid sequence shown in SEQ ID NO: 30 is the amino acid sequence of the protein isolated as a chicken homolog of said canine polypeptide (see Example 1 described below). CAPRIN-1 is known to be expressed when activation or cell division of normal resting-phase cells occurs.

[0026] It is known that CAPRIN-1 is not expressed on the surface of cells. However, as a result of the examination together with the present invention, it has now been revealed that certain portions of the CAPRIN-1 protein are expressed on the surfaces of various cancer cells. According to the present invention, an antibody that binds to a portion of the CAPRIN-1 protein expressed on cancer cell surfaces is preferably used. Examples of partial peptides in the CAPRIN-1 protein expressed on cancer cell surfaces include polypeptides consisting of a sequence of 7 or more consecutive amino acids in the region of amino acid residue no. (or amino acids (aa)) 50 to 98 or amino acid residue no. (aa) 233 to 305 in an amino acid sequence shown in any of the even-numbered SEQ ID NOs: 2 to 30, except for SEQ ID NOs: 6 and 18, in the Sequence Listing.Specific examples of these include the amino acid sequence shown in SEQ ID NO: 37 or 136 (preferably, the region of the amino acid sequence shown in SEQ ID NO: 137 or 138 in the amino acid sequence shown in SEQ ID NO: 136), or an amino acid sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, and with even greater preference, 95% or more. Petition 870260065851, dated 03 / 07 / 2026, p. 23 / 105 13 / 83 more sequence identity with the so-called amino acid sequences. The antibodies of the present invention include all antibodies capable of binding to the above peptides and having antitumor activity.

[0027] Antibodies to CAPRIN-1 useful in the present invention as described above may be any type thereof, provided they exhibit antitumor activity. Examples thereof 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 may be prepared by methods known to those skilled in the art. In the present invention, antibodies capable of specifically binding to a CAPRIN-1 protein are desirable. Such antibodies are preferably monoclonal antibodies; however, provided homogeneous antibodies can be stably produced, polyclonal antibodies may also be used.Furthermore, if the individual is human, a human antibody or a humanized antibody is desirable in order to prevent or inhibit immunorejection.

[0028] The expression that binds specifically to a CAPRIN-1 protein as used in this document means that an antibody of interest binds specifically to the CAPRIN-1 protein and does not bind substantially to other proteins.

[0029] As described below, the antitumor activity of an antibody used in the present invention can be evaluated by examining in vivo the inhibition of tumor growth in an animal with a tumor, or by examining in vitro to determine whether or not complement- or immunocyte-mediated cytotoxic activity against tumor cells expressing the polypeptide is exhibited. Petition 870260065851, dated 03 / 07 / 2026, page 24 / 105 14 / 83

[0030] Furthermore, the individuals in need of cancer treatment and / or prevention according to the present invention are mammals such as humans, domestic animals, farm animals or sporting animals. The preferred individual is a human.

[0031] The production of antigens, the production of antibodies, and the pharmaceutical compositions related to the present invention will be explained below. Production of antigens used for antibody production.

[0032] The proteins or fragments thereof used as sensitization antigens to obtain antibodies to CAPRIN-1 used in the present invention are not limited in terms of origin, such as from animals including, for example, human, canine, bovine, equine, mouse, rat, and avian proteins. However, such proteins or fragments thereof are preferably selected for compatibility with parental cells used for cell fusion. Mammalian-derived proteins are generally preferred, and human-derived proteins are particularly preferred. For example, if CAPRIN-1 is human CAPRIN-1, a human CAPRIN-1 protein, a partial peptide thereof, or cells capable of expressing human CAPRIN-1 may be used.

[0033] Nucleotide sequences and amino acid sequences of human CAPRIN-1 and its homologs can be obtained, for example, by accessing GenBank (NCBI, USA) and using 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).

[0034] 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 a base sequence, the targets are nucleic acids or proteins consisting of a sequence Petition 870260065851, dated 03 / 07 / 2026, p. 25 / 105 15 / 83 having 70% to 100%, preferably 80% to 100%, more preferably 90% to 100%, and with additional preference 95% to 100% (for example, 97% to 100%, 98% to 100%, 99% to 100%, or 99.5% to 100%) sequence identity with the nucleotide sequence or amino acid sequence of the ORF or mature portion of the nucleotide base sequence or amino acid sequence. The term % sequence identity as used herein means a percentage (%) of the number of identical amino acids (or nucleotides) to the total number of amino acids (or nucleotides) in the case where the two sequences are aligned in such a way that maximum similarity can be achieved with or without the introduction of gaps.

[0035] Fragments of a CAPRIN-1 protein have lengths in the range of the amino acid length of an epitope (or an antigenic determinant), which is the smallest unit of an antigen recognized by an antibody, to less than the total length of the protein. An epitope refers to a polypeptide fragment that has antigenicity or immunogenicity in mammals and preferably in humans. The smallest unit of a polypeptide fragment consists of approximately 7 to 12 amino acids and, for example, 8 to 11 amino acids. A specific example thereof is the amino acid sequence shown in SEQ ID NO: 37, SEQ ID NO: 137, or SEQ ID NO: 138, or an amino acid sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, and with further preference 95% or more sequence identity with said amino acid sequence.

[0036] The polypeptides comprising the aforementioned human CAPRIN-1 protein and partial peptides thereof can be synthesized according to chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method) (the Japanese Biochemical Society (ed.), Biochemical Petition 870260065851, dated 03 / 07 / 2026, page 26 / 105 16 / 83 Experimentation Course (Seikagaku Jikken Koza) 1, Protein Chemistry IV, Chemical Modification and Peptide Synthesis, Kagaku-dojin Publishing Company, Inc. (Japan), 1981). Furthermore, they can be synthesized by general methods using a variety of commercially available polypeptide synthesizers. Furthermore, the polypeptides of interest can be obtained by preparing polynucleotides that encode the above polypeptides using known genetic engineering methods (Sambrook et al., Molecular Cloning, 2nd edition, Current Protocols in Molecular Biology (1989), Cold Spring Harbor Laboratory Press; Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, A Compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons, etc.), which incorporate each of the polynucleotides into an expression vector and introduce the vector into a host cell, thus allowing the host cell to produce the polypeptide.Through this, the desired polypeptides can be obtained.

[0037] The polynucleotides encoding the aforementioned polypeptides can be readily prepared by known genetic engineering techniques or general methods using commercially available nucleic acid synthesizers. For example, DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 can be prepared by PCR using a human chromosome DNA or cDNA library as a template and a pair of primers designed to allow amplification of the nucleotide sequence shown in SEQ ID NO: 1. PCR conditions can be appropriately determined. For example, such conditions may comprise conducting 30 cycles of the reaction steps (as one cycle) consisting of: 94°C, 30 seconds (denaturation); 55°C, 30 seconds to 1 minute (annealing); and 72°C, 2 minutes (elongation) using a thermostable DNA polymerase. Petition 870260065851, dated 03 / 07 / 2026, p. 27 / 105 17 / 83 (e.g., Taq polymerase) and a PCR buffer containing Mg2+, followed by reaction at 72°C for 7 minutes after completion of the 30 cycles. However, the present invention is not limited to the PCR conditions exemplified 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 (Chapter 15, in particular).

[0038] Furthermore, the desired DNA can be isolated by preparing appropriate probes and primers with information on the amino acid and nucleotide sequences shown in SEQ ID Nos: 1 to 30 in the Sequence Listing described in this document, and by screening a human cDNA library or similar using such probes and primers. Preferably, such a cDNA library is produced from a cell, organ, or tissue in which the protein with any of the even-numbered SEQ ID Nos: 2 to 30 is expressed. Examples of cells or tissues include cells or tissues from testes and from cancers or tumors such as leukemia, breast cancer, lymphoma, brain tumor, lung cancer, and colon cancer.Operations such as the preparation of probes or primers, construction of cDNA libraries, screening of cDNA libraries, and cloning of genes of interest, as described above, are known to those skilled in the art, and can be performed according to, for example, the methods described in Sambrook et al., Molecular Cloning, 2nd edition, Current Protocols in Molecular Biology (1989) and Ausbel et al. (ibid.). DNAs encoding human CAPRIN-1 protein and partial peptides thereof can be obtained from DNAs obtained in this way.

[0039] The host cells described above can be any cells, provided they can express the polypeptides described above. An example of a prokaryotic host cell includes, but is not limited to. Petition 870260065851, dated 03 / 07 / 2026, page 28 / 105 18 / 83 a, Escherichia coli. Examples of eukaryotic host cells include, but are not limited to, mammalian cells such as monkey kidney cell (COS1), Chinese hamster ovary cell (CHO), human embryo kidney cell line (HEK293), and mouse embryo hair cell line (NIH3T3), yeast cells such as germinating yeast and dividing yeast cells, silkworm cells, and Xenopus egg cells.

[0040] When prokaryotic cells are used as host cells, an expression vector that has a replicable origin in prokaryotic cells, a promoter, a ribosome binding site, a multicloning site, a terminator, a drug resistance gene, an auxotrophic complementary gene, or similar can be used. As expression vectors for Escherichia coli, pUC vectors, pBluescriptII, pET expression systems, pGEX expression systems, and similar vectors can be exemplified. A DNA encoding the above polypeptide is incorporated into such an expression vector, a prokaryotic host cell is transformed with the vector, and then the transformed cell obtained in this way is cultured so that the polypeptide encoded by the DNA can be expressed in the prokaryotic host cell. At this point, the polypeptide can also be expressed as a fusion protein with another protein.

[0041] When eukaryotic cells are used as host cells, eukaryotic expression vectors that have a promoter, a division region, a poly(A) addition site, or similar can be used. Examples of such expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, and pYES2. Through procedures similar to those mentioned above, DNA encoding the aforementioned polypeptide is incorporated into such an expression vector, a eukaryotic host cell is transformed with the vector, and Petition 870260065851, dated 03 / 07 / 2026, page 29 / 105 19 / 83 then, the transformed cell obtained in this way is cultured, so that the polypeptide encoded by the above DNA can be expressed in the eukaryotic host cell. When pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or similar is used as an expression vector, the above polypeptide can be expressed as a fusion protein with a tag, such as a His tag (e.g., (His)6 to (His)io), FLAG tag, myc tag, HA tag or GFP.

[0042] To introduce an expression vector into a host cell, known methods can be employed, such as electroporation, a calcium phosphate method, a liposome method, a dextran DEAE method, microinjection, viral infection, lipofection, and binding with a permeable peptide in a cell membrane.

[0043] The isolation and purification of a polypeptide of interest from host cells can be performed using known isolation techniques in combination. Examples of such known techniques include, but are not limited to, treatment with a denaturing agent such as urea or a surfactant, ultrasonication, enzymatic digestion, salinization, solvent fractionation and precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing electrophoresis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse-phase chromatography. Antibody structure

[0044] In general, antibodies are heteromultimeric glycoproteins comprising at least two heavy chains and two light chains. However, antibodies except for IgM are heterotetrameric glycoproteins (approximately 150 kDa) comprising two identical light chains (L) and two identical heavy chains (H). Typically, each light chain is connected to a heavy chain. Petition 870260065851, dated 03 / 07 / 2026, page 30 / 105 20 / 83 through a single covalent disulfide bond. However, the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy chain and light chain also has an intra-chain disulfide bond. Each heavy chain has a variable domain (VH region) at one end, to which several constant regions are linked in series. 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 a light chain is aligned with the first constant region of a heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. A specific region of an antibody variable domain, called the "complementary determinant region (CDR)," exhibits specific variability in order to confer binding specificity to an antibody.A relatively conserved portion in a variable region is called a structural region (FR). A complete heavy chain or light chain variable domain comprises 4 FRs connected to each other through 3 CDRs. These CDRs are called CDRH1, CDRH2, and CDRH3, respectively, in that order starting from the N-terminus in a heavy chain. Similarly for a light chain, they are called CDRL1, CDRL2, and CDRL3, respectively. CDRH3 plays the most important role in terms of antibody-antigen binding specificity. Furthermore, the CDRs in each chain are retained by FR regions in the state where they are close to each other, and contribute to the formation of antibody-antigen binding sites with CDRs in a corresponding chain. The constant regions do not directly contribute to antibody-antigen binding.However, they exhibit several effector functions such as association with antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis through binding to an Fcg receptor, and half-life / resolution rate via an Fc receptor. Petition 870260065851, dated 03 / 07 / 2026, p. 31 / 105 21 / 83 neonatal (FcRn), and complement-dependent cytotoxicity (CDC) via a C1q component in the complement cascade. Antibody production

[0045] The term anti-CAPRIN-1 antibody used in the present invention refers to an antibody having an immunological reactivity with a normal-sized CAPRIN-1 protein or a fragment thereof.

[0046] The term immunological reactivity used in the present invention indicates the characteristics of an antibody binding in vivo to a CAPRIN-1 antigen. The tumor-damaging function (e.g., death, inhibition, or regression) can be expressed as a result of this binding. Specifically, any type of antibody can be used in the present invention provided that the antibody can bind to a CAPRIN-1 protein to damage a tumor or cancer such as leukemia, lymphoma, breast cancer, brain tumor, lung cancer, esophageal cancer, gastric cancer, kidney cancer, or colon cancer.

[0047] 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). In addition, examples of arbitrary immunoglobulin classes of these antibodies include IgG, IgE, IgM, IgA, IgD, and IgY, and examples of arbitrary immunoglobulin subclasses include IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0048] Antibodies can be further modified by acetylation, formylation, amidation, phosphorylation, or pegylation (PEG), in addition to glycosylation.

[0049] Production examples for a variety of antibodies are described below. Petition 870260065851, dated 03 / 07 / 2026, page 32 / 105 22 / 83

[0050] In a case where an antibody of interest is a monoclonal antibody, an SK-BR-3 breast cancer cell line expressing CAPRIN-1 or similar antibodies is administered to mice for immunization, followed by extraction of the mice's spleen. Cells are separated from each spleen and then fused with mouse myeloma cells. Clones capable of producing an antibody with cancer cell growth inhibition action are selected from the resulting fused cells (hybridomas). A monoclonal antibody-producing hybridoma with cancer cell growth inhibition action is isolated and cultured. An antibody of interest can be prepared by purifying the culture supernatant using a general affinity purification method.

[0051] Furthermore, a monoclonal antibody-producing hybridoma can be produced in the manner described below, for example. First, an animal is immunized with a sensitizing antigen by a known method. In one general method, immunization is performed by intraperitoneal or subcutaneous injection of a sensitizing antigen into a mammal. Specifically, a sensitizing antigen is diluted with, or suspended in, PBS (phosphate-buffered saline), physiological saline, or similar solutions to an appropriate amount. If desired, an appropriate amount of a conventional adjuvant (e.g., Freund's complete adjuvant) is mixed with this. After emulsification, the result is administered to a mammal several times every 4 to 21 days. Additionally, an appropriate carrier can be used for immunization with a sensitizing antigen.

[0052] As described above, after immunization of a mammal and confirmation of an increase to a desired antibody level in serum, immunocytes are collected from the mammal and subjected to cell fusion. Petition 870260065851, dated 03 / 07 / 2026, page 33 / 105 23 / 83 Specifically, the preferred examples of immunocytes are splenocytes.

[0053] Mammalian myeloma cells are used as relevant stem cells that undergo fusion with the above immunocytes. For these myeloma cells, the following various examples of known cell lines are preferably used: P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548 to 1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1 to 7), NS-1 (Kohler, G. and Milstein, C. Eur. J. Immunol. (1976). 6, 511 to 519), MPC-11 (Margulies, d. H. et al., Cell (1976) 8, 405 to 415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269 and 270), FO (from St. Groth, SF et al., J. Immunol. Methods (1980) 35, 1 to 21), S194 (Trowbridge, I. 58. J. Exp. Med. (1978) 148, 313 to 323), and R210 (Galfre, G. et al., Nature (1979) 277, 131 to 133).

[0054] Basically, the cell fusion of immunocytes and myeloma cells described above can be performed according to a method known as the Kohler and Milstein et al. method (Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3 to 46).

[0055] More specifically, the cell fusion described above is performed in the presence of a cell fusion promoter in a culture solution containing conventional nutrients, for example. Examples of a fusion promoter to be used include polyethylene glycol (PEG) and Sendai virus (JHV: Japan hemagglutinating virus). If desired, an adjuvant such as dimethyl sulfoxide may additionally be added to enhance fusion efficiency.

[0056] The ratio of immunocytes used in these myeloma cells can be determined arbitrarily. For example, the ratio of immunocytes to myeloma cells is preferably 1:1 to 10:1. Examples of a culture solution that can be used in the cell fusion described above include an RPMI1640 culture solution and a solution of Petition 870260065851, dated 03 / 07 / 2026, page 34 / 105 24 / 83 MEM culture is suitable for the growth of the above myeloma cell lines, as well as other conventional culture solutions used for this type of cell culture. Additionally, a serum replacement, such as fetal calf serum (FCS), can be used in combination with this.

[0057] For cell fusion, the above immunocytes and myeloma cells are sufficiently mixed in predetermined quantities in the culture solution. A PEG solution (e.g., with an average molecular weight of approximately 1000 to 6000) that has been previously heated to approximately 37°C is added to this at a concentration of generally 30% to 60% (w / v), followed by mixing. This results in the formation of hybridomas of interest. Subsequently, operational steps of sequential addition of a suitable culture solution and removal of the supernatant by centrifugation are performed repeatedly to remove the cell fusion agent(s) and similar substances that are not preferential for the growth of the hybridomas.

[0058] Hybridomas obtained in this manner are cultured in a conventional selection culture solution such as a HAT culture solution (a culture solution comprising hypoxanthine, aminopterin, and thymidine) for selection. Culture in this HAT culture solution is continuously performed for a sufficient period of time (generally several days to several weeks) to kill cells (unfused cells) other than the hybridomas of interest. Then, a conventional limiting dilution method is employed to screen the hybridomas that produce antibodies of interest and to perform a single cloning.

[0059] Additionally, it is also possible to obtain human antibody-producing hybridomas having a desired activity (e.g., cell growth inhibition activity) in the following manner, as well as to obtain the above hybridomas through animal immunization. Petition 870260065851, dated 03 / 07 / 2026, page 35 / 105 25 / 83 non-human with antigens. Human lymphocytes (e.g., human lymphocytes infected with EB virus) are sensitized in vitro with a protein, protein-expressing cells, or a lysate thereof, and sensitized lymphocytes are fused with human-derived myeloma cells that have the ability to divide permanently (e.g., U266) (registration number TIB196).

[0060] Monoclonal antibody-producing hybridomas produced as mentioned above can be subcultured in a conventional culture solution. Furthermore, they can be preserved in liquid nitrogen for an extended period of time.

[0061] Specifically, immunization is performed using a desired antigen or cells expressing a desired antigen, such as sensitizing antigen(s), according to a conventional immunization method. The obtained immunocytes are fused with known stem cells through a conventional cell fusion method. Then, the monoclonal antibody-producing cells (hybridomas) are screened using a conventional screening method. In this way, antibody production can be performed.

[0062] Other examples of antibodies that can be used in the present invention include polyclonal antibodies. For example, polyclonal antibodies can be used in the manner described below.

[0063] Serum is obtained by immunizing small animals such as mice, human antibody-producing mice, or rabbits with a naturally occurring CAPRIN-1 protein, a recombinant CAPRIN-1 protein that has been expressed as a GST-fused protein or similar in a microorganism such as Escherichia coli, or a partial peptide thereof. The serum is purified by ammonium sulfate precipitation, protein column chromatography. Petition 870260065851, dated 03 / 07 / 2026, page 36 / 105 26 / 83 A / G protein, DEAE ion-exchange chromatography, affinity column chromatography with a column to which a CAPRIN-1 protein or a synthetic peptide is coupled, or a similar technique for the preparation of polyclonal antibodies. In the examples described below, a rabbit polyclonal antibody was produced, and its antitumor effects were confirmed, as an antibody acting against a partial peptide (with the sequence shown in SEQ ID NO: 37) of a domain in an amino acid sequence of CAPRIN-1 protein that is expressed on the surfaces of cancer cells.

[0064] A mouse for the production of known human antibodies used in the present invention, for example, is a KM Mouse (Kirin Pharma / Medarex) or a XenoMouse (Amgen) (e.g., WO02 / 43478 and WO02 / 092812). When these mice are immunized with CAPRIN-1 proteins or fragments thereof, complete human polyclonal antibodies can be obtained from the blood. In addition, human monoclonal antibodies can be produced by a splenocyte fusion method collected from mice immunized with myeloma cells.

[0065] Antigen preparation can be performed according to a method such as a method in which animal cells are used (Patent Publication JP (Kohyo) No. 2007-530068) or a method that uses a baculovirus (e.g., WO98 / 46777). If the immunogenicity of an antigen is low, an antigen will be linked to a macromolecule having immunogenicity, such as albumin. Then, the antigen can be used in immunization.

[0066] Additionally, it is possible to use a recombinant genetic antibody produced by cloning an antibody gene from a hybridoma, incorporating the clone into a suitable vector, introducing the vector into a host, and using a technique of Petition 870260065851, dated 03 / 07 / 2026, page 37 / 105 27 / 83 recombinant genetic. (See, for example, Carl, A.K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, published in the UK by MACMILLAN PUBLISHERS LTD, 1990.) Specifically, the cDNA of a variable region (V region) of an antibody is synthesized from the mRNA of a hybridoma using a reverse transcriptase. After obtaining DNA encoding a V region of an antibody of interest, this DNA is ligated to the desired DNA encoding a constant region of the antibody (C region). The result is incorporated into an expression vector. Alternatively, DNA encoding a V region of antibody can be incorporated into an expression vector comprising DNA from a C region of antibody. This DNA is incorporated into an expression vector in such a way that it is expressed under the control of an expression control region such as an enhancer or a promoter.Next, the host cells are transformed with this expression vector, thereby allowing the antibody to be expressed.

[0067] The anti-CAPRIN-1 antibodies of the present invention are preferably monoclonal antibodies. However, they may be polyclonal antibodies, gene-modified antibodies (such as chimeric antibodies and humanized antibodies), and the like.

[0068] 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 produced by culturing hybridomas obtained by fusing myeloma cells and splenocytes from non-human mammals (e.g., mice or mice for human antibody production) immunized with CAPRIN-1 proteins. In the examples described below, the monoclonal antibodies of Petition 870260065851, dated 03 / 07 / 2026, p. 38 / 105 28 / 83 mice were produced and their antitumor effects were confirmed. This monoclonal antibody comprises a variable heavy chain (VH) region having the amino acid sequence 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 variable light chain (VL) region having the amino acid sequence 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. Herein, the VH region comprises: CDR1 represented by the amino acid sequence in 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 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 sequence 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 sequence 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 CDR3 represented by the amino acid sequence 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: 106, SEQ ID NO: 116, or SEQ ID NO: 126.

[0069] A chimeric antibody is an antibody produced by combining sequences from different animals. An example of this is Petition 870260065851, dated 03 / 07 / 2026, page 39 / 105 29 / 83 an antibody consisting of variable light chain and heavy chain regions of mouse antibody and constant light chain and heavy chain regions of human antibody. This chimeric antibody can be produced by a known method. For example, it can be obtained by linking DNA encoding a V region of antibody to DNA encoding a C region of human antibody, incorporating the result into an expression vector, and introducing the vector into a host for antibody production.

[0070] Polyclonal antibodies include antibodies obtained by immunizing animals that produce human antibodies (e.g., mice) with CAPRIN-1 proteins.

[0071] A humanized antibody is a modified antibody, and it is sometimes called a reconformed human antibody. A humanized antibody is known to be constructed by transplanting the CDRs of an antibody derived from an immunized animal into complementarity-determining regions of a human antibody. Furthermore, a general recombinant genetic technique is then known.

[0072] Specifically, a DNA sequence designed to allow mouse antibody CDRs to be linked to human antibody framework regions (FRs) is synthesized by PCR using several oligonucleotides prepared in such a way that the oligonucleotides have overlapping portions at one end of each. A humanized antibody can be obtained by linking the DNA obtained above to DNA encoding a human antibody constant region, incorporating the result into an expression vector, and introducing the vector into a host for antibody production (see documents EP-A-239400 and WO96 / 02576). Human antibody FRs linked together via CDRs are Petition 870260065851, dated 03 / 07 / 2026, page 40 / 105 30 / 83 selected assuming that complementarity-determining regions can form a satisfactory antigen-binding site. If necessary, amino acids in the structure regions of a variable antibody region can be substituted in such a way that complementarity-determining regions in a reshaped human antibody form a suitable antigen-binding site (Sato K. et al., Cancer Research 1993, 53: 851-856). Furthermore, structure regions can be substituted with structure regions from a different human antibody (see WO99 / 51743).

[0073] Human antibody structure regions linked together via CDRs are selected on the assumption that complementarity-determining regions can form satisfactory antigen-binding sites. If necessary, amino acids in the structure regions of a variable antibody region can be substituted in such a way that the complementarity-determining regions in the reshaped human antibody form suitable antigen-binding sites (Sato K. et al., Cancer Research 1993, 53: 851-856).

[0074] After the production of a chimeric antibody or a humanized antibody, the amino acids in a variable region (e.g., FR) or a constant region can be replaced, for example, by different amino acids.

[0075] Here, an amino acid substitution is a substitution of, for example, less than 15, less than 10, not more than 8, not more than 7, not more than 6, not more than 5, not more than 4, not more than 3, or not more than 2 amino acids, preferably from 1 to 5 amino acids, and more preferably 1 or 2 amino acids. A substituted antibody should be functionally equivalent to an unsubstituted antibody. The substitution is preferably a conservative amino acid substitution, which is a substitution between the Petition 870260065851, dated 03 / 07 / 2026, p. 41 / 105 31 / 83 amino acids that have similar characteristics in terms of charge, side chains, polarity, aromaticity, and the like. For example, characteristically similar amino acids can be classified according to the following types: basic amino acids (arginine, lysine, and histidine); acidic amino acids (aspartic acid and glutamic acid); uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, and tyrosine); nonpolar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, and methionine); branched-chain amino acids (threonine, valine, isoleucine); and aromatic amino acids (phenylalanine, tyrosine, tryptophan, and histidine).

[0076] An example of an antibody modifier is an antibody linked to a molecule such as polyethylene glycol (PEG). With respect to the antibody modifiers of the present invention, the substances that bind to an antibody are not limited. This antibody modifier can be obtained by chemically modifying an existing antibody. A method for this modification has already been established in the field related to the present invention.

[0077] The term functionally equivalent used in the present invention indicates a situation where an antibody of interest has a biological or biochemical activity similar to that of an antibody of the present invention. Specifically, this antibody has a function of damaging tumors and causing essentially no rejection reaction when applied to humans. An example of this activity is growth inhibition activity or cell binding activity.

[0078] A known method for preparing a polypeptide functionally equivalent to a given polypeptide that is well known to those skilled in the art is a method comprising introducing a mutation into a polypeptide. For example, one skilled in the art may introduce Petition 870260065851, dated 03 / 07 / 2026, page 42 / 105 32 / 83 suitably mutate an antibody of the present invention using a site-specific mutagenesis method (Hashimoto-Gotoh, T. et al., (1995) Gene 152, 271 to 275; Zoller, MJ., and Smith, M. (1983) Methods Enzymol. 100, 468 to 500; Kramer, W. et al., (1984) Nucleic Acids Res. 12, 9441 to 9456; Kramer, W. and Fritz, HJ., (1987) Methods Enzymol. 154, 350 to 367; Kunkel, TA., (1985) Proc. Natl. Acad. Sci. USA. 82, 488 to 492; or Kunkel (1988) Methods Enzymol. 85, 2763 to 2766) or a similar method. In this way, an antibody functionality equivalent to the antibody of the present invention can be prepared.

[0079] A aforementioned antibody capable of recognizing an epitope of a CAPRIN-1 protein recognized by an anti-CAPRIN-1 antibody can be obtained through a method known to those skilled in the art. For example, it can be obtained through: a method comprising determining an epitope of a CAPRIN-1 protein recognized by an anti-CAPRIN-1 antibody through a general method (e.g., epitope mapping) and producing an antibody using a polypeptide that has an amino acid sequence contained in the epitope as an immunogen; or a method comprising determining an epitope of an antibody produced through a general method and selecting an antibody that has an epitope identical to an epitope of an anti-CAPRIN-1 antibody. Herein, the term epitope refers to a polypeptide fragment that has antigenicity or immunogenicity in mammals and, preferably, in humans.The smallest unit of the same consists of approximately 7 to 12 amino acids, and preferably 8 to 11 amino acids.

[0080] The affinity constant Ka (kon / koff) of an antibody of the present invention is preferably at least 107M-1, at least 108M-1, at least 5 χ 108M-1, at least 109M-1, at least 5 χ 109M-1, at least 1010M-1, at least 5 χ 1010M-1, at least 1011M-1, at least 5 χ Petition 870260065851, dated 03 / 07 / 2026, page 43 / 105 33 / 83 1011M-1, at least 1012M-1, or at least 1013M-1.

[0081] An antibody of the present invention can be conjugated with an antitumor agent. The linkage between an antibody and an antitumor agent can be performed through a spacer that has a reactive group to an amino group, a carboxyl group, a hydroxyl group, a thiol group, or the like (for example, an imidyl succinate group, a formyl group, a 2-pyridyldithio group, a maleimidyl group, an alkoxycarbonyl group, or a hydroxyl group).

[0082] Examples of anti-tumor agents include the following known anti-tumor agents from references or similar sources: paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bulatacin, bulatacinone, camptothecin, briostatin, calistatin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictin, spongistatin, chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, oxide hydrochloride mechlorethamine, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, dynemycin, clodronate, esperamycin, aclacinomycin, actinomycin, authenticamycin, azaserine, bleomycin,cactinomycin, carabicin, carminomycin, carzinophylline, cromomycin, dactinomycin, detorbicin, 6-diazo-5-oxo-Lnorleucine, ADRIAMYCIN, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, chelamicin, rhodorubicin, streptonigrine, streptozocin, tubercidine, ubenimexa, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, Petition 870260065851, dated 03 / 07 / 2026, page. 44 / 105 34 / 83 thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldofosfamide glycoside, aminolevulinic acid, enyluracil, amsacrine, bestrabucil, bisanthrene, edatrataxate, defofamine, demecolcine, diaziquone, elfornithine, eliptinium acetate, epothilone, etoglucid, lentinan, lonidamine, maitansine, ansamitocin, mitoguanine, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pyrarubicin, losoxantrone, podophylinic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxin, schizophyllan, spirogermanium, tenuazonic acid, triaziquone, roridin A, anguidine, urethane, vindesine, dacarbazine, manomustine, mitobronitol, mitolactol, pipobromane, gacitosine, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine,cisplatin, oxaliplatin, carboplatin, vinblastine, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novanthrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan, topoisomerase inhibitor, difluoromethylornithine (DMFO), retinoic acid, capecitabine and pharmacologically acceptable salts or derivatives thereof.

[0083] Alternatively, it is also possible to link a radioactive isotope, such as 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 175Lu, or 176Lu known from references and similar to an antibody of the present invention. It is desirable that such radioactive isotopes be effective for the treatment or diagnosis of tumors.

[0084] An antibody of the present invention is an antibody having immunological reactivity with CAPRIN-1 or an antibody capable of specifically recognizing CAPRIN-1. Such an antibody must have a structure that allows an animal in question, to which the antibody is administered, to completely or almost completely prevent a Petition 870260065851, dated 03 / 07 / 2026, p. 45 / 105 35 / 83 rejection reaction. If the animal in question is a human being, examples of the above antibodies include human antibodies, humanized antibodies, chimeric antibodies (e.g., human-mouse chimeric antibodies), single-chain antibodies, and bispecific antibodies. Such an antibody is a recombinant antibody that has variable heavy chain and light chain regions derived from human antibody, a recombinant antibody that has variable heavy chain and light chain regions, each consisting of complementarity-determining regions derived from non-human animal antibody (CDR1, CDR2, and CDR3) and structure regions derived from human antibody, or a recombinant antibody that has variable heavy chain and light chain regions derived from non-human animal antibody and constant heavy chain and light chain regions derived from human antibody. The first two antibodies are preferred.

[0085] The recombinant antibody described above can be produced as described below. The DNA encoding a monoclonal antibody against human CAPRIN-1 (e.g., a human monoclonal antibody, a mouse monoclonal antibody, a rat monoclonal antibody, a rabbit monoclonal antibody, or a chicken monoclonal antibody) is cloned from an antibody-producing cell, such as a hybridoma. The DNAs encoding a variable light chain region and a variable heavy chain region of the antibody are produced by an RT-PCR or similar method using the obtained clone as a template. Then, the sequences of a variable light chain region and a variable heavy chain region, or the CDR1, CDR2, and CDR3 sequences, are determined by the EU Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institute of Health, Bethesda, Md. (1991)). Petition 870260065851, dated 03 / 07 / 2026, p. 46 / 105 36 / 83

[0085] Furthermore, such DNAs encoding variable regions or DNAs encoding CDRs are produced by a recombinant gene technique (Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) or a DNA synthesizer. Here, the above human monoclonal antibody-producing hybridoma can be produced by immunizing a human antibody-producing animal (e.g., a mouse) with human CAPRIN-1 and fusing splenocytes from the animal's spleen with myeloma cells. In addition to the aforementioned, if necessary, DNAs encoding variable regions and constant heavy chain or light chain regions derived from human antibodies are produced through a recombinant gene technique or a DNA synthesizer.

[0087] In the case of a humanized antibody, DNA is produced in which the CDR coding sequences in DNA encoding a variable region of a human antibody-derived heavy or light chain have been replaced by corresponding CDR coding sequences from an antibody derived from a non-human animal (e.g., a mouse, a rat, or a chicken). The DNA obtained as described above is ligated to DNA encoding a constant region of a human antibody-derived heavy and light chain. Thus, DNA encoding a humanized antibody can be produced.

[0088] In the case of a chimeric antibody, DNA encoding a variable region of heavy chain and light chain antibody derived from a non-human animal (e.g., a mouse, a rat, or a chicken) is ligated to DNA encoding a constant region of heavy chain or light chain derived from a human antibody. Therefore, DNA encoding a chimeric antibody can be produced.

[0089] A single-chain antibody is an antibody in which a Petition 870260065851, dated 03 / 07 / 2026, p. 47 / 105 37 / 83 A variable heavy chain region and a variable light chain region are linearly linked together by means of a linker. The DNA encoding a single-stranded antibody can be produced by linking DNA encoding a variable heavy chain region, DNA encoding a linker, and DNA encoding a variable light chain region. Here, a variable heavy chain region and a variable light chain region are those derived from a human antibody or those derived from a human antibody in which only the CDRs have been replaced by CDRs from an antibody derived from a non-human animal (e.g., a mouse, a rat, or a chicken). Furthermore, the linker consists of 12 to 19 amino acids. An example of this is (G4S)3 which consists of 15 amino acids (GB Kim et al., Protein Engineering Design and Selection 2007, 20 (9): 425 to 432).

[0090] A bispecific antibody (diabody) is an antibody capable of specifically binding to two different epitopes in which, for example, DNA encoding a variable heavy chain region A, DNA encoding a variable light chain region B, DNA encoding a variable heavy chain region B, and DNA encoding a variable light chain region A are linked together in such an order (provided that DNA encoding a variable light chain region B and DNA encoding a variable heavy chain region B are linked together via DNA encoding a ligand described above). Therefore, DNA encoding a bispecific antibody can be produced.Here, both a variable heavy chain region and a variable light chain region are those derived from a human antibody or those derived from a human antibody in which only the CDRs have been replaced by CDRs from an antibody derived from a non-human animal (e.g., a mouse, a rat, or a chicken).

[0091] The recombinant DNA produced, as mentioned Petition 870260065851, dated 03 / 07 / 2026, page 48 / 105 38 / 83 above, is incorporated into one or a plurality of appropriate vector(s). Each of these vectors is introduced into a host cell (e.g., a mammalian cell, a yeast cell, or an insect cell) for (co)expression. Therefore, a recombinant antibody can be produced (PJ Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997; WILEY, P. Shepherd and C. Dean., Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; JW Goding, Monoclonal Antibodies: Principles and Practice., 1993 ACADEMIC PRESS).

[0092] Examples of an antibody of the present invention produced by the above method include the following antibodies (a) to (k). (a) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41, and 42 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 44, 45, and 46 (and preferably an antibody composed of a variable heavy chain region of SEQ ID NO: 43 and a variable light chain region of SEQ ID NO: 47); (b) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41, and 42 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 50, 51, and 52 (and preferably an antibody composed of a variable heavy chain region of SEQ ID NO: 43 and a variable light chain region of SEQ ID NO: 53); (c) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41, and 42 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 55, 56, and 57 (and preferably an antibody composed of a variable heavy chain region of SEQ ID NO: 43 and a variable light chain region of SEQ ID NO: 58); Petition 870260065851, dated 03 / 07 / 2026, page 49 / 105 39 / 83 (d) An antibody comprising a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41, and 42 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 60, 61, and 62 (and preferably an antibody composed of a variable heavy chain region of SEQ ID NO: 43 and a variable light chain region of SEQ ID NO: 63); (e) An antibody comprises a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41 and 42 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 65, 66 and 67 (and preferably an antibody composed of a variable heavy chain region of SEQ ID NO: 43 and a variable light chain region of SEQ ID NO: 68); (f) An antibody comprises a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 70, 71 and 72 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 74, 75 and 76 (and preferably an antibody composed of a variable heavy chain region of SEQ ID NO: 73 and a variable light chain region of SEQ ID NO: 77); (g) An antibody comprises a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 80, 81 and 82 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 84, 85 and 86 (and preferably an antibody composed of a variable heavy chain region of SEQ ID NO: 83 and a variable light chain region of SEQ ID NO: 87); (h) An antibody comprises a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 90, 91 and 92 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 94, 95 and 96 (and preferably an antibody Petition 870260065851, dated 03 / 07 / 2026, page 50 / 105 40 / 83 composed of a variable heavy chain region with SEQ ID NO: 93 and a variable light chain region with SEQ ID NO: 97); (i) An antibody comprises a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 100, 101 and 102 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 104, 105 and 106 (and preferably an antibody composed of a variable heavy chain region of SEQ ID NO: 103 and a variable light chain region of SEQ ID NO: 107); (j) An antibody comprises a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 110, 111 and 112 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 114, 115 and 116 (and preferably an antibody composed of a variable heavy chain region of SEQ ID NO: 113 and a variable light chain region of SEQ ID NO: 117); (k) An antibody comprises a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 120, 121 and 122 and a variable light chain region comprising the sequences shown in SEQ ID NOs: 124, 125 and 126 (and preferably an antibody composed of a variable heavy chain region of SEQ ID NO: 123 and a variable light chain region of SEQ ID NO: 127). [00 93] Here, the amino acid sequences shown in SEQ ID NOs: 40, 41 and 42, amino acid sequences shown in SEQ ID NOs: 70, 71 and 72, amino acid sequences shown in SEQ ID NOs: 80, 81 and 82, amino acid sequences shown in SEQ ID NOs: 90, 91 and 92, amino acid sequences shown in SEQ ID NOs: 100, 101 and 102, amino acid sequences shown in SEQ ID NOs: 110, 111 and 112 or amino acid sequences shown in SEQ ID NOs: 120, 121 and 122 correspond to CDR1, CDR2 and CDR3 of variable heavy chain regions. Petition 870260065851, dated 03 / 07 / 2026, p. 51 / 105 41 / 83 of mouse antibody, respectively.

[0094] In addition, the amino acid sequences shown in SEQ ID NOs: 44, 45 and 46, the amino acid sequences shown in SEQ ID NOs: 50, 51 and 52, the amino acid sequences shown in SEQ ID NOs: 55, 56 and 57, the amino acid sequences shown in SEQ ID NOs: 60, 61 and 62, the amino acid sequences shown in SEQ ID NOs: 65, 66 and 67, the amino acid sequences shown in SEQ ID NOs: 74, 75 and 76, the amino acid sequences shown in SEQ ID NOs: 84, 85 and 86, the amino acid sequences shown in SEQ ID NOs: 94, 95 and 96, the amino acid sequences shown in SEQ ID NOs: 104, 105 and 106, the The amino acid sequences shown in SEQ ID NOs: 114, 115, and 116, or the amino acid sequences shown in SEQ ID NOs: 124, 125, and 126, correspond to CDR1, CDR2, and CDR3 of variable regions of mouse antibody light chain, respectively.

[0095] In addition, a humanized antibody, a chimeric antibody, a single-chain antibody or a bi-specific antibody of the present invention is, for example, the following antibody (i) or (ii) (an example of antibody (a) is described below). (i) An antibody comprising: a variable heavy chain region comprising the amino acid sequences of SEQ ID NOs: 40, 41 and 42 and an amino acid sequence from a human antibody-derived structure region; and a variable light chain region comprising the amino acid sequences of SEQ ID NOs: 44, 45 and 46 and the amino acid sequences from the human antibody-derived structure region (and preferably an antibody comprising the amino acid sequence of SEQ ID NO: 43 in a variable heavy chain region and the amino acid sequence of SEQ ID NO: 47 in a variable light chain region); (ii) An antibody comprising: a variable chain region Petition 870260065851, dated 03 / 07 / 2026, p. 52 / 105 42 / 83 heavy chain comprising the amino acid sequences of SEQ ID Nos: 40, 41, and 42 and the amino acid sequences of the human antibody-derived structure regions; a heavy chain constant region comprising a human antibody-derived amino acid sequence; a light chain variable region comprising the amino acid sequences of SEQ ID Nos: 44, 45, and 46 and the amino acid sequences of the human antibody-derived structure regions; and a light chain constant region comprising a human antibody-derived amino acid sequence (and preferably an antibody comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 43; a heavy chain constant region comprising a human antibody-derived amino acid sequence;a variable light chain region comprising the amino acid sequence of SEQ ID No: 47 and a constant light chain region comprising an amino acid sequence derived from a human antibody.

[0096] In addition, sequences of variable and constant regions of human antibody light and heavy chains can be obtained from, for example, NCBI (USA: GenBank, UniGene, etc.). For example, the following sequences can be used as reference sequences for the corresponding regions: the sequence with registry number J00228 for a constant region of human IgG1 heavy chain; the sequence with registry number J00230 for a constant region of human IgG2 heavy chain; the sequence with registry number X03604 for a constant region of human IgG3 heavy chain; a sequence with registry number K01316 for a constant region of human IgG4 heavy chain; the sequence with registry number V00557, X64135 or X64133 for a constant κ region of human light chain; and the sequence with registry number X64132 or X64134 for a constant λ region of Petition 870260065851, dated 03 / 07 / 2026, page 53 / 105 43 / 83 human light chain.

[0097] The antibodies above preferably have cytotoxic activity, thus exhibiting antitumor effects.

[0098] Furthermore, the above-mentioned specific sequences of heavy chain and variable light chain regions and CDRs in an antibody are merely described for illustrative purposes. It is clear that the present invention is not limited to particular sequences. A hybridoma capable of producing a different human antibody or a non-human animal antibody (e.g., a mouse antibody) as opposed to human CAPRIN-1 is produced. A monoclonal antibody produced by the hybridoma is collected. Then, it is determined whether or not the obtained antibody is an antibody of interest using, as indicators, immunological binding activity and cytotoxic activity relative to a human CAPRIN-1. In this way, a hybridoma that produces a monoclonal antibody of interest is identified. Therefore, as described above, the DNAs encoding the variable light chain and heavy chain regions of an antibody of interest are produced from the hybridoma for sequence determination.DNAs are used to produce different antibodies.

[0099] Additionally, the antibody above the present invention may be any of the antibodies (i) to (iv) above that have a substitution, deletion or addition of one or more (and preferably 1 or 2) amino acid(s), particularly a structure region sequence and / or a constant region sequence, provided that it has a specifically recognizable CAPRIN-1 specific property. Herein, the term more amino acids indicates 2 to 5 and preferably 2 or 3 amino acids.

[0100] Furthermore, according to the present invention, the DNA encoding the antibody above the present invention, the DNA encoding a heavy chain or light chain of the antibody, or DNA encoding a region Petition 870260065851, dated 03 / 07 / 2026, p. 54 / 105 44 / 83 variable light chain or heavy chain of the antibody is also provided. For example, in the case of antibody (a), examples of such DNAs include: DNA encoding a variable heavy chain region comprising nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 40, 41 and 42; and DNA encoding a variable light chain region comprising nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 44, 45 and 46.

[0101] The complementarity-determining regions (CDRs) encoded by the DNAs of the above sequences are regions that determine antibody specificity. Therefore, the sequences encoding the other regions (i.e., constant regions and structure regions) in an antibody may be sequences from a different antibody. Here, different antibodies include antibodies derived from non-human organisms. However, in view of reducing side effects, human-derived antibodies are preferred. That is, in the case above, the DNA regions encoding the structure regions and constant regions of light and heavy chains preferably comprise the nucleotide sequences encoding the relevant amino acid sequences from a human antibody.

[0102] Additionally, different examples of DNA encoding an antibody of the present invention, such as antibody (a), include DNA encoding a variable heavy chain region comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43 and DNA in which a region encoding a variable light chain 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 Petition 870260065851, dated 03 / 07 / 2026, page 55 / 105 45 / 83 nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 47 is the nucleotide sequence of SEQ ID NO: 49. Furthermore, the above DNAs encoding the heavy chain and constant light chain regions preferably comprise the nucleotide sequences encoding the corresponding human antibody-derived amino acid sequences.

[0103] The DNA of the present invention can be obtained by, for example, the aforementioned methods or by the following methods. First, total RNA is prepared from a hybridoma for an antibody of the present invention using a commercially available RNA extraction kit. Then, cDNA is synthesized with a reverse transcriptase using random indicators and the like. Next, the cDNA encoding an antibody is amplified by a PCR method using, as indicators, oligonucleotides that have conserved sequences in various regions of known mouse antibody light and heavy chain genes. The sequences encoding the conserved regions can be obtained by amplifying known sequences using a PCR method. The nucleotide sequence of the DNA can be determined by a general method involving, for example, incorporation into a plasmid or phage for sequence determination.

[0104] The antitumor effects of an anti-CAPRIN-1 antibody used in the present invention on cancer cells expressing CAPRIN-1 are believed to be exhibited through cytotoxicity mechanisms described below.

[0105] Cytotoxicities are antibody-dependent cellular cytotoxicity (ADCC) mediated by effector cells against cells expressing CAPRIN-1, and complement-dependent cytotoxicity (CDC) against cells expressing CAPRIN-1.

[0106] Consequently, the activity of an anti-Petition antibody 870260065851, dated 03 / 07 / 2026, page 56 / 105 46 / 83 The CAPRIN-1 used in the present invention can be evaluated through ex vivo determination of ADCC activity or CDC activity in cancer cells expressing CAPRIN-1, as specifically described in the examples mentioned below.

[0107] An anti-CAPRIN-1 antibody used in the present invention binds to a CAPRIN-1 protein in a cancer cell and exhibits anti-tumor effects based on the above activity. Therefore, such an antibody is believed to be useful in the treatment or prevention of cancer. Specifically, according to the present invention, a pharmaceutical composition for the treatment and / or prevention of cancer comprising, as an active ingredient, an anti-CAPRIN-1 antibody is provided. When an anti-CAPRIN-1 antibody is used for the purpose of administering an antibody to humans (antibody treatment), it is preferably used in the form of a human antibody or a humanized antibody in order to reduce immunogenicity.

[0108] Furthermore, as the binding affinity between an anti-CAPRIN-1 antibody and a CAPRIN-1 protein on a cancer cell surface becomes higher, stronger antitumor activity can be exhibited by an anti-CAPRIN-1 antibody. Therefore, if an anti-CAPRIN-1 antibody with high binding affinity for a CAPRIN-1 protein can be obtained, even stronger antitumor effects can be expected. Consequently, it becomes possible to use such an antibody as a pharmaceutical composition for the treatment and / or prevention of cancer. As described above, for high binding affinity, the affinity constant Ka (kon / koff) is preferably at least 10⁷M⁻¹, at least 10⁸M⁻¹, at least 5χ¹⁰⁸M⁻¹, at least 10⁹M⁻¹, at least 5χ¹⁰⁹M⁻¹, at least 10¹⁰M⁻¹, at least 5χ¹⁰M⁻¹, at least 10¹¹M⁻¹, at least 5χ¹⁰M⁻¹, by Petition 870260065851, dated 03 / 07 / 2026, page 57 / 105 47 / 83 minus 1012M-1 or at least 1013M-1. Binding to cells that express antigen

[0109] The ability of an antibody to bind to a CAPRIN-1 can be specified by a binding assay using, for example, ELISA, a Western blot method, immunofluorescence or flow cytometry analysis, as described in the Examples. Immunohistochemical staining

[0110] An antibody that recognizes CAPRIN-1 can be tested for reactivity with CAPRIN-1 by an immunohistochemical method known to those skilled in the art using a frozen tissue section fixed with paraformaldehyde or acetone or a paraffin-embedded tissue section fixed with paraformaldehyde. Such a section is prepared from tissue obtained from a patient during surgery or an animal bearing xenotransplanted tissue is inoculated with a natural cell or transfected cell line expressing CAPRIN1.

[0111] For immunohistochemical labeling, a CAPRIN-1 reactive antibody can be labeled by a variety of methods. For example, it can be visualized by reaction with a mouse horseradish peroxidase-conjugated goat antibody or rabbit and goat antiantibody. Pharmaceutical composition

[0112] A target of the pharmaceutical composition for the treatment and / or prevention of cancer of the present invention is not particularly limited as long as the target is a cancer (cell) that expresses the CAPRIN-1 gene.

[0113] Both terms tumor and cancer used in the present invention refer to malignant neoplasm and, in this way, they are used interchangeably. Petition 870260065851, dated 03 / 07 / 2026, page 58 / 105 48 / 83

[0114] A cancer that may be a target in the present invention is a cancer that expresses a gene encoding a polypeptide comprising an amino acid sequence of any of the even-numbered SEQ IDs: 2 to 30 or a partial sequence consisting of 7 or more consecutive amino acids of said amino acid sequence. Preferred examples thereof include breast cancer, brain tumor, leukemia, lung cancer, lymphoma, mast cell tumor, esophageal cancer, and colon cancer.

[0115] Examples of these specific cancers include, but are not limited to, breast adenocarcinoma, composite breast adenocarcinoma, malignant mixed breast tumor, intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, glioma which is a neuroepithelial tissue tumor, ependymoma, neuronal tumor, embryonal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, digestive lymphoma, medium to small cell lymphoma, cecal cancer, ascending colon cancer, descending colon cancer, transverse colon cancer, sigmoid colon cancer, and rectal cancer.

[0116] Furthermore, the animal object of the present invention is a mammal. Examples include mammals such as primates, domestic animals, farm animals and sport animals. Humans, dogs and cats are particularly preferred.

[0117] When an antibody used in the present invention is used as a pharmaceutical composition, it may be formulated by a method known to those skilled in the art. For example, it may be used parenterally in the form of a parenteral injection of: an aseptic solution comprising water or a pharmaceutically acceptable non-aqueous solution; or a suspension liquid. For example, in Petition 870260065851, dated 03 / 07 / 2026, page 59 / 105 49 / 83 In one possible case, it may be formulated using a combination of a pharmacologically acceptable carrier or medium, specifically sterile water, physiological saline, vegetable oil, an emulsifier, a suspension, a surfactant, a stabilizer, a flavoring agent, an excipient, a vehicle, a preservative, or a binding agent, in a suitable manner by mixing a dosage unit form required for a generally acceptable pharmaceutical formulation. The amount of an active ingredient in a formulation is determined so that an appropriate dosage within the indicated range can be achieved.

[0118] An aseptic composition for injection purposes may be formulated in accordance with general formulation practice using a vehicle such as distilled water for injection purposes.

[0119] Examples of an aqueous solution for injection purposes include isotonic and physiological saline solutions comprising glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Such a solution may be used with a suitable dissolving aid. Examples of such dissolving aids include alcohols such as ethanol and polyalcohol, propylene glycol, polyethylene glycol, and nonionic surfactants such as polysorbate 80(TM) and HCO-60.

[0120] Examples of oily liquids include sesame oil and soybean oil. Such an oily liquid may be used in combination with a dissolving aid such as benzyl benzoate or benzyl alcohol. Additionally, it may be mixed with a buffering agent such as a phosphate buffer solution, a sodium acetate buffer solution, a tranquilizing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, or an antioxidant. In general, a formulated injection solution is introduced into a suitable wide container.

[0121] The above pharmaceutical composition is for oral or Petition 870260065851, dated 03 / 07 / 2026, pages 60 / 105 50 / 83 administered parenterally. Preferably, it is administered parenterally. Specific examples of dosage forms include injectable agents, intranasally administered agents, transpulmonarily administered agents, and percutaneously administered agents. For example, injectable agents can be administered systemically or locally via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0122] Furthermore, the method of administration may be appropriately determined depending on the patient's age, weight, gender, and symptoms. A single dose of a pharmaceutical composition comprising an antibody or a polynucleotide encoding an antibody may be selected within a range of, for example, 0.0001 mg to 1000 mg per kg of body weight. Alternatively, the dose may be selected within a range of, for example, 0.001 to 100000 mg per kg of body weight; however, it is not necessary to limit it to the same. The dose and method of administration are changed depending on the patient's age, weight, gender, and symptoms. However, persons skilled in the art may appropriately select the dose and method. POLYPEPTIDE AND DNA

[0123] According to the present invention, the following polypeptides and DNAs for antibodies (a) to (k) described above are additionally provided. (i) A polypeptide comprising the amino acid sequences SEQ ID NO: 43, SEQ ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 93, SEQ ID NO: 103, SEQ ID NO: 113, and SEQ ID NO: 123, and DNA encoding the polypeptide; (ii) A polypeptide comprising the amino acid sequences SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: Petition 870260065851, dated 03 / 07 / 2026, page 61 / 105 51 / 83 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 DNA encoding the polypeptide; (iii) DNA comprising the nucleotide sequences 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; (iv) DNA comprising 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; (v) A heavy chain CDR polypeptide comprising amino acid sequences selected from the group consisting of amino acid sequences with SEQ ID NOs: 40, 41 and 42, amino acid sequences with SEQ ID NOs: 70, 71 and 72, amino acid sequences with SEQ ID NOs: 80, 81 and 82, amino acid sequences with SEQ ID NOs: 90, 91 and 92, amino acid sequences with SEQ ID NOs: 100, 101 and 102, amino acid sequences with SEQ ID NOs: 110, 111 and 112, and amino acid sequences with SEQ ID NOs: 120, 121, and 122 and DNA encoding the polypeptide; (vi) A CDR light chain polypeptide comprising amino acid sequences selected from the group consisting of amino acid sequences with SEQ ID NOs: 44, 45 and 46, amino acid sequences with SEQ ID NOs: 50, 51 and 52, amino acid sequences with SEQ ID NOs: 55, 56 and 57, amino acid sequences with SEQ ID NOs: 60, 61 and 62, amino acid sequences with SEQ ID NOs: 65, 66 and 67, amino acid sequences with SEQ ID NOs: 74, 75 and 76, amino acid sequences with SEQ ID NOs: 84, 85 and 86, amino acid sequences with SEQ ID NOs: 94, 95 and 96, amino acid sequences with SEQ ID NOs: 104, 105 and 106, amino acid sequences with SEQ ID Nos: 114, 115 and 116, and Petition 870260065851, dated 03 / 07 / 2026, p. 62 / 105 52 / 83 amino acid sequences of SEQ ID NOS: 124, 125 and 126, and DNA encoding the polypeptide.

[0124] These polypeptides and DNAs can be produced using a recombinant gene technique as described above. Examples

[0125] The present invention is hereinafter described in greater detail with reference to the following examples, although the scope of the present invention is not limited to them. Example 1: Identification of a new cancer antigen protein using the SEREX method (1) cDNA library construction.

[0126] Total RNA was extracted from a healthy dog ​​testicular tissue using a Guanidium-Phenol-Chloroform method and then a polyA RNA was purified according to the protocols included with an Oligotex-dT30 mRNA Purification Kit (Takara Shuzo Co., Ltd.).

[0127] A canine testis cDNA phage library was synthesized using mRNA obtained in this way (5 pg). The cDNA phage library was constructed using a cDNA Synthesis Kit, a ZAP-cDNA Synthesis Kit, and a ZAP-cDNA GigapackIII Gold Cloning Kit (STRATAGENE) according to the protocols included in the kits. The size of the cDNA phage library obtained in this way was 7.73χ 105 pfu / ml. (2) cDNA library scan using serum.

[0128] Immunoscavenging was performed using the canine testis cDNA phage library constructed above. Specifically, host Escherichia coli (XL1-Blue MRF') was infected with the phage on an NZY agarose plate (Φ90 x 15 mm) to obtain 2210 clones. E. coli cells were cultured at 42°C for 3–4 hours to form plates. The slide was covered with a nitrocellulose membrane (Hybond C Extra: GE Petition 870260065851, dated 03 / 07 / 2026, page 63 / 105 53 / 83 (Healthcare Bio-Science) impregnated with IPTG (isopropyl-δ-D-thiogalactoside) at 37°C for 4 hours, so that the protein was induced, expressed, and then transferred to the membrane. Subsequently, the membrane was collected and then immersed in TBS (10 mM Tris-HCl, 150 mM NaCl, and pH 7.5) containing 0.5% skimmed milk powder, followed by overnight agitation at 4°C, thus suppressing non-specific reaction. The filter was reacted with a 500-fold diluted serum from a canine patient at room temperature for 2–3 hours.

[0129] As with the serum above from a canine patient, serum collected from a canine patient with breast cancer was used. These sera were stored at -80°C and then subjected to pretreatment immediately before use. One method for serum pretreatment is as follows. Specifically, host Escherichia coli (XL1-Blue MRF') was infected with a λ ZAP Express phage into which no foreign genes were inserted and then cultured overnight in an NZY slide medium at 37°C. Subsequently, buffer (0.2 M NaHCO3 and pH 8.3) containing 0.5 M NaCl was added to the slide, the slide was left to stand at 4°C for 15 hours, and then a supernatant was collected as a phage / Escherichia coli extract. Then, the phage / Escherichia coli extract collected in this way was applied to an NHS (GE Healthcare BioScience) column, so that a phage-derived protein from Escherichia coli was immobilized.Serum from a canine patient was applied to the immobilized protein column for reaction, and then Escherichia coli and an absorbed antibody to the phage were removed from the serum. The serum fraction that passed through the column was diluted 500 times in TBS containing 0.5% skimmed milk powder. The resulting solution was used as an immunoscavenging material.

[0130] A membrane, onto which the treated serum and fusion protein were transferred, was washed 4 times with TBS-T (0.05% of Petition 870260065851, dated 03 / 07 / 2026, page 64 / 105 54 / 83 Tween20 / TBS) and then induced to react with goat-produced anti-dog IgG (dog anti-IgG-h conjugate + goat-produced I HRP) (BETHYL Laboratories) diluted 5000 times in TBS containing 0.5% skimmed milk powder as a secondary antibody for 1 hour at room temperature. Detection was performed using an enzyme that provides color to the reaction with the use of an NBT / BCIP reaction solution (Roche). Colonies that had positively matching sites for a staining reaction were collected from the NZY agarose plate (Φ90 x 15 mm) and then lysed in 500 µl of SM buffer (100 mM NaCl, 10 mM MgClSO4, 50 mM Tris-HCl, 0.01% gelatin and pH 7.5). Until the colonies that tested positive for staining were unified, secondary and tertiary screening were repeated so that 30,940 phage clones reacting with serum IgG were screened using a similar method as described above. Thus, 5 positive clones were isolated. (3) Homology search for isolated antigen gene.

[0131] For nucleotide sequence analysis of the 5 positive clones isolated by the above method, a procedure for converting phage vectors into plasmid vectors was performed. Specifically, 200 µl of a solution were prepared to contain host Escherichia coli (XL1-Blue MRF') so that the absorbance OD600 was 1.0. The solution was mixed with 250 µl of a purified phage solution and then with 1 µl of an ExAssist helper phage (STRATAGENE), followed by a 15-minute reaction at 37°C. Three (3) ml of LB medium were added and then the culture was carried out at 37°C for 2.5 to 3 hours. Immediately after cultivation, the solution temperature was maintained at 70°C using a water bath for 20 minutes, centrifugation was performed at 4°C and 1000 x g for 15 minutes, and then the supernatant was collected as a phagomid solution. Subsequently, 200 µl of a solution were prepared to contain Petition 870260065851, dated 03 / 07 / 2026, page 65 / 105 55 / 83 host phagomid Escherichia coli (SOLR) so that the absorbance OD600 was 1.0. The solution was mixed with 10 μl of a purified phage solution, followed by a 15-minute reaction at 37°C. The solution (50 μl) was seeded onto LB Agar medium containing ampicillin (final concentration of 50 μg / ml) and then cultured overnight at 37°C. The single transformed SOLR colony was collected and then cultured onto LB medium containing ampicillin (final concentration: 50 μg / ml) at 37°C. A plasmid DNA containing the insert of interest was purified using a QIAGEN Plasmid Minipreparation Kit (QIAGEN).

[0132] The purified plasmid was subjected to full-length insert sequence analysis using a primer walking method with the T3 primer from SEQ ID NO: 31 and the T7 primer from SEQ ID NO: 32. As a result of the sequence analysis, gene sequences from SEQ ID NOs: 5, 7, 9, 11, and 13 were obtained. A homology search program, BLAST search (http: / / www.ncbi.nlm.nih.gov / BLAST / ), was performed using the nucleotide sequences of the genes and the corresponding amino acid sequences (SEQ ID NOs: 6, 8, 10, 12, and 14). As a result of this homology search with known genes, it was revealed that all 5 genes obtained encoded CAPRIN-1. Regarding the regions to be translated into proteins, the sequence identity among the 5 genes was 100% in terms of nucleotide sequence and 99% in terms of amino acid sequence.Furthermore, regarding the regions to be translated into proteins, the sequence identity between the genes and genes encoding homologous human factors (human homologs) was 94% in terms of nucleotide sequence and 98% in terms of amino acid sequence. The nucleotide sequences of the human homologs are shown in SEQ ID NOs: 1 and 3, and their amino acid sequences are shown in SEQ ID NOs: 2 and 4. Additionally, regarding... Petition 870260065851, dated 03 / 07 / 2026, p. 66 / 105 Of the 56 / 83 regions to be translated into proteins, sequence identity between the canine genes obtained in this way and a gene encoding a cattle homolog was 94% in terms of nucleotide sequence and 97% in terms of amino acid sequence. The nucleotide sequence of the cattle homolog is shown in SEQ ID No. 15 and its amino acid sequence is shown in SEQ ID No. 16. In addition, regarding the regions to be translated into proteins, the sequence identity between the genes encoding the human homologs and the gene encoding the cattle homolog was 94% in terms of nucleotide sequence and ranged from 93% to 97% in terms of amino acid sequence. Furthermore, regarding the regions to be translated into proteins, the sequence identity between the canine genes obtained and a gene encoding an equine homolog was 93% in terms of nucleotide sequence and 97% in terms of amino acid sequence.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. In addition, regarding the regions to be translated into proteins, the sequence identity between the genes encoding the human homologs and the gene encoding the equine homolog was 93% in terms of nucleotide sequence and 96% in terms of amino acid sequence. Furthermore, regarding the regions to be translated into proteins, the sequence identity between the obtained canine genes and the genes encoding rat homologs ranges from 87% to 89% in terms of nucleotide sequence and from 95% to 97% in terms of amino acid sequence. The nucleotide sequences of the rat homologs are shown in SEQ ID NOs: 19, 21, 23, 25 and 27, and the amino acid sequences of the same are shown in SEQ ID NOs: 20, 22, 24, 26 and 28.In addition, with respect to the regions to be translated into proteins, the sequence identity between the genes encoding the human homologs and the genes encoding the mouse homologs lies... Petition 870260065851, dated 03 / 07 / 2026, p. 67 / 105 57 / 83 of 89% to 91% in terms of nucleotide sequence and 95% to 96% in terms of amino acid sequence. Furthermore, regarding the regions to be translated into proteins, the sequence identity between the obtained canine genes and a gene encoding a chicken homolog was 82% in terms of nucleotide sequence and 87% in terms of amino acid sequence. 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. In addition, regarding the regions to be translated into proteins, the sequence identity between the genes encoding the human homologs and the gene encoding the chicken homolog ranged from 81% to 82% in terms of nucleotide sequence and was 86% in terms of amino acid sequence. (4) Gene expression analysis in each tissue.

[0133] The expression of genes obtained by the above method in normal canine and human tissues and different cell lines was analyzed by an RT-PCR method. The reverse transcription reaction was performed as follows. Specifically, total RNA was extracted from each tissue (50 mg to 100 mg) and each cell line (5 to 10 x 106 cells) using a TRIZOL reagent (Invitrogen) according to protocols also included. cDNA was synthesized using total RNA and the Superscript First Strand Synthesis System for RT-PCR (Invitrogen) according to protocols also included. PCR was performed as follows using specific primers for the obtained genes (SEQ ID NOs: 33 and 34).Specifically, PCR was performed by repeating 30 cycles at 94°C / 30 seconds, 60°C / 30 seconds, and 72°C / 30 seconds using a thermocycler (BIO RAD) and a reaction solution adjusted to a total quantity of 25 μL by adding each reagent and an attached buffer (0.25 μL of a sample prepared by reverse transcription reaction, the primers above (2. Petition 870260065851, dated 03 / 07 / 2026, page 68 / 105 58 / 83 μM each), dNTPs (0.2 mM each), and 0.65 U of ExTaq polymerase (Takara Shuzo). In addition, the specific gene primers mentioned above were used to extend the region between nucleotide number 206 and nucleotide number 632 in the nucleotide sequence (canine CAPRIN1 gene) of SEQ ID NO: 5 and in the region between nucleotide number 698 and nucleotide number 1124 in the nucleotide sequence (human gene). CAPRIN-1), from SEQ ID NO: 1. For comparison control, the specific primers GAPDH (SEQ ID NO: 35 and 36) were used simultaneously. As a result, as shown in Fig. 1, strong expression was observed in the testis in the case of healthy dog ​​tissues, while expression was also observed in canine breast cancer and adenocarcinoma tissues. Furthermore, the expression of human homologs homologous to the obtained genes was also confirmed. As a result, similar to the canine CAPRIN-1 genes, expression could only be confirmed in the testis in the case of normal tissues. However, in the case of cancer cells, expression was detected in several types of cancer cell lines, such as breast cancer, brain tumor, leukemia, lung cancer, and esophageal cancer cell lines. Expression was confirmed in a particularly large number of breast cancer cell lines.Based on the results, it was confirmed that CAPRIN-1 expression was not observed in normal tissues, with the exception of the testes, while CAPRIN-1 was expressed in many cancer cells, and particularly in breast cancer cell lines.

[0134] Furthermore, in Fig. 1, reference no. 1, along the longitudinal axis indicates the expression pattern of each of the genes identified above, and reference no. 2, along the same axis, indicates the expression pattern of the GAPDH gene for the comparison control. (5) Preparation of polyclonal antibodies against the peptide Petition 870260065851, dated 03 / 07 / 2026, page 69 / 105 59 / 83 derived from CAPRIN-1.

[0135] To obtain an antibody binding to CAPRIN-1, the CAPRIN-1-derived peptide (Arg-Asn-Glu-Leu-Lys-Lys-Lys-Gly-Lys-LeuAsp-Asp-Tyr-Gln (SEQ ID NO: 37)) was synthesized. The peptide (1 mg) as antigen was mixed with an incomplete Freund's adjuvant (IFA) solution in an amount equivalent to the peptide. The mixture was administered subcutaneously to a rabbit four times every two weeks. Subsequently, blood was collected, so that an antiserum containing a polyclonal antibody was obtained. Furthermore, the antiserum was purified using a G protein support (GE Healthcare Biosciences), and then a polyclonal antibody against the derived CAPRIN-1 peptide was obtained. Furthermore, an antibody obtained from purified rabbit serum was designated as the control antibody, and no antigen was administered using a G protein support as described above. (6) Analysis of antigen protein expression in the cancer cell.

[0136] Next, it was analyzed whether the CAPRIN-1 protein was expressed on the cell surfaces of 7 types of 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. Each human breast cancer cell line in which gene expression was confirmed (106 cells), as described above, was centrifuged in a 1.5-ml microcentrifuge tube. The polyclonal antibody against the CAPRIN-1 peptide derivative (2μg)(5μl) prepared in (5) above was added there. The result was again suspended in PBS containing 0.1% fetal bovine serum (95 μl) and then left to stand on ice for 1 hour. After washing with PBS, the resulting sample was suspended in PBS containing a FITC-labeled goat anti-rabbit IgG antibody (Santa Cruz). Petition 870260065851, dated 03 / 07 / 2026, pp. 70 / 105 60 / 83Biotechnology, Inc.) (5μI) and 0.1% fetal bovine serum (FBS) (95 μI) and then left to stand on ice for 1 hour. After washing with PBS, the fluorescence intensity was measured using FACSCalibur (Becton Dickinson and Company). Meanwhile, a similar procedure to the previous one was performed using the control antibody prepared in (5) above, instead of the polyclonal antibody against a CAPRIN-1 derived peptide, so that a control was prepared. As a result, the fluorescence intensity was at least 30% stronger in all cells in which the anti-human CAPRIN-1 antibody was added than in the control cells. Specifically, the following increases in fluorescence intensity were confirmed: MDA-MB-157: 184%, T47D: 221%, MRK-nu1: 115%, MDA-MB-231V: 82%, BT20: 32%, SK-BR-3: 279%, and MDA-MB-231T: 80%.Based on the above, it was confirmed that the CAPRIN-1 protein was expressed on the surfaces of cells in the human cancer cell line. Furthermore, the rate of increase in fluorescence intensity is represented by the rate of growth in the medium fluorescence intensity (MFI value) in the cells. It was calculated using the following equation.

[0137] Rate of increase in fluorescence intensity medium (rate of increase in fluorescence intensity) (%) = ((MFI value of cells reacted with an anti-human CAPRIN-1 antibody) - (control MFI value)) / (control MFI value) *100 (7) Immunohistochemical staining (7) -1 CAPRIN-1 expression in canine and normal mouse tissues.

[0138] Mice (Balb / c, female) and dogs (beagle dogs, female) were exsanguinated under ether anesthesia and ketamine / isoflurane anesthesia. After laparotomy, the organs (stomach, liver, eyeball, thymus gland, muscles, bone marrow, uterus, intestine, Petition 870260065851, dated 03 / 07 / 2026, page 71 / 105 61 / 83 esophagus, heart, kidneys, salivary glands, large intestine (colon), mammary gland, brain, lungs, skin, adrenal gland, ovary, pancreas, spleen, and bladder) were transferred to each 10 cm dish containing PBS. Each organ was opened in PBS and then subjected to overnight perfusion fixation with a 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde (PFA). The perfusate was discarded, the tissue surface of each organ was washed with PBS, and then a 10% sucrose-containing PBS solution was added to a 50 ml centrifuge tube. Each tissue was then placed in each tube and then agitated with a rotor at 4 °C for 2 hours. Each solution was replaced with a 20% sucrose-containing PBS solution and then left at 4 °C until the tissues precipitated. Each solution was replaced with a PBS solution containing 30% sucrose and then left at 4°C until the tissues precipitated.Each tissue sample was removed, and a necessary portion was removed with a surgical scalpel. Then, an OCT (Tissue-Tek) compound was applied and spread over the surface of each tissue, and the tissues were placed in a cryomold. The cryomold was placed on dry ice for rapid freezing. The tissues were cut into sections 10 to 20 μm thick using a cryostat (Leica), and then the cut tissue sections were air-dried on glass slides for 30 minutes using a hairdryer, so that the glass slides on which the cut tissue sections were placed were prepared. Next, each glass slide was placed in a staining vial with PBS-T (saline containing 0.05% Tween20), so that a process involving exchange with PBS-T was performed three times every 5 minutes. Excess water around each sample was removed using Kimwipes, and then each section was surrounded using DAKOPEN (DAKO).As blocking solutions, a mouse lg MOM blocking reagent (VECTASTAIN) was used. Petition 870260065851, dated 03 / 07 / 2026, page 72 / 105 62 / 83 was applied to mouse tissue and a PBS-T solution containing 10% FBS was applied to canine tissue. The resulting samples were left to stand in a humid chamber at room temperature for one hour. Then, a solution was prepared containing a monoclonal antibody (monoclonal antibody #6) against CAPRIN-1 with the variable region of the heavy chain of SEQ ID NO: 73 and the variable region of the light chain of SEQ ID NO: 77 and reacted with the surfaces of the cancer cells prepared in Example 4, where the antibody was dried at a concentration of 10 μg / ml in the blocking solution. The solution was applied to each glass slide and then left in a humid chamber at 4°C overnight. After 3 washes, each 10 minutes with PBS-T, the biotin-labeled anti-IgG antibody MOM (VECTASTAIN) diluted 250 times with the blocking solution was applied to each glass slide and then placed in a humid chamber at room temperature for one hour.After 3 washes, each 10 minutes with PBS-T, an ABC avidin-biotin reagent (Vectastain) was applied, and then the slides were placed in a humid chamber at room temperature for 5 minutes. After 3 washes, each 10 minutes with PBS-T, a DAB staining solution (DAB 10 mg + 30% H2O2 10 μl / 0.05 M Tris-HCl (pH 7.6) 50 ml) was applied, and then the glass slides were left in a humid chamber at room temperature for 30 minutes. The glass slides were washed with distilled water, and then a hematoxylin reagent (DAKO) was applied. After being left at room temperature for 1 minute, the glass slides were washed with distilled water. The slides were immersed in solutions of 70%, 80%, 90%, 95%, and 100% ethanol in that order for 1 minute each, and then left in xylene overnight.

[0139] The glass slides were removed, covered with coverslips with Glycergel Mounting Medium (DAKO), and then observed. As a result, CAPRIN-1 expression was observed to a slight degree. Petition 870260065851, dated 03 / 07 / 2026, page 73 / 105 63 / 83 inside cells in all salivary glands, kidney, colon, stomach, and tissues, but CAPRIN-1 expression was never observed on the cell surface. Furthermore, absolutely no CAPRIN-1 expression was observed in tissues of other organs. In addition, similar results were obtained when the monoclonal antibody was used against CAPRIN-1 having the variable heavy chain region of SEQ ID NO: 103 and the variable light chain region of SEQ ID NO: 107 (monoclonal antibody #9). (7) -2 Expression CAPRIN- 1 in canine tissues with breast cancer. [01 40] Using 108 frozen canine mammary cancer tissue samples from dogs diagnosed by pathological diagnosis as having malignant mammary cancer, the frozen slides were prepared by a method similar to the method above and immunohistochemical staining was performed using monoclonal antibody #6 prepared in Example 4. As a result, CAPRIN-1 expression was confirmed in 100 of 108 specimens (92.5%). CAPRIN-1 was primarily expressed on the surface of highly atypical cancer cells. Furthermore, similar results were obtained when monoclonal antibody #9 produced in Example 4 was used; (7) -3 Expression CAPRIN- 1 IN human breast cancer tissue.

[0141] Immunohistochemical staining was performed using 188 breast cancer tissue samples from a paraffin-embedded human breast cancer tissue matrix (BIOMAX). After 3 hours of treatment at 60 °C, the human breast cancer tissue matrix was added to a staining vial filled with xylene, then the xylene was replaced three times every 5 minutes. Subsequently, a similar procedure was performed using ethanol and PBS-T instead of xylene. The matrix Petition 870260065851, dated 03 / 07 / 2026, pp. 74 / 105 64 / 83 of human breast cancer tissue was added to a staining vial with 10 mM citrate buffer (pH 6.0) containing 0.05% Tween20, treated for 5 minutes at 125°C, and then left at room temperature for 40 minutes or more. Excess water around each sample was removed using Kimwipes, each section was surrounded using DAKOPEN, and then an appropriate amount of Peroxidase Block (DAKO) was added dropwise. The resulting solution was left to stand at room temperature for 5 minutes and then added to a staining vial with PBS-T. PBS-T replacement was performed three times every 5 minutes. As a blocking solution, a PBS-T solution containing 10% FBS was applied and then left in a humid chamber at room temperature for one hour.Next, a solution was prepared containing monoclonal antibody #6 that reacts with the surfaces of cancer cells prepared in Example 4 at a concentration of 10 μg / ml, adjusted with a PBS-T solution containing 5% PBS. The solution was applied and then left overnight in a humid chamber at 4°C. After 3 washes, each lasting 10 minutes, with PBS-T, an appropriate amount of Peroxidase-Labeled Conjugated Polymer (DAKO) was added dropwise, and then the glass slides were left to stand at room temperature for 30 minutes in a humid chamber. After 3 washes, each lasting 10 minutes, with PBS-T, a DAB staining solution (DAKO) was applied and then left to stand at room temperature for 10 minutes. The DAB staining solution was discarded, and then 10-minute washes with PBS-T were performed 3 times.The glass slides were washed with distilled water and immersed in solutions of 70%, 80%, 90%, 95%, and 100% ethanol in that order for 1 minute each time, and then left to stand in xylene overnight. The glass slides were removed, covered with coverslips, and coated with Glycergel Mounting Medium. Petition 870260065851, dated 03 / 07 / 2026, pages 75 / 105 65 / 83 (DAKO), and then observed. As a result, strong CAPRIN-1 expression was observed in 138 (73%) of the total 188 breast cancer tissue samples. Furthermore, similar results were obtained when monoclonal antibody #9 prepared in Example 4 was used. (7) -4 Expression of CAPRIN-1 in malignant human brain tumor.

[0142] Using 247 malignant brain tumor specimens from paraffin-embedded malignant brain tumor tissue matrix (BIOMAX), immunohistochemical staining was performed by a method similar to that of (7)-3 above, using monoclonal antibody #6 prepared in Example 4. As a result, strong CAPRIN-1 expression was observed in 227 (92%) of the total 247 malignant brain tumor tissue samples. Furthermore, similar results were obtained when monoclonal antibody #9 prepared in Example 4 was used. (7) -5 Expression of CAPRIN-1 in metastatic lymph node of human breast cancer.

[0143] Using 150 samples of metastatic lymph node tissue from human breast cancer from paraffin-embedded metastatic breast cancer lymph node tissue matrices (BIOMAX), immunohistochemical staining was performed by a method similar to that of (7)-3 above, using monoclonal antibody #6 prepared in Example 4. As a result, strong CAPRIN-1 expression was observed in 136 (90%) of the total 150 samples of metastatic lymph node tissue from human breast cancer. Specifically, it was revealed that CAPRIN-1 is also strongly expressed in breast cancer metastasis tissue. Furthermore, similar results were obtained when monoclonal antibody #9 prepared in Example 4 was used. Petition 870260065851, dated 03 / 07 / 2026, page 76 / 105 66 / 83 Example 2: Anti-tumor effects (ADCC activity) of antibody against CAPRIN-1 in cancer cells

[0144] Next, it was analyzed whether an antibody against CAPRIN-1 would be able to damage tumor cells expressing CAPRIN-1. The evaluation was performed using the polyclonal antibody against a peptide derived from human CAPRIN-1 prepared in Example 1. Two types of human breast cancer cell lines (T47D and MDA-MB-157) (106 cells each), in which CAPRIN-1 expression was confirmed, were collected separately in a 50 ml centrifuge tube. Chromium 51 (100 μCi) was added, followed by incubation at 37 °C for 2 hours. Subsequently, the cells were washed three times with RPMI1640 medium containing 10% fetal bovine serum and added to the well (103 cells per well) in a 96-well V-bottom plate. The above polyclonal antibodies against a human-derived peptide CAPRIN-1 were added (1 μg per well).Furthermore, lymphocytes separated from rabbit peripheral blood were added to the same (2x105 cells per well), followed by culture under conditions of 37°C and 5% CO2 for 4 hours. After culture, the level of chromium (Cr) 51 released by damaged tumor cells was determined in each culture supernatant. Then, the ADCC activity of the polyclonal antibody against a human peptide derived from CAPRIN-1 for cancer cells was calculated. As a result, ADCC activities against T47D (15.4%) and MDA-MB-157 (17.3%) were confirmed (See Figures 2 and 3). Meanwhile, substantially no activity was observed in a case where a similar process to the previous one was performed with the control antibody prepared from peripheral blood of a rabbit that had not been immunized with an antigen (Example 1(5)) or in a case where the antibody was not added (see Figures 2 and 3).Thus, it was revealed that tumor cells expressing CAPRIN-1 can be damaged by induction of ADCC activity with its use. Petition 870260065851, dated 03 / 07 / 2026, page 77 / 105 67 / 83 of an antibody against CAPRIN-1.

[0145] Furthermore, for cytotoxic activity, an antibody against CAPRIN-1 used in the present invention, mouse lymphocytes and 103 cells incorporating chromium 51 from a leukemia cell line were mixed and cultured for 4 hours. After that, the level of chromium 51 released into the medium was determined. Then, the cytotoxic activity of the leukemia cell line was calculated by the following equation*. * Equation: Cytotoxic activity (%) = [(level of chromium 51 released from T47D or MDA-MB-157 cells to which an antibody against CAPRIN-1 and mouse lymphocytes were added) / (level of chromium 51 released by target cells to which hydrochloric acid was added)] x 100. Example 3: Preparation of new human cancer antigen proteins (1) Recombinant protein preparation.

[0146] A recombinant protein from a human homologous gene was engineered by the following method based on the gene from SEQ ID NO: 1 obtained in Example 1. PCR was performed by repeating 30 cycles at 98 °C / 10 seconds and 68 °C / 2.5 minutes using a Thermocycler (BIO RAD) and a reaction solution adjusted to a total quantity of 50 μl by adding each reagent and a bound buffer (1 μl of cDNA (which was from a tissue / cell-derived variety of cDNA prepared in Example 1 and observed for its RT-PCR expression), 2 types of primers (0.4 μM each; SEQ ID NO: 38 and 39), containing restriction enzyme cleavage sequences Sac I and Xho I, 0.2 mM dNTP, 1.25 U polymerase Primestar HS (Takara Shuzo)). The two types of primers mentioned above were used to extend the region encoding the complete amino acid sequence of SEQ ID NO: 2.After PCR, the amplified DNA was subjected to 1% agarose gel electrophoresis, and then a DNA fragment of approximately 2.1 kbp was purified using the QIAquick Gel Extraction Kit (QIAGEN). Petition 870260065851, dated 03 / 07 / 2026, pp. 78 / 105 68 / 83

[0147] The purified DNA fragment was ligated into a PCR-Blunt cloning vector (Invitrogen), the vector was transformed into Escherichia coli, and then the plasmid was collected. It was confirmed based on the sequence that the amplified gene fragment matched the target sequence. The plasmid that matched the sequence of interest was treated with Sac I and Xho I restriction enzymes, and then the result was purified using the QIAquick Gel Extraction Kit. The gene sequence of interest was then inserted into a pET30a expression vector (Novagen) by Escherichia coli treated with Sac I and Xho I restriction enzymes. A recombinant fused His protein could be produced using the vector. The plasmid was transformed into Escherichia coli BL21 (DE3) for expression, and then expression induction was performed using 1 mM IPTG, so that the target protein was expressed in Escherichia coli. (2) Purification of recombinant protein.

[0148] Each recombinant Escherichia coli obtained above expressing SEQ ID NO: 1 was cultured at 37 °C in LB medium containing 30 μg / ml kanamycin until the absorbance at 600 nm reached approximately 0.7. Then isopropyl-eD-1 thiogalactopyranoside was added at a final concentration of 1 mM, followed by 4 hours of culture at 37 °C. Subsequently, the cells were collected by 10 minutes of centrifugation at 4800 rpm. The cell pellet was suspended in a phosphate-buffered saline solution and centrifuged at 4800 rpm for 10 minutes to wash the cells.

[0149] The cells were suspended in phosphate buffer and then subjected to ultrasound on ice. The sonicated lysate of Escherichia coli was centrifuged at 6000 rpm for 20 minutes. The resulting supernatant was used as a soluble fraction and the resulting precipitate was used as an insoluble fraction.

[0150] The soluble fraction was added to a chelate column of Petition 870260065851, dated 03 / 07 / 2026, p. 79 / 105 69 / 83 nickel (stationary: Chelating Sepharose (registered trademark) Fast Flow (GE Healthcare), column capacity: 5 ml, 50 mM hydrochloric acid buffer (pH 8.0) as balanced buffer)) prepared according to the conventional method. The unadsorbed fraction was washed with 50 mM hydrochloric acid buffer (pH 8.0) in an amount 10 times the column capacity and 20 mM phosphate buffer (pH 8.0) containing 20 mM imidazole. Immediately after washing, 6 beds were eluted with 20 mM phosphate buffer (pH 8.0) containing 100 mM imidazole. A 20 mM elution fraction of phosphate buffer (pH 8.0) containing 100 mM imidazole (for which elution of the protein of interest was confirmed by Coomassie staining) was added to a strong anion exchange column (carrier: Q Sepharose (registered trademark) Fast Flow (GE Healthcare), column capacity: 5 ml, and 20 mM phosphate buffer (pH 8.0) as balanced buffer).

[0151] The unabsorbed fraction was washed with 20 mM phosphate buffer (pH 7.0) in an amount 10 times the column capacity and 20 mM phosphate buffer (pH 7.0) containing 200 mM sodium chloride. Immediately after washing, 5 beds were eluted using 20 mM phosphate buffer (pH 7.0) containing 400 mM sodium chloride. Thus, purified protein fractions were obtained, each having the amino acid sequence shown in SEQ ID NO: 2.

[0152] 200 μl of each purified preparation obtained by the above method was distributed in 1 ml of reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2 pH 7.4) and then 2 μl of enteropeptidase (Novagen) was added. The preparation was left to stand at room temperature overnight for reaction, the His tag was segmented, and then purification was performed according to the attached protocols using an Enteropeptidase Capture and Segmentation Kit (Novagen). Then, 1.2 ml of each Petition 870260065851, dated 03 / 07 / 2026, pages 80 / 105 The purified preparation obtained by the above method was replaced with physiological phosphate buffer (Nissui Pharmaceutical Co., Ltd.) using NANOSEP 10K OMEGA ultrafiltration (PALL). Sterile filtration was performed using 0.22-μm HT Tuffryn Acrodisc (PALL), and the results were used for the following experiments. Example 4: Preparation of a monoclonal antibody against CAPRIN-1

[0153] The antigen protein (human CAPRIN-1) (100 μg) shown in SEQ ID NO: 2 prepared in Example 3 was mixed with an MPL+TDM adjuvant (Sigma) in an amount equivalent to that of the antigen protein. The mixture was used with an antigen solution according to the mouse. The antigen solution was administered intraperitoneally to 6-week-old Balb / c mice (Japan SLC Inc.) and then subsequently administered 3 times or 24 times weekly for complete immunization. The spleen was removed on day 3 after final immunization and then crushed between two sterile glass slides. Each result was washed with PBS (-) (Nissui) and then centrifuged at 1500 RPM for 10 minutes; this procedure for supernatant removal was repeated 3 times. Thus, spleen cells were obtained. The spleen cells thus obtained were mixed with SP2 / 0 mouse myeloma cells (purchased from ATCC) at a ratio of 10:1.The PEG solution was prepared by mixing 200 μl of RPMI1640 medium containing 10% FBS heated to 37°C and adding 800 μl of PEG1500 (Boehringer) to the cells. The solution was allowed to stand for 5 minutes for cell fusion. Centrifugation was performed at 1700 RPM for 5 minutes to remove supernatants. The cells were suspended in 150 ml of RPMI1640 medium (HAT selective medium) containing 15% FBS, to which the equivalent of 2% HAT solution (Gibco) was added, and then seeded in fifteen 96-well plates (Nunc) at 100 μl per well. The cells were cultured for 7 days under 37°C conditions. Petition 870260065851, dated 03 / 07 / 2026, page 81 / 105 71 / 83 and 5% CO2, so that hybridomas resulting from the fusion of spleen cells with myeloma cells were obtained.

[0154] Hybridomas were selected using as an indicator the binding affinity of the antibody produced by the thus prepared hybridomas for the CAPRIN-1 protein. The CAPRIN-1 protein solution (1 μg / ml) prepared in Example 3 was added at 100 μl per well of 96-well plates and then left to stand at 4°C for 18 hours. Each well was washed 3 times with PBS-T, 0.5% Bovine Serum Albumin (BSA) solution (Sigma) was added at 400 μl per well, and then the plates were left to stand at room temperature for 3 hours. The solution was removed, and then each well was washed 3 times with 400 μl of PBS-T. Each supernatant from the hybridoma cultures obtained above was added at 100 μl per well and then left to stand at room temperature for 2 hours.Each well was washed 3 times with PBS-T, a 5000-fold diluted HRP-sorted anti-mouse IgG (H+L) antibody with PBS was added at 100 μl per well and then left to stand at room temperature for 1 hour. Each well was washed 3 times with PBS-T, a TMB substrate solution (Thermo) was added at 100 μl per well and left to stand for 15 to 30 minutes until a color reaction occurred. After color development, 1N sulfuric acid was added at 100 μl per well to stop the reaction. Absorbance at 450 nm and absorbance at 595 nm were measured using an absorption spectrometer. As a result, a plurality of hybridomas producing antibodies with high absorbance were selected.

[0155] The hybridomas thus selected were added to 0.5 hybridomas per well of 96-well plates and then cultured. After 1 week, the hybridomas were observed forming a single colony in the wells. The cells in these wells were further cultured. The hybridomas were Petition 870260065851, dated 03 / 07 / 2026, page 82 / 105 72 / 83 were selected using a binding affinity indicator (of the antibody produced by cloned hybridomas) for the CAPRIN-1n protein. The CAPRIN-1 protein solution (1 μg / ml) prepared in Example 3 was added at 100 μl per well in 96-well plates and incubated at 4°C for 18 hours. Each well was washed 3 times with PBS-T, a 0.5% BSA solution was added at 400 μl per well, and then incubated at room temperature for 3 hours. The solution was removed, and then each well was washed 3 times with 400 μl of PBS-T. Each hybridoma culture supernatant obtained above was added at 100 μl per well and incubated at room temperature for 2 hours. Each well was washed 3 times with PBS-T, an HRP-labeled anti-IgG (H+L) mouse antibody (Invitrogen) diluted 5000 times with PBS was added at 100 μl per well and then left to stand at room temperature for 1 hour.Each well was washed 3 times with PBS-T, a TMB substrate solution (Thermo) was added at 100 μl per well and left to stand for 15 to 30 minutes until a color reaction occurred. After color development, 1N sulfuric acid was added at 100 μl per well to stop the reaction. Absorbance at 450 nm and absorbance at 595 nm were measured using an absorption spectrometer. As a result, 150 hybridoma cell lines producing monoclonal antibodies exhibiting reactivity with the CAPRIN-1 protein were obtained.

[0156] Next, among these monoclonal antibodies, monoclonal antibodies exhibiting reactivity with the surfaces of breast cancer cells expressing CAPRIN-1 were selected. Specifically, 106 cells of the human breast cancer cell line MDA-MB-231V were centrifuged using a 1.5-ml microcentrifuge tube. The supernatant (100 μl) from each hybridoma was added and left to stand on ice for 1 hour. After washing with PBS, an anti-CAPRIN IgG antibody was added. Petition 870260065851, dated 03 / 07 / 2026, page 83 / 105 73 / 83 goat mice labeled with FITC (Invitrogen) diluted 500-fold with PBS containing 0.1% FBS were added and left to stand on ice for 1 hour. After washing with PBS, fluorescence intensity was measured using FACSCalibur (Becton, Dickinson and Company). Meanwhile, a similar procedure was performed using untreated serum from 6-week-old Balb / c mice diluted 500-fold with a hybridoma culture medium instead of the antibody until a control was prepared. As a result, 11 monoclonal antibodies (#1 to #11) having stronger fluorescence intensity than the control, i.e., reacting with the surfaces of breast cancer cells, were selected. Example 5: Characterization of selected antibodies (1) Cloning of a variable region gene of anti-CAPRIN-1 monoclonal antibody mRNAs was extracted from hybridoma cell lines producing the 11 monoclonal antibodies selected in Example 4. The variable region heavy chain (VH) gene and the variable region light chain (VL) gene for each anti-CAPRIN-1 monoclonal antibody were obtained by RT-PCR using specific primers for a mouse FR1-derived sequence and a mouse FR4-derived sequence. For sequencing, the genes were separately cloned into pCR2.1 vectors (Invitrogen). (1)-1 RT-PCR.

[0157] mRNA was prepared from each hybridoma cell line (106 cells) using an mRNA micropurification kit (GE Healthcare). Each mRNA obtained was subjected to reverse transcription using a SuperScriptII first-strand synthesis kit (Invitrogen) for cDNA synthesis. The above procedures were performed according to the protocols attached to the kits. Petition 870260065851, dated 03 / 07 / 2026, page 84 / 105 74 / 83

[0158] Each cDNA obtained was used for antibody gene amplification by PCR.

[0159] In order to obtain the VH region gene, a primer specific for a mouse FR1 heavy chain sequence (SEQ ID NO: 130) and a primer specific for a mouse FR4 heavy chain sequence (SEQ ID NO: 131) were used. Additionally, in order to obtain the VL region gene, a primer specific for a mouse FR1 light chain sequence (SEQ ID NO: 132) and a mouse FR4 light chain sequence (SEQ ID NO: 133) were used. These primers were determined with reference to Jones, ST and Bending, MM Bio / Technology 9, 88-89 (1991). For PCR, a thermostable primer (Takara Bio Inc.) was used. Each cDNA sample was mixed with a 10x thermostable buffer (5 μl), dNTP mixture (2.5 mM) (4 μl), primers (1.0 pM) (2 pl each), and thermostable (5 U / μl) (0.25 μl). The total volume was adjusted to 50 pl with sterile water.PCR was performed under conditions comprising, after treatment at 94°C for 2 minutes, 30 cycles of a combination of denaturation at 94°C for 1 minute, tempering at 58°C for 30 seconds, and a prolongation reaction at 72°C for 1 minute. (1)-2 Cloning.

[0160] Each previously obtained PCR product was subjected to agarose gel electrophoresis, followed by DNA band excision from the VH and VL regions. The DNA was purified using a QIAquick Gel Purification Kit (QIAGEN) according to the protocols included with the kit. Each purified DNA was cloned into a pCR2.1 vector using a TA cloning kit (Invitrogen). Each DBA-linked vector was transformed into competent DH5a cells (TOYOBO) according to a conventional method. Each transformant (10 clones) was cultured overnight in a medium (100 pg / ml ampicillin) at 37°C. The resulting plasmid DNA was Petition 870260065851, dated 03 / 07 / 2026, pages 85 / 105 75 / 83 purified using a Qiaspin Miniprep kit (QIAGEN). (1)-3 Sequencing.

[0161] Gene sequence analysis of the VH and VL regions in each previously obtained plasmid was performed using a forward primer M13 (SEQ ID NO: 134) and a reverse primer M13 (SEQ ID NO: 135) with a fluorescent sequencer (ABI; DNA sequencer 3130XL) and a BigDye cycle sequencing kit (ABI) Version 3.1 terminator according to the protocols attached to the kit. As a result, each gene sequence (identical in 10 clones) was determined.

[0162] The amino acid sequences obtained from the variable regions of the monoclonal antibody heavy chain 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 obtained from the variable regions of the light chain 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.

[0163] Specifically, a monoclonal antibody #1 comprises the variable heavy chain region of SEQ ID NO: 47. A monoclonal antibody #2 comprises the variable heavy chain region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 53. A monoclonal antibody #3 comprises the variable heavy chain region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 58. A monoclonal antibody #4 comprises the variable heavy chain region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 63. A monoclonal antibody #5 comprises the variable heavy chain region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 68. A monoclonal antibody #6 comprises the variable heavy chain region of SEQ ID NO: Petition 870260065851, dated 03 / 07 / 2026, page 86 / 105 Monoclonal antibody #7 comprises the variable heavy chain region of SEQ ID NO: 83 and the variable light chain region of SEQ ID NO: 87. Monoclonal antibody #8 comprises the variable heavy chain region of SEQ ID NO: 93 and the variable light chain region of SEQ ID NO: 97. Monoclonal antibody #9 comprises the variable heavy chain region of SEQ ID NO: 103 and the variable light chain region of SEQ ID NO: 107. Monoclonal antibody #10 comprises the variable heavy chain region of SEQ ID NO: 113 and the variable light chain region of SEQ ID NO: 117. Monoclonal antibody #11 comprises the variable heavy chain region of SEQ ID NO: 123 and the variable light chain region of SEQ ID NO: 127. (2) Expressions of CAPRIN-1 on the surface of different cells caused with the use of the obtained monoclonal antibodies.

[0164] Next, it was examined whether or not the CAPRIN-1 protein was expressed on the cell surfaces of 7 types of breast cancer cell lines (MDA-MB-157, T47D, MRK-nu-1, MDA-MB-231V, BT20, SKBR-3, and DA-MB-231T) in which the expression of the CAPRIN-1 gene was confirmed, 3 types of other breast cancer cell lines (MDAMB-231C, MCF-7, and ZR75-1), 6 types of glioma cell lines (T98G, SNB19, U251, and U87G), 3 types of kidney cancer cell lines (Caki-1, Caki2, and A498), 1 type of stomach cancer cell line (MKN45), 1 type of colon cancer cell line (Caco2), 3 types of cell lines Lung cancer cell lines (A549, QG56 and PC8), and 3 types of leukemia cell lines (Namalwa, BDCM and RPI1788). Each cell line (106 cells) was centrifuged in a 1.5-ml microcentrifuge tube.Hybridoma supernatants (100 pl each) containing monoclonal antibodies #1 to #10 against CAPRIN-1, prepared in Example 4, and which react with the surface of cancer cells, were separately analyzed. Petition 870260065851, dated 03 / 07 / 2026, page 87 / 105 77 / 83 added, and then left to stand on ice for 1 hour. After washing with PBS, each resulting sample was suspended in a FITC-labeled goat anti-mouse IgG antibody (Invitrogen Corporation) diluted 500 times with PBS containing 0.1% FBS and then left to stand on ice for 1 hour. After washing with PBS, the fluorescence intensity was measured using FACSCalibur (Becton, Dickinson and Company). Meanwhile, a similar procedure to the previous one was performed using, as a control, the control antibody prepared in (5) previously instead of the hybridoma supernatants containing monoclonal antibodies #1 to #11 against CAPRIN-1, until the control was prepared. As a result, the fluorescence intensity found was at least 30% stronger in all cells to which monoclonal antibodies #1 to #11 were added than in the control cells.Specifically, the following increases in fluorescence intensity were confirmed when, for example, monoclonal antibody #9 was used: MDA-MB-157: 211%; T47D: 145%; MRK-nu-1: 123%; MDA-MB-231V: 251%; BT20: 168%; and MDA-MB-231T: 94%. Based on the above, it was confirmed that the CAPRIN-1 protein was expressed on the cell surfaces of the previous human cancer cell lines. Additionally, the rate of increase in fluorescence intensity is represented in medium fluorescence intensity (MFI value) in cells. This was calculated using the following equation.

[0165] Rate of increase in fluorescence intensity of medium (rate of increase in fluorescence intensity) (%) = ((MFI value of cells that reacted with an anti-human CAPRIN-1 antibody) - (control MFI value)) / (control MFI value) x 100 (3) Antitumor effects (ADCC activity) of antibodies against CAPRIN-1 on cancer cells.

[0166] The previously selected monoclonal antibodies #1 Petition 870260065851, dated 03 / 07 / 2026, pages 88 / 105 Monoclonal antibodies #1 to #11 against CAPRIN-1 were evaluated in terms of cytotoxic activity (ADCC activity) against cancer cells. Hybridomas producing monoclonal antibodies #1 to #11 were cultured using SFM hybridoma medium (Invitrogen). Each supernatant obtained was purified using Hitrap Protein A Sepharose FF (GE Healthcare), followed by PBS (-) replacement and purification with a 0.22-μm filter (Millipore). Each resulting supernatant was used as an antibody for activity determination. The human breast cancer cell line MDA-MB-157 (106 cells) was collected in a 50-ml centrifuge tube. Chromium 51 (100 μCi) was added to this, followed by incubation at 37°C for 2 hours. From there, the cells were washed 3 times with an RPMI1640 medium containing 10% FBS. The cells were added to the wells (103 cells per well) in 96-well V-bottom plates. Thus, target cells were prepared.The previously purified antibodies were added to these (1 μg per well). Next, mouse lymphocytes separated from mouse spleen (2 χ¹⁰⁵ cells) were added to this, followed by culture under conditions of 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. Then, the ADCC activity of each polyclonal antibody against a peptide derived by human CAPRIN-1 for cancer cells was calculated. As a result, all monoclinal antibodies #1 to #11 exhibited ADCC activity against MDA-MB-157 (20% or more).

[0167] Specifically, for example, the following cytotoxic activity results were obtained: No. 1: 22.1%; No. 2: 29.1%; No. 6: 30.2%; and No. 9: 32.4% (see Fig. 4). Meanwhile, no cytotoxic activity was confirmed in a case where a procedure similar to the above was performed using the monoclonal antibody reactive to a CAPRIN-1 protein itself, but not to cancerous cell surfaces prepared in Example 4 (see Fig. 4). The Petition 870260065851, dated 03 / 07 / 2026, pages 89 / 105 79 / 83 results above showed that the anti-CAPRIN-1 obtained monoclonal antibodies (No. 1 to No. 11) damaged CAPRIN-1-expressing cancer cells exhibiting ADCC activity (4) Antitumor effects (CDC activity) of antibodies against CAPRIN-1 on cancer cells.

[0168] Selected monoclonal antibodies No. 1 to No. 11 against CAPRIN-1 were evaluated in terms of cytotoxic activity (CDC activity) against cancer cells. Blood collected from rabbits by blood sampling was added to an Eppendorf tube and then left at room temperature for 60 minutes, followed by centrifugation at 3000 rpm for 5 minutes. Thus, serum for CDC activity determination was prepared. Human breast cancer MDA-MB-231V cell line (105 cells) was collected in a 50-ml centrifuge tube. Chromium 51 (100 μCi) was added to this, followed by incubation at 37°C for 2 hours. Then, cells were washed 3 times with an RPMI medium containing 10% FBS and then suspended in an RPMI containing 50% rabbit serum prepared above. The cells were added to the wells (103 cells per well) in 96-well V-bottom plates.Antibodies No. 1 to No. 11 obtained in (3) above were separately added to the wells (1 pg per well), followed by culture under conditions of 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 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 No. 1 to No. 11 exhibited CDC activity (30% or more). Meanwhile, cytotoxic activity was not confirmed in a case where a procedure similar to the above was performed using the monoclonal antibody reactive to the CAPRIN1 protein itself, but not to cancerous cell surfaces prepared in. Petition 870260065851, dated 03 / 07 / 2026, pages 90 / 105 80 / 83 Example 4 (see Fig. 4). Consequently, it was revealed that monoclonal antibodies against CAPRIN-1 (No. 1 to No. 11) can damage tumor cells that express CAPRIN-1, also exhibiting CDC activity. Example 6: In vivo antitumor effects of monoclonal antibodies-1 in MICE.

[0169] Next, in vivo antitumor effects of monoclonal antibodies No. 1 to No. 11 against CAPRIN-1 in tumor-bearing mice were evaluated. Antibodies used in this Example were obtained after subjecting the supernatant of each hybridoma to column purification as described above.

[0170] Antitumor effects of monoclonal antibodies No. 1 to No. 11 against CAPRIN-1 were examined using tumor-bearing mice in which a mouse-derived cancer cell line expressing CAPRIN-1 was transplanted. CT26 cells (acquired from ATCC) were transplanted subcutaneously into the dorsal parts of 70 Balb / c mice (Japan SLC, Inc.) (106 cells per mouse). Each tumor was allowed to grow to approximately 7 mm in diameter. Tumor-bearing mice (60 out of 70) were subjected to peritoneal administration of monoclonal antibodies No. 1 to No. 11 against CAPRIN-1 and a type of monoclonal antibody (reactive to the CAPRIN-1 protein itself, but not to cancer cell surfaces) prepared in Example 4 (5 mice per antibody) at a dose of 300 μg (300 μl) per mouse.Next, each antibody was administered intraperitoneally at the same dose to mice bearing relevant tumors 3 times in total over 2 days. Tumor size was measured daily to observe antitumor effects. The remaining 10 tumor-bearing mice were given PBS (-) instead of an antibody. This group of mice was designated as a control group. As a result of... Petition 870260065851, dated 03 / 07 / 2026, pp. 91 / 105 81 / 83 observation of antitumor effects, in the case of the test group to which monoclonal antibodies No. 1 to No. 11 against CAPRIN-1 were administered, tumor regression occurred to such an extent that the tumor volume at the beginning of antibody administration (100%) decreased to 50% on Day 4, approximately 10% on Day 6, and to many percent on Day 8. Substantially complete tumor regression occurred from Day 11 to 14 (see Fig. 5). On the other hand, in the control group, the tumor volume increased to approximately 260%, 350%, 550%, and 800% of the original volume on Days 4, 6, 8, and 11, respectively (see Fig. 5). Furthermore, in the group of mice in which a monoclonal antibody (reactive to the CAPRIN-1 protein itself, but not to the surfaces of cancerous cells) was administered, no antitumor effects could be observed and tumor growth occurred as in the control group.The results indicate that monoclonal antibodies No. 1 to No. 11 obtained against CAPRIN-1 exhibited a strong antitumor effect in vivo on cancer cells expressing CAPRIN-1. Furthermore, tumor size was obtained by calculating tumor volume using the following formula: major diameter x minor diameter x minor diameter x 0.5.

[0171] In addition, monoclonal antibodies No. 1 to No. 11 against CAPRIN-1 were administered as described above to tumor-bearing mice (Balb / c) into which mouse N1E cancer cells (acquired from ATCC) were transplanted. This resulted in complete tumor regression on Day 15 after antibody administration. On the other hand, in the control group, the tumor volume grew to approximately 950% of the original volume (see Fig. 6). Example 7: The identification of a peptide in the CAPRIN-1 protein, to which an ANTIBODY AGAINST CAPRIN-1 REACTIVE ON THE SURFACE OF CANCER CELLS LEAGUE

[0172] Using monoclonal antibodies No. 1 to No. 11 against CAPRIN-1, reacting with the surfaces of cancerous cells (obtained Petition 870260065851, dated 03 / 07 / 2026, pages 92 / 105 82 / 83 above), partial sequences in the CAPRIN-1 protein were identified as being recognized by these monoclonal antibodies.

[0173] First, DTT (Fluka) was added to 100 μl of a solution prepared by dissolving recombinant CAPRIN-1 protein at a concentration of 1 μg / μl with PBS to a final concentration of 10 mM, followed by a 5-minute reaction at 95°C, thus reducing disulfide bridges within the CAPRIN-1 protein. Next, iodoacetamide (Wako Pure Chemical Industries, Ltd.) at a final concentration of 20 mM was added, and then an alkylation reaction was carried out to thiol groups at 37°C for 30 minutes under shading conditions. Fifty (50) μg of each monoclonal antibody No. 1 to No. 11 against CAPRIN-1 were added to 40 μg of the thus obtained reduced-alkylated CAPRIN-1 protein, the volume of the mixture was adjusted to 1 mL of 20 mM phosphate buffer (pH 7.0), and then the mixture was left to react overnight at 4°C while stirring each mixture.

[0174] Next, trypsin (Promega) was added to a final concentration of 0.2 μO. After 1 hour, 2 hours, 4 hours, and then 12 hours of reaction at 37°C, the resulting products were mixed with protein A (GE) glass microspheres, which had been previously blocked with PBS containing 1% BSA (Sigma) and then washed with PBS 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), followed by 30 minutes of reaction.

[0175] The reaction mixtures were each washed with 25 mM ammonium carbonate buffer (pH 8.0) and then antibody-antigen complexes were eluted using 100 μl of 0.1% formic acid. LC-MS analysis was conducted on the eluates using the Q-TOF Premier (Waters-MicroMass) according to the protocols attached to the instrument.

[0176] As a result, the polypeptide with SEQ ID NO: 136 was Petition 870260065851, dated 03 / 07 / 2026, pages 93 / 105 83 / 83 was identified as a partial sequence of CAPRIN-1, which was recognized by all monoclonal antibodies No. 1 to No. 11 against CAPRIN-1. Furthermore, the peptide of SEQ ID NO: 137 was identified as a partial sequence in the polypeptide of SEQ ID NO: 136 above, which peptide was recognized by monoclonal antibodies No. 2 to No. 5, No. 6 to No. 8, and No. 10. It was additionally revealed that monoclonal antibodies No. 2 to No. 5 recognized the peptide of SEQ ID NO: 138, which was a partial sequence peptide of the peptide of SEQ ID NO: 137. Industrial Applicability

[0177] The antibodies of the present invention are useful for the treatment and / or prevention of cancers.

[0178] This description includes all or part of the content as revealed in the description and / or drawings of Japanese Patent Application No. 2009-087285, to which the present application claims priority. Furthermore, all publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. PHARMACEUTICAL COMPOSITION FOR TREATMENT AND / OR PREVENTION OF A CANCER EXPRESSING CAPRIN-1, characterized in that it comprises, as an active ingredient, a monoclonal antibody that has immunological reactivity with and specifically binds to a polypeptide having the amino acid sequence shown in SEQ ID NO: 37 or SEQ ID NO: 136, and the monoclonal antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) activity against cancer cells expressing CAPRIN-1, wherein the monoclonal antibody comprises: (i) a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41 and 42, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 44, 45 and 46;(ii) a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41 and 42, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 50, 51 and 52; (iii) a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41 and 42, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 55, 56 and 57; (iv) a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 40, 41 and 42, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 60, 61 and 62; (v) a variable heavy chain region comprising the sequences shown in SEQ ID Nos: 40, 41 and 42, and a variable light chain region comprising the sequences shown in SEQ ID Nos: 65, 66 and 67;(vi) a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 70, 71 and 72, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 74, 75 and 76; (vii) a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 80, 81 and 82, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 84, 85 and 86; (viii) a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 90, 91 and 92, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 94, 95 and 96; (ix) a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 100, 101 and 102, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 104, 105 and 106;(x) a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 110, 111 and 112, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 114, 115 and 116; or (xi) a variable heavy chain region comprising the sequences shown in SEQ ID NOs: 120, 121 and 122, and a variable light chain region comprising the sequences shown in SEQ ID NOs: 124, 125 and 126, and a pharmaceutically acceptable excipient or medium. Petition 870260065851, dated 03 / 07 / 2026, p. 96 / 105 3 / 3; 2. PHARMACEUTICAL COMPOSITION, according to claim 1, characterized in that the cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, esophageal cancer or colon cancer.

Images