Antibody, pharmaceutical composition, pharmaceutical combination, use of an antibody, use of a pharmaceutical composition and use of a pharmaceutical combination

Abstract

Pharmaceutical compositions, antibodies, pharmaceutical combinations, methods for the treatment and / or prevention of cancer, uses of a pharmaceutical composition, use of an antibody, and use of a pharmaceutical combination according to the present invention, a cancer antigen protein has been identified that is specifically expressed on the surfaces of cancer cells and, therefore, the use of an antibody directed against the cancer antigen protein is provided as an agent for the treatment and / or prevention of cancer.Specifically, the present invention provides a pharmaceutical composition for the treatment and / or prevention of cancer, comprising an antibody or fragment thereof as an active ingredient possessing immunological reactivity with a partial polypeptide of the caprin-1 protein, wherein caprin-1 is represented by any of the numbered even sequences from seq id nos: 2 to 30, and wherein the polypeptide comprises the amino acid sequence represented by seq id no: 37 or an amino acid sequence possessing 80% or more sequence identity with the amino acid sequence.

Description

1 / 82 “ANTIBODY, PHARMACEUTICAL COMPOSITION, PHARMACEUTICAL COMBINATION, USE OF AN ANTIBODY, USE OF A PHARMACEUTICAL COMPOSITION AND USE OF A PHARMACEUTICAL COMBINATION” Field of the Invention

[001] The present invention relates to pesticide mixtures comprising chlorfenapyr and teflubenzuron in synergistically effective amounts.

[002] The present invention relates to a novel pharmaceutical use of an antibody against CAPRIN-1 or a fragment thereof, as an agent for the treatment and / or prevention of cancer. Background of the Invention

[003] Cancer is the leading cause of death. Current therapy mainly comprises surgical therapy in combination with radiotherapy and chemotherapy. Despite the development of new surgical procedures and the discovery of new anticancer agents in recent years, with the exception of some types of cancer, treatment outcomes have not improved. Recent advances in the molecular biology or immunology of cancer have led to the identification of antibodies that react specifically with cancer, cancer antigens that are recognized by cytotoxic T cells, genes that encode cancer antigens, and the like. The demand for specific therapies targeting cancer antigens is increasing (non-patent literature 1).

[004] In cancer therapy, it is desirable that peptides, polypeptides, or proteins recognized as antigens be nearly absent in normal cells but specifically present in cancer cells in order to alleviate side effects. In 1991, Boon et al. (Ludwig Institute for Cancer Research in Belgium) isolated a Petition 870230039907, dated 12 / 05 / 2023, page 10 / 100 2 / 82 Human melanoma antigen MAGE1 recognized by CD8-positive T cells by a cDNA expression cloning method using an autologous cancer cell line and cancer-reactive T cells (Non-patent Literature 2). Subsequently, the SEREX method (serological identification of antigens by recombinant expression cloning) was described, which comprised the identification of tumor antigens recognized by antibodies that are produced in vivo in response to autologous cancer from the patient's own cancer by a gene expression cloning technique (Non-patent Literature 3 and Patent Literature 1). Using this method, some cancer antigens, which are almost never expressed in normal cells but are specifically expressed in cancer cells, were isolated (Non-patent Literature 4-9).Furthermore, clinical trials have been conducted based on cell-based therapies targeting specific cancer antigens using immunocytes specifically reactive to cancer antigens, or with cancer-specific immunotherapies using vaccines or similar products containing cancer antigens.

[005] On the other hand, in recent years, several antibody drugs targeting antigenic proteins in cancer cells have emerged worldwide for the treatment of cancer. Antibody drugs exhibit some pharmacological effects as cancer-specific therapeutic agents and thus attract attention. However, most of the antigenic proteins used as targets for the antibody are also expressed in normal cells, so not only cancer cells but also normal cells expressing such antigens are damaged as a result of antibody administration. Side effects Petition 870230039907, dated 12 / 05 / 2023, page 11 / 100 3 / 82 resulting is a cause for concern. Therefore, it is expected that the identification of cancer antigens that are specifically expressed on the surface of a cancer cell and the use of antibodies targeting cancer antigens as pharmaceuticals will enable antibody drug treatment with lower side effects.

[006] Cytoplasmic proliferation-associated protein 1 (CAPRIN-1) is expressed when normal resting-phase cells are activated or undergo cell division, and it is an intracellular protein known to form intracellular stress granules with RNA within cells, thus being involved in mRNA transport and translation regulation. However, there are many other names representing CAPRIN-1, such as GPI-anchored membrane protein 1 or membrane component surface marker protein 1 (M11S1), as if such proteins were known to be cell membrane proteins. These names originated from a report that the CAPRIN-1 gene sequence is a membrane protein that has a GPI-binding region and is expressed in colorectal cancer cells (Non-patent literature 10). However, the CAPRIN-1 gene sequence provided in this report was later revealed to be incorrect.It was subsequently reported that the deletion of a single nucleotide in the sequence of the CAPRIN-1 gene registered in GenBank or a similar database causes a change in the frame, such that 80 amino acids are lost from the C-terminal end, resulting in the generation of an artifact (74 amino acids), which corresponds to the GPI-binding portion in the previous report, and, additionally, another error is also present at 5' of the gene sequence, such that 53 amino acids were lost from there. Petition 870230039907, dated 12 / 05 / 2023, page 12 / 100 4 / 82 of the N-terminal end (Non-patent literature 11). It has also recently been reported that the protein encoded by the CAPRIN-1 gene sequence registered in GenBank or a similar database is not a cell membrane protein (Non-patent literature 11).

[007] Furthermore, based on the non-patent literature report 10 stating that CAPRIN-1 is a cell membrane protein, patent literatures 2 and 3 describe that CAPRIN-1 (as a cell membrane protein), under the name M11S1, can be used as a target for an antibody drug in cancer therapy, although they do not describe practical examples of treatment using an antibody against the protein. However, as reported in non-patent literature 11, it is believed from the filing of patent literature 2 to the present date that CAPRIN-1 is not expressed on the surface of a cell. The content of patent literatures 2 and 3, based solely on the incorrect information that CAPRIN-1 is a cell membrane protein, should not be clearly understood as common general knowledge for those skilled in the art. Literature of the Previous Art Patent Literature Patent Literature 1: US Patent 5,698,396 Patent Literature 2: US2008 / 0075722 Patent literature 3: WO2005 / 100998. Non-patent literature Non-patent literature 1: Tsuyoshi Akiyoshi, “Gan To KagakuRyoho (Cancer and Chemotherapy),” 1997, Vol. 24, pp 551-519 (Cancer and Chemotherapy Publishers, Inc., Japan). Non-Patent Literature 2: Bruggen P. et al., Science, 254:1643-1647 (1991). Petition 870230039907, dated 12 / 05 / 2023, page 13 / 100 5 / 82 Non-Patent Literature 3: Proc. Natl. Acad. Sci. USA, 92:1181011813 (1995). Non-Patent Literature 4: Int. J. Cancer, 72:965-971 (1997). Non-Patent Literature 5: Cancer Res., 58:1034-1041 (1998). Non-Patent Literature 6: Int. J. Cancer, 29:652-658 (1998). Non-Patent Literature 7: Int. J. Oncol., 14:703-708 (1999). Non-Patent Literature 8: Cancer Res., 56:4766-4772 (1996). Non-Patent Literature 9: Hum. Mol. Genet 33-39 (1997). Non-Patent Literature 10: J. Biol Chem., 270:20717-20723 (1995). Non-Patent Literature 11: J. Immunol., 172:2389-2400, 2004. Brief Description of the Invention Problems to be Solved by the Invention

[008] The objects of the present invention are to identify a cancer antigen protein that is specifically expressed on the surface of a cancer cell and to provide the use of an antibody directed against the cancer antigen protein as an agent for the treatment and / or prevention of cancer. Ways to Solve the Problems

[009] As a result of intensive studies, the present inventors have now obtained a cDNA encoding a protein that binds to an antibody present in the serum of dogs with breast cancer by the SEREX method using two cDNA libraries prepared from canine testicular tissue and serum from dogs with breast cancer. The inventors have additionally prepared CAPRIN-1 proteins possessing the even-numbered amino acid sequences of SEQ ID NOS: 2 to 30 and antibodies against such CAPRIN-1 proteins based on the obtained canine gene and the corresponding homologous genes in humans, cattle, Petition 870230039907, dated 12 / 05 / 2023, page 14 / 100 6 / 82 horse, mouse, and chicken. Thus, the inventors of the present invention have now discovered that CAPRIN-1 is specifically expressed in breast cancer, brain tumor, leukemia, lymphoma, lung cancer, cervical cancer, bladder cancer, esophageal cancer, colorectal cancer, gastric cancer, renal cancer, ovarian cancer, prostate cancer, and fibrosarcoma, and that a portion of the CAPRIN-1 protein is specifically expressed on the surface of each cancer cell. The inventors have now verified that an antibody or antibodies against the portion of CAPRIN-1 expressed on the surface of each cancer cell is / are cytotoxic to cancer cells expressing CAPRIN-1. Based on these findings, the present invention as described below has been completed.

[0010] The present invention has the following characteristics.

[0011] The present invention provides a pharmaceutical composition for the treatment and / or prevention of cancer, comprising an antibody or a fragment thereof as an active ingredient possessing immunological reactivity with a partial polypeptide of CAPRIN-1, wherein CAPRIN-1 is represented by any of the even-numbered sequences of SEQ ID NOS: 2 to 30, and wherein the partial polypeptide comprises the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence possessing 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 37.

[0012] In one embodiment, the above cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, cervical cancer, bladder cancer, esophageal cancer, colorectal cancer, gastric cancer, kidney cancer, ovarian cancer, prostate cancer, or fibrosarcoma. Petition 870230039907, dated 12 / 05 / 2023, page 15 / 100 7 / 82

[0013] In another embodiment, the antibody is either a monoclonal antibody or a polyclonal antibody.

[0014] In another embodiment, the antibody is a human antibody, humanized antibody, chimeric antibody, single-chain antibody, or bispecific antibody.

[0015] This description includes all or part of the content as disclosed in the description and / or drawings of Japanese patent applications Nos. 2010-023454 and 2012-183162, from which the present application claims priority. Effects of the Invention

[0016] The antibody against CAPRIN-1 used in the present invention is cytotoxic to cancer cells. Therefore, the antibody against CAPRIN-1 is useful for the treatment or prevention of cancers. Brief Description of the Figures

[0017] Fig. 1 shows the expression patterns of CAPRIN-1 protein-coding genes in normal tissues and tumor cell lines. Reference No. 1 indicates the expression patterns of CAPRIN-1 protein-coding genes, and Reference No. 2 indicates the expression patterns of GAPDH genes.

[0018] Fig. 2 shows the cytotoxicity to the MDA-MB-157 breast cancer cell line expressing CAPRIN-1 by polyclonal anti-CAPRIN-1 antibodies that are reactive with cancer cell surfaces. Reference No. 3 indicates the activity exhibited when polyclonal anti-CAPRIN1 antibody #1 was added. Reference No. 4 indicates the activity exhibited when a control antibody from a non-antigen-immunized rabbit was added. Reference No. 5 indicates the activity exhibited when PBS was added instead of antibodies. Detailed Description of the Present Invention

[0019] The antitumor activity of an antibody against a polypeptide Petition 870230039907, dated 12 / 05 / 2023, p. 16 / 100 8 / 82 represented by any of the even numbering sequences of SEQ ID NOs: 2 to 30 used in the present invention can be evaluated by examining the suppression of tumor growth in vivo, in animals with cancer, or by evaluating whether the antibody exhibits cytotoxicity via immunocytes or complement to tumor cells expressing the polypeptide in vitro, as described further below.

[0020] In this context, protein-coding polynucleotide nucleotide sequences comprising even-numbered amino acid sequences (i.e., SEQ ID NOs: 2, 4, 6, ..., 28, 30) from SEQ ID NOs: 2 to 30 are represented by odd-numbered sequences (i.e., SEQ ID NOs: 1, 3, 5, ..., 27, 29) from SEQ ID NOs: 1 to 29.

[0021] The amino acid sequences represented by SEQ ID NOs: 6, 8, 10, 12, and 14 in the Sequence Listing disclosed in the present invention are the amino acid sequences of CAPRIN-1 isolated as polypeptides that specifically bind to antibodies present in the serum of a dog with cancer, using the SEREX method with a cDNA library from canine testicular tissue and the serum of a dog with breast cancer. The amino acid sequences represented by SEQ ID NOs: 2 and 4 are the CAPRIN-1 amino acid sequences isolated as human homologs. The amino acid sequence represented by SEQ ID NO: 16 is the CAPRIN-1 amino acid sequence isolated as a cattle homolog. The amino acid sequence represented by SEQ ID NO: 18 is the CAPRIN-1 amino acid sequence isolated as a horse homolog.The amino acid sequences represented by SEQ ID NOs: 20 to 28 are the amino acid sequences of CAPRIN-1 isolated as mouse homologs. The amino acid sequence represented by SEQ ID NO: 30 is the amino acid sequence of CAPRIN-1 isolated as a chicken homolog (see Example 1 described later). CAPRIN-1 is known to be expressed when normal cells in the resting phase are activated or initiate cell division. Petition 870230039907, dated 12 / 05 / 2023, p. 17 / 100 9 / 82

[0022] It was known that CAPRIN-1 was not expressed on cell surfaces. However, as a result of examination by the inventors of the present invention, it has now been revealed that a portion of the CAPRIN-1 protein is expressed on the surfaces of cells of various types of cancer. Thus, it has now been revealed that an antibody that recognizes a partial polypeptide of the CAPRIN-1 protein, comprising the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 37, exhibits antitumor activity. Examples of the antibody of the present invention include all antibodies that bind to a fragment of the CAPRIN-1 protein above and exhibit antitumor activity.

[0023] The anti-CAPRIN-1 antibody described above used in the present invention can be any type of antibody, provided it can exhibit antitumor activity. Examples of such antibodies include monoclonal antibodies, polyclonal antibodies, recombinant antibodies, such as synthetic antibodies, multispecific antibodies, humanized antibodies, chimeric antibodies, and single-chain antibodies (scFv), human antibodies, and fragments thereof, such as Fab, F(ab')2, and Fv. These antibodies and fragments thereof can be prepared by methods known to those skilled in the art. In the present invention, antibodies are desired that have immunological reactivity with CAPRIN-1 proteins or partial (poly)peptides thereof (i.e., binding to CAPRIN-1 proteins via antigen-antibody reaction) and, preferably, antibodies capable of specifically binding to CAPRIN-1 proteins. Preferably, they are monoclonal antibodies.Polyclonal antibodies can also be used, provided that homogeneous antibodies can be stably produced. Furthermore, when the subject is a human being... Petition 870230039907, dated 12 / 05 / 2023, page 18 / 100 10 / 82 human antibodies or humanized antibodies are desired in order to avoid or suppress rejection. The term “specifically binds to a CAPRIN1 protein,” as used herein, means that the antibody specifically binds to a CAPRIN-1 protein but does not substantially bind to other proteins besides the CAPRIN-1 protein.

[0024] The antitumor activity of an antibody that can be used in the present invention can be evaluated, as described below, by in vivo analysis of tumor growth suppression in cancer animals, or by evaluating whether or not said antibody exhibits in vitro cytotoxicity activity, which is mediated by immunocytes or complement, in tumor cells expressing the polypeptide.

[0025] In addition, examples of subjects for the treatment and / or prevention of cancer of the present invention include mammals, such as humans, pets, domestic animals and animals for competition. A preferred subject is a human.

[0026] The preparation of antigens, antibodies and pharmaceutical compositions related to the present invention are described below. Antigen Preparation for Antibody Preparation

[0027] The proteins or fragments thereof to be used as sensitization antigens for obtaining the antiCAPRIN-1 antibodies used in the present invention may be derived from any animal species, without particular limitation, such as humans, dogs, cattle, horses, mice, rats, and chickens. However, the proteins or fragments thereof are preferably selected taking into account compatibility with the parent cells used for cell fusion. In general, mammalian-derived proteins are preferred and, in particular, human-derived proteins are preferred. For example, when CAPRIN-1 is human CAPRIN-1, the following may be used; the CAPRIN-1 protein Petition 870230039907, dated 12 / 05 / 2023, page 19 / 100 11 / 82 human, a partial peptide thereof, or cells expressing human CAPRIN-1.

[0028] The nucleotide and amino acid sequences of human CAPRIN-1 and its homologs can be obtained by accessing GenBank (NCBI, USA) and using an algorithm such as BLAST or FASTA (Karlin and Altschul, Proc. Natl. Acad. Sci USA, 90:5873-5877 1993; Altschul et al, Nucleic Acids Res, 25:3389-3402, 1997).

[0029] In the present invention, based on the nucleotide sequence (SEQ ID NO: 1 or 3), or the amino acid sequence (SEQ ID NO: 2 or 4) of human CAPRIN-1, a target nucleic acid or target protein comprises a sequence with about 70% to 100%, preferably 80% to 100%, more preferably 90% to 100%, even more preferably 95% to 100% (e.g., 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 human CAPRIN-1. As used herein, the term “% sequence identity” refers to a percentage (%) of identical amino acids (or nucleotides) relative to the total number of amino acids (or nucleotides) when two sequences are aligned to achieve maximum similarity with or without the introduction of gaps.

[0030] The length of a fragment of the CAPRIN-1 protein varies from the amino acid length of an epitope (antigenic determinant), which is the smallest unit recognized by an antibody, to a length shorter than the total length of the protein. The term "epitope" refers to a polypeptide fragment possessing antigenicity or immunogenicity in mammals, preferably in humans, and the smallest unit of the epitope consists of about 7 to 12 amino acids, for example, 8 to 11 amino acids. Therefore, the antibody of the present invention is Petition 870230039907, dated 12 / 05 / 2023, page 20 / 100 12 / 82 characterized by the recognition of a fragment consisting of about 7 to 12 amino acids (e.g., 8 to 11 amino acids) in the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 37.

[0031] Polypeptides comprising the human CAPRIN-1 protein or the partial protein peptides mentioned above can be synthesized by a chemical synthesis method, such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method) (Edited by the Japanese Society for Biochemistry, Seikagaku Jikken Koza (Biochemical Experimental Lecture Series) 1, Protein Chemistry IV, Chemical Modification and Peptide Synthesis, TOKYO KAGAKU DOZIN (Japan), 1981). Alternatively, the aforementioned polypeptides can also be synthesized by conventional methods using various commercially available peptide synthesizers. Furthermore, using known genetic engineering techniques (e.g., Sambrook et al., Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology (1989), Cold Spring Harbor Laboratory Press, Ausubel et al.), Short Protocols in Molecular Biology, 3rd Edition, A compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons), a polynucleotide encoding the above polypeptide is prepared and then incorporated into an expression vector, which is subsequently introduced into a host cell in order to produce a polypeptide of interest in the host cell, and then this polypeptide is recovered.

[0032] The coding polynucleotides of the above polypeptides can be easily prepared by known genetic engineering techniques or by conventional techniques using a nucleic acid synthesizer. Petition 870230039907, dated 12 / 05 / 2023, page 21 / 100 13 / 82 commercially available. For example, DNA comprising the nucleotide sequence SEQ ID NO: 1 can be prepared by PCR using human chromosomal DNA or a cDNA library as a template, and a pair of primers designed to amplify the nucleotide sequence represented by SEQ ID NO: 1. PCR conditions can be appropriately determined. For example, PCR conditions comprise performing 30 cycles of the reaction cycle: denaturation at 94 °C for 30 seconds; annealing at 55 °C for 30 seconds to 1 minute, and extension at 72 °C for 2 minutes, using a thermostable DNA polymerase (e.g., Taq polymerase or Pfu-polymerase) and PCR buffer containing Mg2+, followed by reaction at 72 °C for 7 minutes. However, PCR conditions are not limited to the above example. PCR techniques, conditions, etc., are described in Ausubel et al., Short Protocols in Molecular Biology, 3aEdição, A compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons (especialmente o Capítulo 15).

[0033] Furthermore, based on the nucleotide sequence and amino acid sequence information represented by SEQ ID NOs: 1 to 30 in the Sequence Listing described in the present invention, appropriate probes or primers are prepared, and then a cDNA library from a human or similar organism is screened using them, so that the desired DNA can be isolated. A cDNA library is preferably constructed from cells, organs, or tissues expressing proteins possessing the even-numbered sequences of SEQ ID NOs: 2 to 30. Examples of such cells or tissues include cells or tissues derived from the testis, cancers or tumors such as leukemia, breast cancer, lymphomas, brain tumors, lung cancer, colorectal cancer, and the like. Procedures such as the preparation of probes or primers, the construction of a cDNA library, the screening of a cDNA library, and the cloning of target genes are known to the present invention. Petition 870230039907, dated 12 / 05 / 2023, page 22 / 100 14 / 82 expert in the technique and such procedures can be performed using the methods described in Sambrook et al. Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology (1989), Ausbel et al., (above), etc. The DNA encoding the human CAPRIN-1 protein or a partial peptide thereof can be obtained from DNA obtained in this way.

[0034] Host cells can be of any cell type, as long as they can express the polypeptide mentioned above. Examples of prokaryotic cells include, but are not limited to, E. coli and similar organisms. Examples of eukaryotic cells include, but are not limited to, mammalian cells such as monkey kidney cells (COS1) and Chinese hamster ovary cells (CHO), the human fetal kidney cell line (HEK293), the mouse fetal skin cell line (NIH3T3), yeast cells such as budding yeast and yeast, silkworm cells, and Xenopus oocytes.

[0035] When prokaryotic cells are used as host cells, an expression vector used in the present invention includes a replicable origin within prokaryotic cells, a promoter, a ribosome binding site, a multiple cloning site, a terminator, a drug resistance gene, a complementary auxotrophic gene, and the like. Examples of expression vectors for Escherichia coli include a pUC-based vector, pBluescript II, a pET expression system, and a pGEX expression system. DNA encoding the above polypeptide is incorporated into such an expression vector, prokaryotic host cells are transformed with the vector, the transformed cells thus obtained are cultured, and thereby the polypeptide encoded by the DNA can be expressed in prokaryotic host cells. At this point, the polypeptide can also be expressed as a fusion protein with another protein.

[0036] When eukaryotic cells are used as cells Petition 870230039907, dated 12 / 05 / 2023, page 23 / 100 15 / 82 host cells, an expression vector used in the present invention is an expression vector for eukaryotic cells, containing a promoter, a splicing region, a poly(A) addition site, and the like. Examples of such expression vectors include pKa1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, and pYES2. In a manner similar to that described above, the DNA encoding the above polypeptide is incorporated into such an expression vector, eukaryotic host cells are transformed with the vector, the transformed cells obtained in this way are cultured, and thus the polypeptide encoded by the DNA can be expressed in eukaryotic host cells.When pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or similar is used as an expression vector, the above polypeptide can be expressed as a fusion protein to which a tag, such as the His tag (e.g., (His)6-(His)10), FLAG tag, myc tag, HA tag or GFP, has been added.

[0037] To introduce an expression vector into host cells, a known method can be employed, such as electroporation, the calcium phosphate method, the liposome method, the DEAE dextran method, microinjection, viral infection, lipofection, and binding to a cell membrane-permeable peptide.

[0038] The polypeptide of interest can be isolated and purified from host cells by a combination of known separation processes. Examples of such known separation processes include, but are not limited to, treatment with a denaturing agent such as urea or a surfactant; ultrasonication; enzymatic digestion; salting-out precipitation or solvent fractionation and precipitation; dialysis; centrifugation; ultrafiltration; gel filtration; SDS-PAGE; isoelectric focusing; ion-exchange chromatography; hydrophobic interaction chromatography; affinity chromatography; and reverse-phase chromatography. Petition 870230039907, dated 12 / 05 / 2023, page 24 / 100 16 / 82 Antibody Structure

[0039] An antibody is a heteromultimeric glycoprotein that generally contains at least two heavy chains and two light chains. Antibodies that are not of the IgM class are heterotetrameric glycoproteins of approximately 150 kDa composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is connected to a heavy chain by a single covalent disulfide bond; however, the number of disulfide bonds between the heavy chains varies among different immunoglobulin isotypes. Each heavy chain or light chain also has an intrachain disulfide bond. Each heavy chain has a variable domain (VH region) at one end followed by several constant regions. Each light chain has a variable domain (VL region) and a constant region at one end opposite the other end.The constant region of a light chain is aligned with the first constant region of a heavy chain, and a variable domain of a light chain is aligned with a variable domain of a heavy chain. The specific region of an antibody variable domain exhibits specific variability that is referred to as the complementarity-determining region (CDR), thus conferring binding specificity to the antibody. A relatively conserved portion of a variable region is referred to as a framework region (FR) (or structural region). The complete heavy chain and variable domains of the light chain separately contain four FRs linked via three CDRs. The three CDRs of a heavy chain are referred to as CDRH1, CDRH2, and CDRH3, in that order from the N-terminal end. Similarly, in the case of a light chain, the CDRLs are referred to as CDRL1, CDRL2, and CDRL3.CDRH3 is the most important for the specificity of an antibody's binding to an antigen. Furthermore, the CDRs of each chain are held together in an adjacent state due to the FR regions. Petition 870230039907, dated 12 / 05 / 2023, p. 25 / 100 17 / 82 contributing to the formation of the antibody antigen-binding site along with the CDRs of the other chain. The constant region does not directly contribute to the binding of an antibody to an antigen, but it has several effector functions, such as antibody participation in antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis via binding to an Fcy receptor, half-life / clearance rate via the neonatal Fc receptor (FcRn), and complement-dependent cytotoxicity (CDC) via a C1q component of the complement system cascade. Antibody Preparation

[0040] The term “anti-CAPRIN-1 antibody”, as used herein, refers to an antibody with immunological reactivity to a full-length CAPRIN-1 protein or a fragment thereof.

[0041] As used in the present invention, the term “immunological reaction” refers to the in vivo binding property of an antibody to a CAPRIN-1 antigen. Through such in vivo binding, the function of harming the tumor (e.g., death, suppression, or degeneration) is exhibited. Specifically, an antibody used in the present invention can be any type of antibody, provided that it binds to a CAPRIN-1 protein in such a way as to be able to damage the tumor, such as leukemia, lymphoma, breast cancer, brain tumor, lung cancer, esophageal cancer, gastric cancer, kidney cancer, colorectal cancer, ovarian cancer, prostate cancer, or fibrosarcoma.

[0042] Examples of antibodies include a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, and an antibody fragment (e.g., Fab and F(ab')2). In addition, the antibody may be an immunoglobulin molecule of any class, such as IgG, IgE, IgM, IgA, IgD, or IgY, or of any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. Petition 870230039907, dated 12 / 05 / 2023, p. 26 / 100 18 / 82

[0043] The antibody may be further modified by glycosylation, acetylation, formylation, amidation, phosphorylation, pegylation (PEG) and / or similar processes.

[0044] Several examples of antibody preparation are as described below.

[0045] When the antibody is a monoclonal antibody, for example, the SK-BR-3 breast cancer cell line expressing CAPRIN-1 is administered to a mouse for immunization, the mouse spleen is removed, the cells are separated, and then the cells and mouse myeloma cells are fused. From among the fusion cells thus obtained (hybridomas), a clone producing an antibody that has the effect of suppressing the proliferation of cancer cells is selected. A hybridoma that produces a monoclonal antibody that has the effect of suppressing the proliferation of cancer cells is isolated, the hybridoma is cultured, and then an antibody is purified from the culture supernatant by general affinity purification, so that the antibody can be prepared.

[0046] The hybridoma that produces a monoclonal antibody can also be prepared as described below, for example. First, an animal is immunized with a sensitizing antigen according to a known method. A general method is performed by injecting a sensitizing antigen into a mammal intraperitoneally or subcutaneously. Specifically, a sensitizing antigen is diluted with PBS (phosphate-buffered saline), saline solution, or similar in an appropriate quantity, followed by suspension. The resulting solution is then mixed with an appropriate amount of a general adjuvant, as needed, such as Freund's complete adjuvant. After emulsification, the solution is administered to a mammal several times every 4 to 21 days. Additionally, a suitable vehicle can also be used after immunization with an antigen. Petition 870230039907, dated 12 / 05 / 2023, page 27 / 100 19 / 82 sensitizing.

[0047] A mammal is immunized as described above. After confirmation of an increase in the level of the desired antibody in the serum, immunized cells are collected from the mammal and then subjected to cell fusion. Preferred immunized cells are particularly splenocytes.

[0048] Mammalian myeloma cells are used as other parent cells to be fused with immunized cells. As myeloma cells, several known cell lines are preferentially employed, such as; P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8. 653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (deSt. Groth, SF et al., J. Immunol.

[0049] The fusion between the immunized cell and the myeloma cell can be performed basically according to a known method, for example, such as the Kohler and Milstein technique (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

[0050] More specifically, the cell fusion described above is performed, for example, in the presence of a cell fusion accelerator in a conventional nutrient medium. Polyethylene glycol (PEG), Sendai virus (HVJ), or similar substances are used as fusion accelerators. If desired, an auxiliary agent, such as dimethyl sulfoxide, can be added and used to increase fusion efficiency.

[0051] The ratio of immunized cells to myeloma cells to be used in the present invention may be arbitrarily established. By Petition 870230039907, dated 12 / 05 / 2023, page 28 / 100 20 / 82 For example, the number of immunized cells that are preferably used is one to ten times the number of myeloma cells. As a culture medium to be used for the cell fusion referred to above, an RPMI1640 culture medium suitable for the proliferation of the aforementioned myeloma cell line, a MEM culture medium, and other culture media normally used for this type of cell culture can be used. In addition, fluid that is supplementary to serum, such as fetal bovine serum (FBS), can be used in conjunction with it.

[0052] Cell fusion can be performed by mixing predetermined quantities of the above immunized cells and myeloma cells in the aforementioned culture medium, and a PEG solution (e.g., having an average molecular weight ranging from about 1000 to 6000), pre-heated to about 37 °C, is usually added at a concentration of 30% to 60% (w / v) and mixed, thus forming a culture containing hybridomas of interest. Then, an appropriate culture medium is successively added to the culture thus obtained, which is then centrifuged to remove the supernatant, and this process is repeated to remove the cell fusion agent or similar, which is not preferable for the growth of hybridomas.

[0053] The hybridomas thus obtained are cultured by selection in a standard selection culture medium (e.g., a HAT culture medium containing hypoxanthine, aminopterin, and thymidine). Culture in this HAT culture medium continues for a sufficient period of time (usually several days to several weeks) so that cells (unfused cells) that are not of the desired hybridoma die. Subsequently, screening and simple cloning of the hybridoma that produces an antibody of interest are performed using the general limiting dilution method.

[0054] The hybridomas above are obtained by immunizing a non-human animal with an antigen. In addition to this method, hybridomas that Petition 870230039907, dated 12 / 05 / 2023, page 29 / 100 21 / 82 producing a human antibody possessing desired activity (e.g., cell proliferation suppression activity) can also be obtained in vitro by sensitizing human lymphocytes, such as human lymphocytes that have been infected with the EB virus, with a protein, a cell expressing the protein, or a lysate thereof, followed by fusion of the sensitized lymphocytes with human myeloma cells possessing a permanently dividing capacity, such as U266 (registration no.: TIB196).

[0055] The hybridoma thus prepared, which produces a monoclonal antibody of interest, can be subcultured in general culture medium and can be stored in liquid nitrogen for a long period of time.

[0056] Specifically, a hybridoma can be prepared by immunization using a general immunization method, employing as a sensitizing antigen a desired antigen or a cell expressing the desired antigen, fusing the immunized cell thus obtained with a known parent cell by a general cell fusion method, and then screening a monoclonal antibody-producing cell (e.g., a hybridoma) by a general screening method.

[0057] Another example of an antibody that can be used in the present invention is a polyclonal antibody. A polyclonal antibody can be obtained as described below, for example.

[0058] A small animal, such as a mouse, a human antibody-producing mouse, or a rabbit, is immunized with a natural CAPRIN-1 protein, a recombinant CAPRIN-1 protein expressed in a microorganism such as Escherichia coli in the form of a GST fusion protein or similar, or a partial peptide thereof, and then serum is obtained. The serum is purified by protein ammonium sulfate precipitation, protein A column, protein G column, chromatography Petition 870230039907, dated 12 / 05 / 2023, page 30 / 100 22 / 82 DEAE ion exchange column, an affinity column to which a CAPRIN-1 protein or a synthetic peptide has been coupled, or similar, so that a polyclonal antibody can be prepared.

[0059] As a mouse producing human antibodies, a KM mouse (Kirin Pharma / Medarex) and a Xeno mouse (Amgen) are known (e.g., International Patent Application Publications WO 02 / 43478 and WO 02 / 092812), for example. When such a mouse is immunized with the CAPRIN-1 protein or a fragment thereof, a complete human polyclonal antibody can be obtained from the blood. In addition, splenocytes are collected from the immunized mouse and then a human-like monoclonal antibody can be prepared by means of a method for fusion with myeloma cells.

[0060] An antigen can be prepared according to a method using animal cells (Patent Publication JP (Kohyo) No. 2007-530068) or baculovirus (e.g., International Publication WO98 / 46777), for example. When an antigen has low immunogenicity, the antigen can be linked to a macromolecule that possesses immunogenicity, such as albumin, and then immunization is performed.

[0061] Additionally, an antibody gene is cloned from the aforementioned hybridoma and then incorporated into an appropriate vector. The vector is then introduced into a host, and the genetically recombined antibody produced using gene recombination techniques can then be used (e.g., see 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 hybridoma mRNA using reverse transcriptase. When the DNA encoding the V region of an antibody of interest can be obtained, the DNA is ligated to the DNA Petition 870230039907, dated 12 / 05 / 2023, page 31 / 100 23 / 82 encoding the constant region (C region) of the desired antibody, and then the resulting fusion product is incorporated into an expression vector. Alternatively, the DNA encoding the V region of an antibody can be incorporated into an expression vector containing the DNA for the C region of an antibody. At this point, the DNA can be incorporated into an expression vector so that it is expressed under the control of expression control regions, such as an enhancer and promoter. Then, host cells are transformed with the expression vector so that the antibody can be expressed.

[0062] The anti-CAPRIN-1 antibody of the present invention is preferably a monoclonal antibody. However, the anti-CAPRIN1 antibody may also be a polyclonal antibody or a genetically modified antibody (e.g., a chimeric antibody or a humanized antibody), for example.

[0063] Examples of a human monoclonal antibody include monoclonal antibodies, monoclonal antibodies from non-human animals (e.g., a mouse monoclonal antibody, a rabbit monoclonal antibody, and a chicken monoclonal antibody), and chimeric monoclonal antibodies. A monoclonal antibody can be prepared by culturing a hybridoma obtained by fusing spleen cells (splenocytes) from a non-human mammal (e.g., a mouse, a human antibody-producing mouse, a chicken, or a rabbit) immunized with a CAPRIN-1 protein, with a myeloma cell. A chimeric antibody is prepared by combining sequences from different animals, such as an antibody heavy chain comprising variable heavy chain and light chain regions from a mouse antibody and the constant heavy chain and light chain regions from a human antibody.A chimeric antibody can be prepared using one. Petition 870230039907, dated 12 / 05 / 2023, page 32 / 100 24 / 82 known method. For example, a chimeric antibody can be obtained by linking the DNA encoding a V region of an antibody to a DNA encoding a C region of a human antibody, incorporating the resulting fusion product into an expression vector, and then introducing the vector into a host for the production of the chimeric antibody.

[0064] In the examples described below, monoclonal antibodies possessing immunological reactivity with a partial polypeptide of CAPRIN-1 were prepared, wherein CAPRIN-1 is represented by any of the even-numbered SEQ ID sequences from 2 to 30, and wherein the partial polypeptide comprises the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 37. The antitumor effects of the monoclonal antibodies were confirmed. These monoclonal antibodies comprise a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID Nos: 43 or 63 and a variable light chain (VL) region comprising the amino acid sequence of SEQ ID Nos: 47, 51 or 67, wherein: the VH region comprises the CDR1 represented by the amino acid sequence SEQ ID NO: 40 or 60,a CDR2 is represented by the amino acid sequence SEQ ID NO: 41 or 61, and CDR3 is represented by the amino acid sequence SEQ ID NO: 42 or 62; and the VL region comprises CDR1, represented by the amino acid sequence SEQ ID NO: 44, 48, or 64, CDR2, represented by the amino acid sequence SEQ ID NO: 45, 49, or 65, and CDR3, represented by the amino acid sequence SEQ ID NO: 46, 50, or 66.

[0065] Examples of a polyclonal antibody include an antibody obtained by immunizing an animal that produces human antibodies (e.g., a mouse) with a CAPRIN-1 protein.

[0066] A humanized antibody is a modified antibody that Petition 870230039907, dated 12 / 05 / 2023, p. 33 / 100 25 / 82 is also referred to as a remodeled human antibody. A humanized antibody can be constructed by transplanting CDRs from an antibody originating from an immunized animal into the complementarity-determining regions of a human antibody. General gene recombination techniques are also known.

[0067] Specifically, DNA sequences designed to have each of the CDRs of a mouse or chicken antibody linked to each of the framework regions (FRs) of a human antibody are synthesized by the PCR method from various oligonucleotides, which are prepared in such a way as to have overlapping portions at their terminal portions, for example. A humanized antibody can be obtained by linking the DNA obtained in this way to DNA encoding the constant region of a human antibody, incorporating the resulting fusion product into an expression vector, introducing the vector into a host, and thus causing the host to produce the gene product (see European Patent Publication No. 239,400 and International Publication WO 96 / 02576). As FRs of a human antibody, to which they are linked via CDRs, the FRs that allow the formation of an antigen-binding site with good complementarity-determining regions are selected.If necessary, to form an antigen-binding site possessing the appropriate complementarity-determining regions of a remodeled human antibody, the amino acids of the scaffold regions of a variable region of the antibody can be substituted (Sato, K. et al., Cancer Research 1993, 53: 851-856). Furthermore, the amino acids of RFs can be substituted with those of the scaffold regions of different human antibodies (see International Patent Publication WO 99 / 51743).

[0068] As the framework regions (FRs) of a human antibody, which are linked via CDRs, are selected the Petition 870230039907, dated 12 / 05 / 2023, page 34 / 100 26 / 82 FRs that allow the formation of an antigen-binding site with good complementarity-determining regions. If necessary, for the formation of an antigen-binding site possessing the appropriate complementarity-determining regions of a remodeled human antibody, the amino acids of the scaffold regions of a variable region of the antibody can be substituted (Sato, K. et al., Cancer Research 1993, 53: 851-856).

[0069] After preparing a chimeric antibody or a humanized antibody, the amino acids in the variable regions (e.g., FR) or in the constant region can be replaced by other amino acids.

[0070] Amino acid substitution is the substitution of, for example, less than 15, less than 10, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less amino acids and is preferably a substitution of 1 to 5 amino acids, and more preferably of 1 or 2 amino acids. A substituted antibody must be functionally equivalent to an unsubstituted antibody. The substitution is desirably a conservative amino acid substitution(s) between amino acids that have analogous properties such as electrical charge, side chain, polarity, aromaticity.Amino acids possessing analogous properties can be classified, for example, into basic amino acids (lysine, arginine, and histidine), acidic amino acids (aspartic acid and glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, and tyrosine), nonpolar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, and methionine), branched-chain amino acids (threonine, valine, and isoleucine), and aromatic amino acids (phenylalanine, tyrosine, tryptophan, and histidine).

[0071] Examples of a modified antibody product include antibodies linked to various molecules such as polyethylene glycol (PEG). The substances to be linked in the modified antibody product of the present Petition 870230039907, dated 12 / 05 / 2023, page 35 / 100 27 / 82 inventions are not limited. Such a modified antibody product can be obtained by subjecting the antibody thus obtained to chemical modification. The methods for this have already been established in the state of the art.

[0072] As used herein, the “functional equivalent” refers to an antibody that has a biological or biochemical activity similar to that of the antibody of the present invention and, specifically, refers to a subject antibody that has the function of essentially harming the tumor without causing rejection after its application to a human, for example. An example of such activity includes cell proliferation suppression activity or binding activity.

[0073] As a method well known to those skilled in the art of preparing a polypeptide functionally equivalent to a polypeptide, a method for introducing mutations into a polypeptide is known. For example, those skilled in the art may prepare an antibody functionally equivalent to the antibody of the present invention by appropriately introducing a mutation into the antibody using site-directed mutagenesis (Hashimoto-Gotoh, T. et al., (1995) Gene 152, 271-275; Zoller, MJ., and Smith, M. (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al., (1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W. and Fritz, HJ., (1987) Methods Enzymol. 154, 350-367; Kunkel, TA., (1985) Proc. Natl. Acad. Sci. USA 82, 488-492; Kunkel (1988) Methods Enzymol. 85, 2763-2766).

[0074] An antibody that recognizes an epitope of a CAPRIN-1 protein recognized by the anti-CAPRIN-1 antibody above can be obtained by a method known to those skilled in the art. For example, such an antibody can be obtained by a method involving the determination of an epitope of a CAPRIN-1 protein recognized by an anti-CAPRIN-1 antibody by a general method (e.g., epitope mapping) and then the preparation of an antibody using a polypeptide possessing a Petition 870230039907, dated 12 / 05 / 2023, page 36 / 100 28 / 82 amino acid sequence contained in the epitope as an immunogen, or a method involving the determination of an epitope of such an antibody prepared by a general method, and then the selection of an antibody possessing the epitope identical to that of an anti-CAPRIN-1 antibody. As used herein, the term “epitope” refers to, in a mammal and preferably in a human, a polypeptide fragment with antigenicity or immunogenicity. The minimum unit size of this consists of about 7 to 12 amino acids, and preferably 8 to 11 amino acids.

[0075] The affinity constant Ka (Kon / Koff) of the antibody of the present invention is preferably at least 107 M-1, at least 108 M-1, at least 5^108 M-1, at least 109 M-1, at least 5x109 M-1, at least 1010 M-1, at least 5x1010 M-1, at least 1011 M-1, at least less than 5x1011 M-1, at least 1012 M-1 or at least 1013 M-1.

[0076] The antibody of the present invention can be conjugated with an antitumor agent. The conjugation of the antibody with an antitumor agent can be carried out by means of a spacer having a reactive group with an amine group, a carboxyl group, a hydroxyl group, a thiol group or the like (for example, a succinimidyl succinate group, a formyl group, a 2-pyridylthio group, a maleimidinyl group, an alkoxy carbonyl group and a hydroxyl group).

[0077] Examples of antitumor agents include the following antitumor agents known in the literature and similar agents, such as paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethioplosylphosphoramide, trimethylolmelamine, bulatacin, bulatacinone, camptothecin, briostatin, calistatin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictin, spongistatin, Petition 870230039907, dated 12 / 05 / 2023, page 37 / 100 29 / 82 chlorambucil, chlornafazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novembicin, fenesterin, prednimustine, trofosfamide, uracil mostards, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, caliceamycin, dinemycin, clodronate, speramycin, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophylline, cromomycin, dactinomycin, detorbicin, 6-diazo-5-oxo-L-norleucine, adriamycin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin C, acid mycophenolic, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptonigrine, streptozocin, tubercidine, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine,enocitabine, floxuridine, androgens (e.g. calusterone, dromostanolone propionate, epithiostanol, mepitiostane and testolactone), aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, enyluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, ethoglyside, lentinan, lonidamine, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerin, pentostatin, fenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxine, scizophyllan, spirogermanium, tenuazonic acid, triaziquone, roridine A, anguidin, urethane, vindesine, dacarbazine, manomustine, mitobronitol, mitolactol, pipobromane, gacytosine, docetaxel, chlorambucil, gemcitabine, thioguanine, mercaptopurine, cisplatin, oxaliplatin, carboplatin, vinblastine, etoposide, ifosfamide, mitoxantrone, vincristinevinorelbine, novantron, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan, topoisomerase inhibitor, difluoromethylornithine (DMFO), acid, Petition 870230039907, dated 12 / 05 / 2023, page 38 / 100 30 / 82 retinoic acid and capecitabine, and pharmaceutically acceptable salts and derivatives thereof.

[0078] By administering the antibody of the present invention in combination with an antitumor agent, even greater therapeutic effects can be obtained. This technique is applicable before and after surgery in a patient with CAPRIN-1 expressing cancer. Especially after surgery, more effective prevention of cancer recurrence or a prolonged survival period can be obtained against CAPRIN-1 expressing cancer that has been conventionally treated with an antitumor agent alone.

[0079] Examples of antitumor agents to be administered in combination with the antibody of the present invention include the following antitumor agents known from the previous literature or similar, such as paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethioplostropramide, trimethylolmelamine, bulatacin, bulatacinone, camptothecin, briostatin, calistatin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictin, spongistatin, chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride oxide, melphalan, novembicine, fenesterin, prednimustine, trofosfamide, uracil mustards, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, caliceamycin, dinemycin, clodronate,waitamycin, aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophylline, chromomycin, dactinomycin, detorbicin, 6-diazo-5oxo-L-norleucine, adriamycin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, Petition 870230039907, dated 12 / 05 / 2023, page 39 / 100 31 / 82 peplomycin, potfiromycin, puromycin, chelamicin, rhodorubicin, streptonigrine, streptozocin, tubercidine, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldofosfamide glycoside, aminolevulinic acid, enyluracil, amsacrine, bestrabucil, bisanthrene, edathraxate defofamine, demecolcine, diaziquone, elfornitine, eliptinium acetate, epothilone, ethoglucide, lentinan, lonidamine, maitansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerin, pentostatin, fenamet, pyrarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxine, scizophyllan, spirogermanium, tenuazonic acidtriaziquone, roridine A, anguidine, urethane, vindesine, dacarbazine, manomustine, mitobronitol, mitolactol, pipobromane, gacytosine, docetaxel, chlorambucil, gemcitabine, thioguanine, mercaptopurine, cisplatin, oxaliplatin, carboplatin, vinblastine, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantron, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan, topoisomerase inhibitor, difluoromethylornithine (DMFO), retinoic acid and capecitabine, and (known) pharmaceutically acceptable salts or (known) derivatives thereof. Of the examples mentioned above, cyclophosphamide, paclitaxel, docetaxel, and vinorelbine are particularly preferred.

[0080] Alternatively, a radioactive isotope known from prior or similar literature, such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Lu175 or Lu176, may be linked to the antibody of the present invention. A desired radioisotope is effective for tumor treatment or diagnosis. Petition 870230039907, dated 12 / 05 / 2023, p. 40 / 100 32 / 82

[0081] The antibody of the present invention is an antibody that has immunological reactivity with CAPRIN-1, an antibody that specifically recognizes CAPRIN-1, or an antibody that specifically binds to CAPRIN-1, which exhibits cytotoxic activity against cancer or a suppressive effect on tumor growth. The antibody must have a structure such that rejection is almost or completely avoided in an animal subject to which the antibody is administered. Examples of such an antibody include, when the animal subject is human, human antibodies, humanized antibodies, chimeric antibodies (e.g., human-mouse chimeric antibody), single-chain antibodies, and bispecific antibodies.These antibodies are: recombinant antibodies possessing the variable regions of the heavy chain and light chain from a human antibody; recombinant antibodies possessing the variable regions of the heavy chain and light chain composed of the complementarity-determining regions (CDRs) (CDR1, CDR2, and CDR3) of an antibody from a non-human animal and the scaffold regions of a human antibody; or recombinant antibodies possessing the variable regions of the heavy chain and light chain from a non-human animal antibody; wherein said recombinant antibodies also possess the constant regions of the heavy chain and light chain of a human antibody. The preferred antibodies are the first two antibodies mentioned.

[0082] These recombinant antibodies can be prepared as follows by cloning the DNA encoding a human anti-CAPRIN-1 monoclonal antibody (e.g., a human monoclonal antibody, a mouse monoclonal antibody, a rat monoclonal antibody, a rabbit monoclonal antibody, or a chicken monoclonal antibody) from an antibody-producing cell, such as a hybridoma, preparing the DNA encoding a variable light chain region and a variable region of Petition 870230039907, dated 12 / 05 / 2023, p. 41 / 100 33 / 82 heavy chain of the antibody by an RT-PCR method using the same as a template, and then determining the sequence of each variable region of the light chain and heavy chain or each sequence of CDR1, CDR2 and CDR3 based on the EU numbering system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md. (1991)).

[0083] In addition, the coding DNA for each of the variable regions or coding DNA for each CDR is prepared using gene recombination techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) or a DNA synthesizer. In the present invention, the human monoclonal antibody-producing hybridoma can be prepared by immunizing a human antibody-producing animal (e.g., a mouse) with human CAPRIN-1, followed by fusion of spleen cells taken from the previously immunized animal with myeloma cells. Alternatively, DNAs encoding a variable light chain region and a constant heavy chain region of a human antibody are prepared as needed, using gene recombination techniques or using a DNA synthesizer.

[0084] In the case of humanized antibody, the DNA is prepared by replacing a CDR coding sequence in DNA encoding a variable light chain or heavy chain region derived from a human antibody with a CDR coding sequence corresponding to that of an antibody derived from a non-human animal (e.g., a mouse, a rat, or a chicken) and then linking the DNA obtained in this way to DNA encoding a constant light chain or heavy chain region derived from a human antibody. Thus, the humanized antibody coding DNA can be prepared.

[0085] In the case of the chimeric antibody, the DNA coding for a Petition 870230039907, dated 12 / 05 / 2023, page 42 / 100 34 / 82 A chimeric antibody can be prepared by linking the DNA encoding a variable light chain or heavy chain region of an antibody from a non-human animal (e.g., a mouse, rat, or chicken) to the DNA encoding a constant light chain or heavy chain region of a human antibody.

[0086] In the case of a single-chain antibody, this antibody is prepared by linearly linking the variable region of the heavy chain to a variable region of the light chain by means of a linker. Thus, the coding DNA of a single-chain antibody can be prepared by linking the coding DNA of a variable region of the heavy chain, and a coding DNA of a linker, and a coding DNA of a variable region of the light chain. In the present invention, a variable region of the heavy chain and a variable region of the light chain are both from a human antibody, or only the CDRs are replaced by CDRs from an antibody derived from a non-human animal (for example, a mouse, a rat, and a chicken), although the other regions originate from a human antibody. Furthermore, the linker has 12 to 19 amino acids, and examples of this include (G4S)3 having 15 amino acids (Kim, GB. et al., Protein Engineering Design and Selection 2007, 20 (9): 425-432).

[0087] In the case of a bispecific antibody (diabody), this antibody is capable of specifically binding to two different epitopes. For example, the coding DNA of a bispecific antibody can be prepared by linking the coding DNA of a variable heavy chain region “A”, a coding DNA of a variable light chain region “B”, a coding DNA of a variable heavy chain region “B”, and a coding DNA of a variable light chain region “A”, in this order (in this case, the coding DNA of a variable light chain region “B” is linked to the coding DNA of a variable heavy chain region “B” via the coding DNA of the linker). Petition 870230039907, dated 12 / 05 / 2023, page 43 / 100 35 / 82 above). In this case, a variable heavy chain region and a variable light chain region are both from a human antibody, or, a human antibody in which only the CDRs have been replaced with CDRs from an antibody from a non-human animal (e.g., a mouse, a rat, or a chicken).

[0088] The recombinant DNA prepared above is incorporated into one or a variety of vectors, these vectors are introduced into host cells (e.g., mammalian cells, yeast or insect cells), then (co)expression is induced, so that the recombinant antibody can be prepared (PJ Delves, ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES, 1997 WILEY; P. Shepherd and C. Dean., Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; JW Goding., Monoclonal Antibodies: principles and practice, 1993 ACADEMIC PRESS).

[0089] Examples of the antibody of the present invention prepared by the method described above include the following antibody (a), (b) or (c) obtained in the Examples below: (a) an antibody (for example, the antibody composed of the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 47) comprising a heavy chain variable region comprising SEQ ID NOs: 40, 41 and 42 and a light chain variable region comprising SEQ ID NOs: 44, 45 and 46; and (b) an antibody (for example, the antibody composed of the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 51) comprising a heavy chain variable region comprising SEQ ID NOs: 40, 41 and 42 and a light chain variable region comprising SEQ ID NOs: 48, 49 and 50. (c) an antibody (for example, the antibody composed of the variable heavy chain region of SEQ ID NO: 63 and the variable light chain region Petition 870230039907, dated 12 / 05 / 2023, p. 44 / 100 36 / 82 of SEQ ID NO: 67) comprising a variable heavy chain region comprising SEQ ID NOs: 60, 61 and 62 and a variable light chain region comprising SEQ ID NOs: 64, 65 and 66.

[0090] The amino acid sequences represented by SEQ ID NOs: 40, 41, and 42, and SEQ ID NOs: 60, 61, and 63 are CDR1, CDR2, and CDR3, respectively, from mouse antibody heavy chain variable regions. Furthermore, the amino acid sequences represented by SEQ ID NOs: 44, 45, and 46, SEQ ID Nos: 48, 49, and 50, and SEQ ID NOs: 64, 65, and 66 are CDR1, CDR2, and CDR3, respectively, from mouse antibody light chain variable regions.

[0091] In addition, the humanized antibody, chimeric antibody, single-chain antibody or bispecific antibody of the present invention is the following antibody (exemplified as “antibody (a)”), for example: (i) an antibody in which the variable heavy chain region comprises the amino acid sequence of SEQ ID NOs: 40, 41, and 42 and the amino acid sequences of the framework regions of a human antibody, and a variable light chain region comprises the amino acid sequences of SEQ ID NOs: 44, 45, and 46 and the amino acid sequences of the framework regions of a human antibody (preferably, the antibody in which the variable heavy chain region comprises the amino acid sequence of SEQ ID NO: 43, and the variable light chain region comprises the amino acid sequence of SEQ ID NO: 47); and (ii) an antibody in which the variable heavy chain region comprises the amino acid sequence of SEQ ID NOs: 40, 41, and 42, and the amino acid sequences of the framework regions of a human antibody; and a constant heavy chain region comprises an amino acid sequence of a human antibody;and a variable light chain region comprising the amino acid sequences of SEQ ID NOS: 44, 45, and 46; Petition 870230039907, dated 12 / 05 / 2023, page 45 / 100 37 / 82 the amino acid sequences of the framework regions of a human antibody and a constant light chain region comprising an amino acid sequence of a human antibody (preferably, the antibody, wherein the variable heavy chain region comprises the amino acid sequence of SEQ ID NO: 43, and the constant heavy chain region comprises the amino acid sequence of a human antibody, as well as 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 of a human antibody).

[0092] In addition, human antibody heavy chain sequences and constant light chain regions and variable regions can be obtained from NCBI (e.g., in the US: GenBank, Unigene), for example.For example, the sequence with accession number J00228 can be referred to a constant region of human IgG1 heavy chain, the sequence with accession number J00230 can be referred to a constant region of human IgG2 heavy chain, the sequence with accession number X03604 can be referred to a constant region of human IgG3 heavy chain, the sequence with accession number K01316 can be referred to a constant region of human IgG4 heavy chain, the sequences with accession numbers V00557, X64135, X64133, and others similar can be referred to constant regions of human κ light chain, and the sequences with accession numbers X64132, X64134, and similar, can be referred to constant regions of human λ light chain.

[0093] The antibodies mentioned above preferably have cytotoxic activity and, therefore, may exhibit antitumor effects.

[0094] In addition, the specific sequences of variable heavy chain and light chain regions or CDRs in the above antibodies are given Petition 870230039907, dated 12 / 05 / 2023, page 46 / 100 Figure 38 / 82 is for illustrative purposes only and therefore is not clearly limited to these specific sequences. A hybridoma capable of producing another human antibody or a non-human animal antibody (e.g., a mouse antibody) against human CAPRIN-1 is prepared, a monoclonal antibody produced by the hybridoma is collected, and then it is determined whether the antibody is the desired antibody or not, using as indicators the immunological binding property with human CAPRIN-1 and cytotoxic activity. After identifying a hybridoma that produces the target monoclonal antibody in this way, the DNA encoding the variable heavy chain and light chain region of the target antibody is prepared from the hybridoma as described above, and sequencing is performed, and then the DNA is used for the preparation of another antibody.

[0095] Furthermore, with respect to the antibody above, the sequence of antibodies (a) to (c) mentioned above, and particularly the sequence of the framework region and / or the sequence of the constant region of each of the antibodies, may have a substitution, a deletion, or addition of one or more amino acids, provided that the specific recognition specificity of CAPRIN-1 is maintained. In this case, the term “more” preferably refers to 2 to 5, and more preferably 2 or 3.

[0096] The present invention further provides a DNA encoding the antibody of the present invention described above, or a DNA encoding the heavy chain or light chain of the antibody above, or a DNA encoding the variable region of the heavy chain or light chain of the antibody above. Examples of such DNAs include, in the case of antibody (a), the DNA encoding a variable region of the heavy chain comprising the nucleotide sequences encoding the amino acid sequences of SEQ ID NOS: 40, 41, and 42, and the DNA encoding a variable region of the light chain comprising the nucleotide sequences encoding the amino acid sequences of Petition 870230039907, dated 12 / 05 / 2023, page 47 / 100 39 / 82 SEQ ID NOS: 44, 45, and 46.

[0097] The complementarity-determining regions (CDRs) encoded by the DNA sequences are regions for determining antibody specificity. Therefore, the coding sequences of regions in an antibody other than the CDRs (more specifically, a constant region and a framework region) may be from other antibodies. In the present invention, examples of such “other antibodies” include antibodies derived from non-human organisms, and are preferably derived from humans to reduce side effects. Thus, in the case of the DNA above, the coding regions of each framework region and each heavy-light chain contact region preferably comprise the nucleotide sequences that encode corresponding amino acid sequences of a human antibody.

[0098] Other examples of alternative antibody-coding DNA of the present invention include, in the case of antibody (a), DNA encoding a variable heavy chain region comprising the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43 and DNA encoding a variable light chain region comprising the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 47. In this case, an example of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43 is the nucleotide sequence of SEQ ID NO: 52. Furthermore, an example of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 47 is the nucleotide sequence of SEQ ID NO: 53. In these DNAs, the coding regions of each constant region of heavy and light chains preferably comprise the nucleotide sequences encoding the corresponding amino acid sequences of a human antibody.

[0099] The DNAs of these antibodies can be obtained by Petition 870230039907, dated 12 / 05 / 2023, p. 48 / 100 40 / 82 methods above or, for example, by the following method. First, total RNA is prepared from a hybridoma related to the antibody of the present invention using a commercial RNA extraction kit, then cDNA is synthesized with a reverse transcriptase using random or similar primers. Subsequently, the cDNA encoding an antibody is amplified by a PCR method using as primers the oligonucleotides of conserved sequences in each variable region of known mouse antibody heavy and light chain genes. The coding sequence of a constant region can be obtained by amplifying a known sequence by the PCR method. The DNA nucleotide sequence can be determined by a conventional method such as insertion into a plasmid or phage for sequencing.

[00100] An anti-CAPRIN-1 antibody to be used in the present invention is considered to be an antibody that exhibits antitumor effects against cancer cells expressing CAPRIN-1 through the following mechanism:

[00101] Antibody-dependent effector cell-mediated cytotoxicity (ADCC) of CAPRIN-1-expressing cells and complement-dependent cytotoxicity (CDC) of CAPRIN-1-expressing cells.

[00102] For this reason, the activity of an anti-CAPRIN1 antibody to be used in the present invention can be evaluated, as specifically described in the following Examples, by measuring the ex vivo activity of ADCC or CDC against cancer cells expressing CAPRIN-1.

[00103] An anti-CAPRIN-1 antibody to be used in the present invention binds to a CAPRIN-1 protein in a cancer cell and exhibits antitumor effects due to the activity cited above, and is thus useful for treating or preventing cancer. Specifically, the present invention provides a pharmaceutical composition for the treatment and / or prevention of cancer, comprising an anti-CAPRIN-1 antibody as the active ingredient. When the Petition 870230039907, dated 12 / 05 / 2023, page 49 / 100 41 / 82 anti-CAPRIN-1 antibody is used for administration to a human body (antibody therapy); it is preferably a human antibody or humanized antibody in order to decrease immunogenicity.

[00104] Furthermore, the higher the binding affinity between an anti-CAPRIN-1 antibody and a CAPRIN-1 protein on the surfaces of cancer cells, the stronger the antitumor activity of the anti-CAPRIN-1 antibody can be obtained. Therefore, when an anti-CAPRIN-1 antibody with high binding affinity for the CAPRIN-1 protein can be acquired, strong antitumor effects can be expected, so that the application of the antibody as a pharmaceutical composition for cancer treatment and / or prevention becomes possible. High binding affinity is desirably as follows. As described above, the linkage constant (affinity constants) Ka (kon / koff) is preferably at least 107 M-1, at least 108 M-1, at least 5 *108 M-1, at least 109 M-1, at least 5 *109 M-1, at least 1010 M-1, at least 5 *1010 M-1, at least 1011 M-1, at least 5 *1011 M-1, at least 1012 M-1 or at least 1013 M-1. Binding to Cells Expressing the Antigen

[00105] The ability of an antibody to bind to CAPRIN-1 can be specified by a binding assay using the ELISA method, a Western blot method, immunofluorescence and flow cytometry analysis, or similar techniques, such as those described in the Examples. Immunohistochemical staining

[00106] An antibody that recognizes CAPRIN-1 can be tested for reactivity to CAPRIN-1 using an immunohistochemical method known to those skilled in the art, using acetone-fixed frozen tissue sections or paraffin-embedded tissue sections, which are prepared from tissue samples obtained from a patient. Petition 870230039907, dated 12 / 05 / 2023, page 50 / 100 42 / 82 during surgery, or tissue samples obtained from an animal possessing a heterotransplant inoculated with a cell line that expresses CAPRIN1, naturally or after transfection.

[00107] A reactive antibody for CAPRIN-1 can be stained by various immunohistochemical staining methods. For example, a goat anti-mouse antibody or a goat anti-chicken antibody conjugated with horseradish peroxidase can perform the reaction, and a target antibody can be visualized. Pharmaceutical Composition

[00108] The present invention further provides a pharmaceutical composition for the treatment and / or prevention of cancer, which is characterized by containing the above antibody or a fragment thereof as an active ingredient that has immunological reactivity with partial polypeptides of CAPRIN1 represented by even-numbered SEQ IDs: 2 to 30, wherein the polypeptide has the amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 37.

[00109] A target of the pharmaceutical composition for the treatment and / or prevention of cancer of the present invention is not particularly limited, provided that it is the cancer (cell) that expresses a CAPRIN1 gene.

[00110] The terms “tumor” and “cancer”, as used in the present invention, refer to malignant neoplasms and are used interchangeably.

[00111] The cancer to be subjected in the present invention is cancer that expresses genes encoding CAPRIN-1 proteins possessing the amino acid sequences NE with even numbering of SEQ ID NOs: 2 to 30. Examples of such cancer preferably include breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mast cell tumor, kidney cancer, Petition 870230039907, dated 12 / 05 / 2023, page 51 / 100 43 / 82 cervical cancer, bladder cancer, esophageal cancer, gastric cancer, colorectal cancer, ovarian cancer, prostate cancer, or fibrosarcoma.

[00112] Examples of such specific cancer include, but are not limited to, breast adenocarcinoma, compound breast adenocarcinoma, malignant mixed mammary gland tumor, intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, glioma which is a neural epithelial tissue tumor, ependymoma, neurocytoma, fetal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, digestive lymphoma, small cell and medium cell lymphoma, cecal cancer, ascending colon, descending colon, transverse colon cancer, sigmoid colon, rectal cancer, epithelial ovarian cancer, germ cell tumor, and interstitial cell tumor.

[00113] In addition, the preferred individuals are mammals, including primates, pets, domestic animals, purebred animals, and the like, and are particularly preferred humans, dogs, and cats.

[00114] When the antibody used in the present invention is used as a pharmaceutical composition, it can be formulated by a method known to those skilled in the art. For example, the antibody can be used parenterally in the form of an injectable preparation, such as an aseptic solution or suspension prepared with water or with a pharmaceutically acceptable solution other than water. For example, it can be formulated by mixing a unit dosage form required by generally accepted pharmaceutical practice, in suitable combination with a vehicle or pharmacologically acceptable medium, specifically, sterile water or physiological saline, vegetable oil, an emulsifier, a suspension, a surfactant, a stabilizer, a flavoring compound, an excipient, a Petition 870230039907, dated 12 / 05 / 2023, page 52 / 100 44 / 82 vehicle, an antiseptic, a binding agent, and the like. The quantities of active ingredients in these preparations are determined so that an adequate dose within the indicated range can be obtained.

[00115] A composition for aseptic injection may be prescribed in accordance with general pharmaceutical practice, using a vehicle such as distilled water for injection.

[00116] Examples of an aqueous solution for injection include saline solution, isotonic solution containing dextrose or other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These examples can be used in combination with a suitable solubilizing agent, such as alcohol, especially ethanol and polyalcohol (e.g., propylene glycol and polyethylene glycol), and a non-ionic surfactant (e.g., polysorbate 80® and HCO-60).

[00117] Examples of oils include sesame oil and soybean oil, which can be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol. Additionally, buffering agents such as phosphate buffer or sodium acetate buffer, calming agents such as procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, or antioxidants can be mixed with it. A suitable ampoule is usually filled with the injection solution thus prepared.

[00118] Administration is oral or parenteral, and parenteral administration is preferred. Specific examples of the route of administration include injection, transnasal administration, pulmonary administration, and transdermal administration. Examples of injection include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection, so systemic or local administration is possible.

[00119] In addition, the methods of administration can be Petition 870230039907, dated 12 / 05 / 2023, page 53 / 100 45 / 82 appropriately selected depending on the patient's age, body weight, sex, symptoms, among other factors. The dosage by administering a pharmaceutical composition containing an antibody or polynucleotide encoding the antibody may be selected from the range between 0.0001 mg and 1000 mg per kg of body weight, for example. Alternatively, the dosage may be selected, for example, from the range between 0.001 mg / body and 100000 mg / body per patient. However, the dosage range is not always limited to these numerical values. The dosage and method of administration vary depending on the patient's body weight, age, sex, symptoms, and the like, but may be appropriately selected by those skilled in the art.

[00120] The above pharmaceutical composition containing the antibody or a fragment thereof of the present invention is administered to a subject, so that cancer, preferably breast cancer, brain tumor, leukemia, lung cancer, lymphoma, mast cell tumor, kidney cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer, colorectal cancer, can be treated and / or prevented.

[00121] The present invention further encompasses a method for the treatment and / or prevention of cancer, comprising administering to a subject the pharmaceutical composition of the present invention in combination with the antitumor agent exemplified above or a pharmaceutical composition containing said antitumor agent. The antibody or fragment thereof of the present invention and the antitumor agent may be administered simultaneously or separately to a subject. They may be administered separately, regardless of the order of administration. The administration intervals, dosage, route of administration, and frequency of administration may be appropriately selected by a specialist. Examples of other pharmaceutical formulations to be administered simultaneously include pharmaceutical compositions obtained by mixing the antibody or fragment thereof of the Petition 870230039907, dated 12 / 05 / 2023, page 54 / 100 46 / 82 same as the present invention with an antitumor agent in a pharmaceutically acceptable vehicle (or medium) followed by the formulation. Furthermore, explanations regarding prescription, formulation, route of administration, dose, cancer to be treated, and similar aspects for the administration of a pharmaceutical composition containing the antibody of the present invention are applicable to the above pharmaceutical composition containing an antitumor agent or formulation; and to the formulation.

[00122] Therefore, the present invention also provides a pharmaceutical combination for the treatment and / or prevention of cancer, comprising the pharmaceutical composition of the present invention and the pharmaceutical composition exemplified above containing an antitumor agent. Furthermore, the present invention provides a pharmaceutical composition for the treatment and / or prevention of cancer, comprising the antibody or fragment thereof of the present invention and an antitumor agent together with a pharmacologically acceptable vehicle. POLYPEPTIDE AND DNA

[00123] The present invention also provides the following antibody-related polypeptides and DNAs (a), (b) or (c) above.

[00124] (i) A polypeptide comprising the amino acid sequence of SEQ ID NOS: 43 and 63, and the polypeptide-coding DNA, wherein the DNA comprises the nucleotide sequences of SEQ ID NOS: 52 and 68.

[00125] (ii) A polypeptide comprising the amino acid sequence of SEQ ID NOs: 47, 51 and 67, and the polypeptide-coding DNA, wherein the DNA comprises the nucleotide sequences of SEQ ID NOs: 70, 53 and 69.

[00126] (iii) A CDR heavy chain polypeptide selected from the group consisting of the amino acid sequences represented Petition 870230039907, dated 12 / 05 / 2023, p. 55 / 100 47 / 82 by SEQ ID NOS: 40, 41 and 42, and SEQ ID NOS: 60, 61 and 62, and the DNA encoding the polypeptide.

[00127] (iv) A CDR light chain polypeptide selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 44, 45 and 46, SEQ ID NOs: 48, 49 and 50, and SEQ ID NOs: 64, 65 and 66, and the polypeptide coding DNA.

[00128] These polypeptides and DNAs can be prepared using gene recombination techniques as described above. Brief Description of the Invention

[00129] The present invention described above is summarized as follows.

[00130] (1) A pharmaceutical composition for the treatment and / or prevention of cancer, comprising an antibody or a fragment thereof as an active ingredient that has immunological reactivity with a partial polypeptide of CAPRIN-1, wherein CAPRIN-1 is represented by any of the even numbered sequences of SEQ ID NOS: 2 to 30, and wherein the partial polypeptide comprises the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 37.

[00131] (2) The pharmaceutical composition according to item (1) wherein the cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mast cell tumor, kidney cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer or colorectal cancer.

[00132] (3) The pharmaceutical composition according to item (1) or (2) wherein said antibody is a monoclonal antibody or a polyclonal antibody.

[00133] (4) The pharmaceutical composition according to any one of items (1) to (3), characterized in that said antibody is a Petition 870230039907, dated 12 / 05 / 2023, page 56 / 100 48 / 82 human antibody, humanized antibody, chimeric antibody, single-chain antibody or a bispecific antibody.

[00134] (5) An antibody possessing immunological reactivity with a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence possessing 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 37.

[00135] (6) The antibody according to item (5) above, which has a cytotoxic activity against the cancer cell that expresses a CAPRIN-1 protein.

[00136] (7) An antibody comprising a variable heavy chain region comprising SEQ IDs NOs: 40, 41, and 42; and a variable light chain region comprising SEQ IDs NOs: 44, 45, and 46, and which has immunological reactivity with a CAPRIN-1 protein.

[00137] (8) An antibody comprising a variable heavy chain region comprising SEQ ID Nos: 40, 41, and 42; and a variable light chain region comprising SEQ ID Nos: 48, 49, and 50, and possessing immunological reactivity with a CAPRIN-1 protein.

[00138] (9) An antibody comprising a variable heavy chain region comprising SEQ ID Nos: 60, 61, and 62; and a variable light chain region comprising SEQ ID Nos: 64, 65, and 66, and having immunological reactivity with a CAPRIN-1 protein.

[00139] (10) The antibody according to any of items (5) to (9) above, characterized in that it is a human antibody, humanized antibody, chimeric antibody, single-chain antibody or bispecific antibody.

[00140] (11) A pharmaceutical composition for the treatment and / or prevention of a cancer, comprising the antibody or fragment thereof of any of the items from (5) to (10) above as the active ingredient. Petition 870230039907, dated 12 / 05 / 2023, page 57 / 100 49 / 82

[00141] (12) The pharmaceutical composition according to item (11) wherein the cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mast cell tumor, kidney cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer or colorectal cancer.

[00142] (13) A pharmaceutical combination for the treatment and / or prevention of a cancer, comprising the pharmaceutical composition of any of the items from (1) to (4) above, or the pharmaceutical composition of item (11) or (12) above, and a pharmaceutical composition containing an antitumor agent.

[00143] (14) A pharmaceutical composition for the treatment and / or prevention of a cancer, comprising administering to a subject the antibody or fragment thereof of any of items (5) to (10) or the pharmaceutical composition according to item (11) or (12) above.

[00144] (15) A method for the treatment and / or prevention of cancer, comprising the use of pharmaceutical compositions of the pharmaceutical combination of item (13) above, in combination, in a subject. Examples

[00145] The present invention is described more specifically based on examples, but the scope of the present invention is not limited by these specific examples. Example 1 Identification of Novel Cancer Antigen Proteins by the SEREX Method (1) cDNA Library Preparation

[00146] Total RNA was extracted from testes tissue of a healthy dog ​​using a guanidino-phenol-chloroform method. PolyA RNA was purified according to protocols provided with an “Oligotex-dT30 mRNA” purification kit (Takara Shuzo Co., Ltd.).

[00147] A canine testes cDNA phage library was Petition 870230039907, dated 12 / 05 / 2023, page 58 / 100 50 / 82 synthesized using the mRNA thus obtained (5 μg). For the preparation of the cDNA phage library, a cDNA synthesis kit, a “ZAPcDNA” synthesis kit, and a “ZAP-cDNA gigapack III gold cloning kit” (Stratagene) were used, and the library was prepared according to protocols attached to the kits. The size of the prepared cDNA phage library was 7.73 x 10⁵ pfu / ml. (2) Selection of the cDNA Library Using Serum

[00148] Immunoselection was performed using the canine testis cDNA phage library prepared above. Specifically, an Escherichia coli host (XL1-Blue MRF') was infected with the phage so that 2210 clones were present in a 0.90 x 15 mm NZY agarose plate. Cells were cultured at 42 °C for 3–4 hours to induce plaque formation. The plate was covered with a nitrocellulose membrane (Hybond C extra: GE Healthcare Bio-Science) impregnated with IPTG (isopropylβ-D-thiogalactoside) at 37 °C for 4 hours. Proteins were induced, expressed, and then transferred to the membrane. Subsequently, the membrane was retrieved, immersed, and agitated in TBS (10 mM Tris-HCl, 150 mM NaCl pH 7.5) containing 0.5% skimmed milk powder at 4 °C overnight, so that the non-specific reaction was suppressed. The filter was then allowed to react with dog serum diluted 500 times at room temperature for 2 to 3 hours.

[00149] As with the above serum from dogs with cancer, sera collected from dogs with breast cancer were used. The sera were stored at -80 °C and then subjected to pretreatment immediately before use. The pretreatment for the serum was performed by the following method. Specifically, the host Escherichia coli (XL1-blure MRF') was infected with the expressed phage “λ ZAP Express” within which no foreign genes were introduced, and then cultured on a plate with NZY medium at 37 °C for Petition 870230039907, dated 12 / 05 / 2023, page 59 / 100 51 / 82 at night. Subsequently, a 0.2 M NaHCO3 buffer (pH 8.3) containing 0.5 M NaCl was added to the plate, and then the plate was left to stand at 4 °C for 15 hours. The supernatants were collected as Escherichia coli / phage extracts. The collected Escherichia coli / phage extracts were then passed through an NHS column (GE Healthcare Bio-Science) to immobilize the Escherichia coli / phage-derived protein. Serum from a dog with cancer was passed through the column, to which the protein was immobilized by reaction, thus removing Escherichia coli and phage-adsorbed antibodies from the serum. Each serum fraction that passed through the column was diluted 500 times with TBS containing 0.5% skimmed milk powder, and the resulting solution was used as immunoselection material.

[00150] A membrane, to which the treated serum and the fusion protein mentioned above were subjected by blotting, was washed 4 times with TBS-T (0.05% Tween 20 / TBS). The membrane was reacted with goat anti-dog IgG antibody (goat anti-dog IgG-h+i conjugated with HRP: BETHYL Laboratories), diluted 5,000 times, as a secondary antibody with TBS containing 0.5% skimmed milk powder at room temperature for 1 hour. Detection was performed by enzymatic color reaction using an NBT / BCIP reaction solution (Roche). Colonies corresponding to the positive site of the color reaction were collected from 090 x 15 mm NZY agarose plates and then dissolved in 500 μl of SM buffer (100 mM NaCl, 10 mM MgClSO4, 50 mM Tris-HCl, 0.01% gelatin, pH 7.5). Until unification of colonies positive for the color reaction, secondary and tertiary selection were repeated using a method similar to that described above.Thus, 30,940 phage clones that reacted with serum IgG were screened so that 5 positive clones were isolated. (3) Homology Search for Isolated Antigen Gene

[00151] A procedure for converting phage vectors to Petition 870230039907, dated 12 / 05 / 2023, page 60 / 100 52 / 82 plasmid vectors were prepared for the 5 positive clones isolated by the method described above for the purpose of subjecting the clones to nucleotide sequence analysis. Specifically, 200 μL of an Escherichia coli host solution (XL1-Blue MRF') prepared to give an absorbance at OD600 of 1.0; 250 μL of a purified phage solution, and 1 μL of ExAssist helper phage (Stratagene) were mixed and allowed to react at 37 °C for 15 minutes. Then 3 ml of LB medium was added, the cells were cultured at 37 °C for 2.5 to 3 hours, and then the resulting culture was immediately placed in a water bath at 70 °C for a 20-minute incubation. Centrifugation was performed at 4 °C, 1000 x g for 15 minutes, and then the supernatant was collected as a phagomid solution.Subsequently, 200 μl of a solution prepared from the host phagomid Escherichia coli SOLR to give an absorbance at OD600 of 1.0; and 10 pL of the purified phage solution were mixed, followed by a 15-minute reaction at 37 °C. 50 μl of the resulting solution was plated onto LB agar medium containing ampicillin (at a final concentration of 50 μg / ml) and then cultured overnight at 37 °C. A single transformed SOLR colony was collected and then cultured on LB medium containing ampicillin (at a final concentration of 50 pg / ml) at 37 °C. After culture, plasmid DNA containing an insertion of interest was purified using a “QIAGEN plasmid Miniprep Kit” (QIAGEN).

[00152] The purified plasmid was subjected to whole-sequence analysis of the inserted segment using the primer walking method with primer T3 for SEQ ID NO: 31 and primer T7 for SEQ ID NO: 32. The gene sequences for SEQ ID NOs: 5, 7, 9, 11, and 13 were obtained through sequence analysis. Using the nucleotide sequences of the genes and their amino acid sequences (SEQ ID NOs: 6, 8, 10, 12, and 14), the BLAST homology research program Petition 870230039907, dated 12 / 05 / 2023, p. 61 / 100 BLAST 53 / 82 (http: / / www.ncbi.nlm.nih.gov / BLAST / ) was conducted to search for homology with known genes. As a result, it was revealed that all five genes obtained were genes encoding CAPRIN-1. Sequence identities among the five genes were 100% with respect to nucleotide sequence and 99% with respect to amino acid sequence in the regions to be translated into proteins. Sequence identities of these genes with homologous human coding genes were 94% with respect to nucleotide sequence and 98% with respect to amino acid sequence in the regions to be translated into proteins. The nucleotide sequences of the human homologs are represented by SEQ ID NOs: 1 and 3, and the amino acid sequences of the same are represented by SEQ ID NOs: 2 and 4.Furthermore, the sequence identities of the obtained canine genes with the genes encoding cattle homologs were 94% with respect to nucleotide sequence and 97% with respect to amino acid sequence in the regions that are translated into proteins. The nucleotide sequence of the cattle homolog is represented by SEQ ID NO: 15 and its amino acid sequence is represented by SEQ ID NO: 16. Additionally, the sequence identities of the human homolog coding genes with the cattle homolog coding genes were 94% with respect to nucleotide sequence and 97% with respect to amino acid sequence in the regions that are translated into proteins. Furthermore, the sequence identities of the obtained canine genes with the horse homolog coding genes were 93% with respect to nucleotide sequence and 97% with respect to amino acid sequence in the regions that are translated into proteins.The nucleotide sequence of the horse homolog is represented by SEQ ID NO: 17 and its amino acid sequence is represented by SEQ ID NO: 18. Furthermore, the sequence identities of genes encoding the human homolog with genes encoding the horse homolog were... Petition 870230039907, dated 12 / 05 / 2023, p. 62 / 100 54 / 82 93% with respect to nucleotide sequence and 97% with respect to amino acid sequence in the regions that are translated into proteins. Furthermore, the sequence identities of the obtained dog genes with the genes encoding mouse homologs were 87% to 89% with respect to nucleotide sequence and 95% to 97% with respect to amino acid sequence in the regions that are translated into proteins. The nucleotide sequences of the mouse homologs are represented by SEQ ID NOs: 19, 21, 23, 25, and 37; and their amino acid sequences are represented by the SEQ ID NOs: 20, 22, 24, 26, and 28. Furthermore, the sequence identities of the human homolog coding genes with the mouse homolog coding genes were 89% to 91% with respect to nucleotide sequence and 95% to 96% with respect to amino acid sequence in the regions that are translated into proteins.Furthermore, the sequence identities of the obtained dog genes with the genes encoding chicken homologs were 82% with respect to nucleotide sequence and 87% with respect to amino acid sequence in the regions that are translated into proteins. The nucleotide sequence of the chicken homolog is represented by SEQ ID NO: 29 and its amino acid sequence is represented by SEQ ID NO: 30. Additionally, the sequence identities of the genes encoding the human homolog with the genes encoding the chicken homolog were 81% to 82% with respect to nucleotide sequence; and 86% with respect to amino acid sequence in the regions that are translated into proteins. (4) Analysis of Gene Expression in Each Tissue

[00153] Gene expression obtained by the method described above was examined in normal dog and human tissues and in different cell lines by RT-PCR. Reverse transcription was performed Petition 870230039907, dated 12 / 05 / 2023, p. 63 / 100 55 / 82 as follows. Specifically, total RNA was extracted from 50 mg to 100 mg of tissue or 5 to 10 x 10⁶ cells of the cell line using a Trizol reagent (Invitrogen) according to the attached protocols. cDNA was synthesized from the total RNA using a “Superscript First-Strand Synthesis System” RT-PCR synthesis system (Invitrogen) according to the manufacturer's protocol. PCR was performed as follows using primers with SEQ ID NOs: 33 and 34 specific for the genes obtained. Specifically, the reagents and a monitoring buffer were added to 0.25 μL of the sample, prepared by reverse transcription reaction in a total volume of 25 μL, so that the resulting solution contained the above primers at 2 μM each, 0.2 mM dNTPs each, and 0.65U of ExTaq polymerase (Takara Shuzo Co., Ltd.).PCR was performed by repeating a cycle of 94 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds, for 30 cycles, using a Thermal Cycler (BIO RAD). The gene-specific primers above are capable of amplifying the region from nucleotides 206 to 632 in the nucleotide sequence SEQ ID NO: 5 (dog CAPRIN-1 gene) and the region from nucleotides 698 to 1124 in the nucleotide sequence SEQ ID NO: 1 (human CAPRIN-1 gene). As a control for comparison, primers specific for GAPDH of SEQ ID NOs: 35 and 36 were also used simultaneously. As a result, as shown in Fig. 1, strong expression was observed in the testicle among normal canine tissues, while expression was also observed in canine breast cancer and adenocarcinoma tissues. Furthermore, the expression of the human homolog from the obtained genes was also observed.As a result, similarly to the case of the canine CAPRIN-1 gene, expression could only be observed in the testicle among normal tissues. However, in the case of cancer cells, expression was detected in many types of cancer cell lines, including breast cancer cell lines and tumor cell lines. Petition 870230039907, dated 12 / 05 / 2023, page 64 / 100 56 / 82 brain, leukemia, lung cancer, and esophageal cancer. Expression was observed especially in many breast cancer cell lines. These results were confirmed by findings that, with the exception of testicular tissue, CAPRIN-1 expression is not observed in normal tissues, while CAPRIN-1 was expressed in cancer cells and, specifically, in many breast cancer cell lines.

[00154] In Fig. 1, reference number 1 on each vertical axis indicates the gene expression patterns identified above, and reference number 2 indicates the expression patterns of the GAPDH gene as a control.

[00155] (5) Preparation of Polyclonal Antibody Against Caprin-1 Derived Peptide.

[00156] To obtain an antibody that binds to CAPRIN-1, the CAPRIN-1-derived peptide represented by SEQ ID NO: 37 was synthesized. 1 mg of the peptide as antigen was mixed with an equivalent volume of an incomplete Freund's adjuvant (IFA) solution. The mixture was administered subcutaneously to rabbits 4 times every 2 weeks. Blood was then collected and antiserum containing polyclonal antibodies was obtained. Furthermore, the antiserum was purified using a G protein carrier (GE Healthcare Bio-Sciences) so that polyclonal antibodies against the CAPRIN-1-derived peptide were obtained. Additionally, serum from a rabbit that was not administered the antigen was purified using a G protein carrier in a similar manner, and the resulting serum was used as a control antibody. (6) Analysis of Antigen Protein Expression on Cancer Cells

[00157] Next, 7 breast cancer cell lines (MDA-MB-157, T47D, MRK-nu-1, MDA-MB-231V, BT20, SK-BR-3 and MDA-MB231T), in which the expression of the CAPRIN-1 gene was observed at levels Petition 870230039907, dated 12 / 05 / 2023, p. 65 / 100 57 / 82 elevated, were examined for CAPRIN-1 protein expression on cell surfaces. 106 cells from each human breast cancer cell line for gene expression that had been previously observed were centrifuged in a 1.5 ml microcentrifuge tube. 2 μg (5 μL) of polyclonal antibodies against the CAPRIN-1-derived peptide prepared in item (5) above were added to the tube. After suspension with 95 μL of PBS containing 0.1% fetal bovine serum, the cells were left to stand on ice for 1 hour. After washing with PBS, the resulting product was suspended in PBS containing 5 μL of FITC-labeled goat anti-rabbit IgG antibody (SantaCruz) and 95 μL of 0.1% fetal bovine serum (FBS), and then the result was left to stand on ice for 1 hour. After washing with PBS, the fluorescence intensity was measured using a FACScalibur (Becton, Dickinson & Company).Meanwhile, procedures similar to the above were performed using the control antibody prepared in item (5) above, instead of polyclonal antibodies against the CAPRIN-1 derived peptide so that a control sample was obtained. As a result, all cells in which the human anti-CAPRIN-1 antibody was added exhibited increased fluorescence intensity compared to the control. Specifically, the fluorescence intensity increased by 193% in the case of MDA-MB-231V and 169% in the case of 3-SK-BR. With these results it was revealed that the CAPRIN-1 protein was expressed on the cell membrane surfaces of the human cancer cell lines above. The percentage increase in fluorescence intensity above was expressed as a percentage of the increase in mean fluorescence intensity (MFI level) in each cell type and was calculated by the following formula.

[00158] Percentage increase in mean fluorescence intensity (percentage increase in fluorescence intensity) (%) = Petition 870230039907, dated 12 / 05 / 2023, page 66 / 100 58 / 82 ((MFI level in cells that reacted with the anti-human CAPRIN-1 antibody) (MFI level of the control)) / (MFI level of the control) x 100.

[00159] Using a technique similar to the one above, CAPRIN-1 expression was also analyzed by 3 renal cancer cell lines (Caki-1, Caki-2, and A498), one ovarian cancer cell line (SKOV3), one lung cancer cell line (QG56), one prostate cancer cell line (PC3), one cervical cancer cell line (Hela), one fibrosarcoma cell line (HT1080), 2 brain tumor cell lines (T98G and U87MG), two mouse colorectal cancer cell lines (CT26 and colon 26), one mouse breast cancer cell line (4T1), one mouse melanoma cell line (B16), and two mouse neuroblastoma cell lines (N1E-115 and Neuro2a). As a result, CAPRIN-1 expression was confirmed in all cell lines.Furthermore, similar results were obtained in the case of using the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody #1) comprising the variable heavy chain region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 47 or the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody #2) comprising the variable heavy chain region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 51, or the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody #3), comprising the variable heavy chain region of SEQ ID NO: 63, and the variable light chain region of SEQ ID NO: 67 (obtained in Example 3). (7) Immunohistochemical Staining (7)-1 Expression of CAPRIN-1 in Normal Mouse and Dog Tissues

[00160] Mice (Balb / c, females) and dogs (beagles, females) were exsanguinated under ether anesthesia and ketamine / isoflurane anesthesia. After laparotomy, each organ (stomach, liver, eyeball, thymus, muscle, bone marrow, uterus, small intestine, esophagus, heart, Petition 870230039907, dated 12 / 05 / 2023, page 67 / 100 59 / 82 kidney, salivary gland, large intestine, mammary gland, brain, lung, skin, adrenal gland, ovary, pancreas, spleen, and bladder) were transferred to a 10 cm plate containing PBS. Each organ was sectioned in PBS and then subjected to overnight fixation perfusion in 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde (PFA). The perfusion solution was discarded, the tissue surface of the organs was washed with PBS, a PBS solution containing 10% sucrose was added to a 50 ml centrifuge tube, each tissue was added to the tube, and then the tube was agitated using a rotor at 4 °C for 2 hours. The solution was replaced with a PBS solution containing 20% ​​sucrose, and then left to stand at 4 °C until the tissue sank. The solution was replaced with a PBS solution containing 30% sucrose and then left to stand at 4°C until the tissue sank.The tissue was removed, and then necessary portions were excised with a surgical scalpel. Next, an OCT (Tissue Tek) compound was added to the tissue to ensure complete application to the tissue surface, and then the tissue was placed in a cryomold. The cryomold was placed on dry ice for rapid freezing. Then, the tissue was cut into 10 μm to 20 μm sections using a cryostat (Leica). The sections were air-dried on glass slides using a hairdryer for 30 minutes to prepare the sectioned tissue mounted on a glass slide. Each sample was then placed in a staining bottle filled with PBS-T (saline solution containing 0.05% Tween 20) and the PBS-T was replaced three times every 5 minutes. Excess water around the cuts was removed with Kimwipes, and then the cuts were circled using a “DAKOPEN” (DAKO) pen.As blocking solutions, a mouse Ig blocking reagent MOM (Vectastain) and a 10% SBF-containing PBS-T solution were placed on slides over mouse and dog tissue, respectively. Petition 870230039907, dated 12 / 05 / 2023, p. 68 / 100 60 / 82 and then left to stand in a humid chamber at room temperature for 1 hour. Then, a solution of polyclonal antibodies (reactive with cancer cell surfaces, prepared according to item (5) above), against the CAPRIN-1 derived peptide (SEQ ID NO: 37), at 10 μg / ml adjusted with a blocking solution was placed on the slides and then left to stand overnight in a humid chamber at 4 °C. 10-minute washes with PBS-T were performed 3 times, and then a biotin-labeled anti-IgG MOM antibody (Vectastain) diluted 250 times with the blocking solution was placed, and then the slides were incubated at room temperature for 1 hour in a humid chamber. After ten (10) minutes of washing with PBS-T 3 times, an avidin-biotin ABC reagent (Vectastain) was placed on the slides, and then the sample was left to stand in a humid chamber at room temperature for 5 minutes.After ten (10) minutes of washing with PBS-T three times, a DAB staining solution (10 mg DAB + 30% H2O2 10 μl / 0.05 M Tris-HCl (pH 7.6) 50 ml) was added, and then the sample was left to stand in a humid chamber at room temperature for 30 minutes. After washing with distilled water, a hematoxylin reagent (DAKO) was placed on the sample and the sample was left to stand at room temperature for 1 minute and then washed with distilled water. The glass slide was immersed in ethanol solutions at 70%, 80%, 90%, 95%, and finally, 100%, for 1 minute each, then the slides were left to stand overnight in xylene. The glass slides were removed, sealed with Glycergel Mounting Medium (DAKO), and then analyzed.As a result, caprin-1 expression was observed slightly within cells of each tissue from salivary glands, kidney, colon, and stomach, but no expression was observed on cell surfaces. Furthermore, no expression was observed in tissues from other organs. In addition, [further information was obtained]. Petition 870230039907, dated 12 / 05 / 2023, page 69 / 100 61 / 82 similar results in the case of using the anti-CAPRIN1 monoclonal antibody (monoclonal antibody #1) comprising the variable heavy chain region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 47, the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody #2) comprising the variable heavy chain region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 51, or the anti-CAPRIN-1 monoclonal antibody (monoclonal antibody #3), comprising the variable heavy chain region of SEQ ID NO: 63, and the variable light chain region of SEQ ID NO: 67 (obtained in Example 3). (7)-2 Expression of CAPRIN-1 in Canine Breast Cancer Tissue.

[00161] Slides with frozen sections were prepared by a method similar to that described above using 108 frozen breast cancer specimens from dogs pathologically diagnosed as having malignant breast cancer, and immunohistochemical staining was performed using the polyclonal antibody against the CAPRIN-1 derived peptide (SEQ ID NO: 37) prepared in item (5) above. As a result, CAPRIN-1 expression was observed in 100 of the 108 samples (92.5%) and CAPRIN-1 was strongly expressed on the surface of cancer cells with a particularly high degree of atypicality. Furthermore, similar results were obtained when using monoclonal antibody #1, #2 or #3 obtained in Example 3. (7)-3 Expression of CAPRIN-1 in Human Breast Cancer Tissue

[00162] Immunohistochemical staining was performed using 188 human breast cancer tissue specimens in a paraffin-embedded human breast cancer tissue array (BIOMAX). After 3 hours of treatment of the breast cancer tissue array at 60 °C, the array was placed in a xylene staining bottle, followed by xylene replacement three times each time. Petition 870230039907, dated 12 / 05 / 2023, page 70 / 100 62 / 82 minutes. Subsequently, a similar process was performed with ethanol and PBST instead of xylene. The human breast cancer tissue matrix was placed in a staining bottle with 10 mM citrate buffer (pH 6.0) containing 0.05% Tween 20. After 5 minutes of treatment at 125 °C, the matrix was left to stand at room temperature for 40 minutes or more. Excess water around the sections was removed with Kimwipes, and the sections were circled with a DAKOPEN pen, and an endogenous peroxidase blocker (DAKO) was added dropwise in appropriate amounts. The tissue samples in matrix were then left to stand at room temperature for 5 minutes, then placed in a staining bottle with PBS-T, followed by replacement of the PBS-T three times every 5 minutes.As a blocking solution, a 10% SBF-containing PBS-T solution was placed on the matrix, and then the matrix was left to stand in a humid chamber at room temperature for 1 hour. Then, a solution of the polyclonal antibody against the 1-CAPRIN-derived peptide (SEQ ID NO: 37) prepared according to item (5) above, having a concentration of 10 μg / ml adjusted with a 5% SBF-containing PBS-T solution was placed on the slides and the tissue matrix was incubated overnight in a humid chamber at 4 °C. After ten (10) minutes of washing with PBS-T 3 times, a peroxidase-labeled polymer conjugate “Peroxidase Labeled Polymer Conjugate (DAKO)” was added dropwise onto the slides in appropriate amounts, and then the tissue matrix arrangement was left to stand in a humid chamber at room temperature for 30 minutes.After ten (10) minutes following 3 washes with PBS-T, a DAB (DAKO) staining solution was placed on the slides and then incubated at room temperature for approximately 10 minutes. The staining solution was discarded, and 3 washes of 10 minutes with PBS-T were performed, followed by washes with distilled water. The tissue arrangement. Petition 870230039907, dated 12 / 05 / 2023, page 71 / 100 63 / 82 specimens in a matrix were immersed in ethanol solutions at 70%, 80%, 90%, 95%, and finally, 100%, for 1 minute each, then left to stand overnight in xylene. The glass slides were removed, sealed with Glycergel Mounting Medium (DAKO), and then analyzed. As a result, strong expression of CAPRIN-1 was observed in a total of 138 of the 188 breast cancer tissue specimens (73%). Furthermore, similar results were obtained when using monoclonal antibodies #1, #2, or #3 obtained in Example 3. (7)-4 Expression of CAPRIN-1 in Human Malignant Brain Tumor

[00163] Immunohistochemical staining was performed according to a method similar to that used in item (7)-3 above with 247 malignant brain tumor tissue specimens in a paraffin-embedded human malignant brain tumor tissue array (BIOMAX), using the polyclonal antibody against the CAPRIN-1 derived peptide (SEQ ID NO: 37) prepared in item (5) above. As a result, strong CAPRIN-1 expression was observed in a total of 227 of the 247 human malignant brain tumor tissue specimens (92%). Furthermore, similar results were obtained when using monoclonal antibody #1, #2 or #3 obtained in Example 3. (7)-5 Expression of CAPRIN-1 in Lymph Node with Metastasis of Human Breast Cancer

[00164] Immunohistochemical staining was performed according to a method similar to that described in item (7)-3 above, with 150 metastatic breast cancer lymph node tissue specimens embedded in a paraffin-embedded human breast cancer metastatic lymph node tissue microarray (BIOMAX), using the polyclonal antibody against the CAPRIN-1 derived peptide (SEQ ID NO: 37) prepared in item (5) above. As a result, strong expression of Petition 870230039907, dated 12 / 05 / 2023, page 72 / 100 64 / 82 CAPRIN-1 was found in 136 out of a total of 150 lymph node tissue specimens with breast cancer metastasis (90%). Specifically, CAPRIN1 was also found to be strongly expressed in cancer tissues that had breast cancer metastases. Furthermore, similar results were obtained when using monoclonal antibodies #1, #2, or #3 obtained in Example 3. (7)-6 Caprin-1 Expression in Various Human Cancer Tissues

[00165] Immunohistochemical staining was performed according to a method similar to the previous one, with specimens in different tissue arrays embedded in human cancer tissue paraffin (BIOMAX), using the polyclonal antibody against the CAPRIN-1 derived peptide (SEQ ID NO: 37) prepared in item (5) above. As a result, strong CAPRIN-1 expression was observed in esophageal cancer, colon cancer, rectal cancer, lung cancer, renal cancer, bladder cancer, and cervical cancer. Furthermore, similar results were obtained when using monoclonal antibody #1, #2, or #3 obtained in Example 3. Example 2 Preparation of Human CAPRIN-1 (1) Recombinant Protein Preparation

[00166] Based on the SEQ ID NO: 1 gene obtained in Example 1, a recombinant human CAPRIN-1 protein was prepared using the following method. A PCR was performed on a total volume of 50 μL, with 1 μL of cDNA, two primers (SEQ ID NOs: 38 and 39, comprising the sequences for cleavage by the restriction enzymes SacI and XhoI) of 0.4 μM each, 0.2 mM dNTP, and 1.25 U of PrimeStar HS polymerase (Takara Shuzo Co., Ltd.), prepared by adding the reagents and a monitoring buffer. Expression was confirmed by an RT-PCR method for the cDNA used from among several cDNAs derived from human tissues or cells prepared in Example 1. The PCR was performed by repeating thermocycling at 98 °C for 10 seconds. Petition 870230039907, dated 12 / 05 / 2023, page 73 / 100 65 / 82 and 68 °C for 2.5 minutes for 30 cycles using a Thermal Cycler (BIO RAD). The two primers above are capable of amplifying a coding region of the entire amino acid sequence of SEQ ID NO: 2. After PCR, the amplified DNA was subjected to electrophoresis on a 1% agarose gel, then a DNA fragment of approximately 2.1 kbp was purified using a “QIAquick Gel Extraction Kit” (QIAGEN).

[00167] The purified DNA fragment was ligated into a PCR-Blunt cloning vector (Invitrogen). After transformation of Escherichia coli with it, the plasmid was collected. Sequencing of the fragment confirmed that the amplified gene possesses the sequence of interest. The plasmid with a sequence corresponding to the sequence of interest was treated with the restriction enzymes SacI and XhoI and then purified using a QIAquick gel extraction kit. The sequence of the gene of interest was introduced into an Escherichia coli pET30a expression vector (Novagen) and treated with the restriction enzymes SacI and XhoI. The recombinant protein fused to the His-tag marker could be produced using the vector. The plasmid was transformed into E. coli for recombinant expression, BL21(DE3), and then expression was induced with 1 mM IPTG, so that the protein of interest was expressed in Escherichia coli. (2) Recombinant Protein Purification

[00168] The recombinant Escherichia coli obtained above, expressing the SEQ ID NO:1 gene, was cultured in LB medium containing 30 μg / ml kanamycin at 37 °C until the absorbance at 600 nm reached approximately 0.7. Isopropyl-e-D-thiogalactopyranoside was added to a final concentration of 1 mM, and the cells were then cultured at 37 °C for 4 hours. Subsequently, centrifugation was performed at 4800 rpm for 10 minutes, after which the cells were collected. The resulting cell precipitate was suspended in phosphate-buffered saline and centrifuged at 4800 rpm. Petition 870230039907, dated 12 / 05 / 2023, page 74 / 100 66 / 82 rpm for 10 minutes, then the cells were washed.

[00169] The cells were suspended in phosphate-buffered saline solution and then disrupted using ultrasound on ice. The resulting lysate of ultrasonically treated Escherichia coli was centrifuged at 6000 rpm for 20 minutes, and then the resulting supernatant was considered the soluble fraction and the precipitate was considered the insoluble fraction.

[00170] The soluble fraction was added to a nickel chelate column set up according to a conventional method (carrier: “Chelating Sepharose® Fast Flow” resin (GE Healthcare), 5 ml column capacity, and an equilibrium buffer: 50 mM hydrochloride buffer (pH 8.0)). The unadsorbed fractions were washed with 50 mM hydrochloride buffer (pH 8.0) in an amount 10 times the column capacity and 20 mM phosphate buffer (pH 8.0) containing 20 mM imidazole. Immediately after washing, 6 beds were eluted with 20 mM phosphate buffer (pH 8.0) containing 100 mM imidazole.Elution of the protein of interest was confirmed by Coomassie staining of the elution fraction with 20 mM phosphate buffer (pH 8.0) containing 100 mM imidazole. The elution fraction was then added to a strong anion exchange column (carrier: “Q Sepharose® Fast Flow” resin (GE Healthcare), 5 mL column capacity, and 20 mM phosphate buffer (pH 8.0) as equilibration buffer). An unadsorbed fraction was washed with 20 mM phosphate buffer (pH 7.0) in an amount 10 times the column capacity and 20 mM phosphate buffer (pH 7.0) containing 200 mM sodium chloride. Immediately after washing, 5 beds were eluted with 20 mM phosphate buffer (pH 7.0) containing 400 mM sodium chloride, thus obtaining the purified protein fraction with the amino acid sequence represented by SEQ ID NO: 2.

[00171] 200 μL of each purified sample obtained by the method Petition 870230039907, dated 12 / 05 / 2023, page 75 / 100 The 67 / 82 sample described above was distributed in 1 ml of reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2, pH 7.4), followed by the addition of 2 μL of enterokinase (Novagen). The resulting mixture was then left to stand overnight at room temperature to allow for His-tag cleaving, and purification was performed using an Enterokinase Cleavage Capture Kit (Novagen), according to the manufacturer's protocols. Subsequently, 1.2 ml of the purified sample obtained by the method described above was subjected to buffer replacement with physiological phosphate buffer (Nissui Pharmaceutical Co., Ltd.) using NANOSEP 10K OMEGA ultrafiltration (PALL). Additionally, sterile filtration was performed using a 0.22 μm HT Tuffryn Acrodisc (PALL), and the resulting material was then used in the following experiment. Example 3 Preparation of Mouse Monoclonal Antibody Against CAPRIN-1

[00172] 100 μg of the antigen protein (human CAPRIN-1) comprising the amino acid sequence SEQ ID NO: 2 prepared in Example 2 was mixed with an equivalent amount of MPL+TDM adjuvant (Sigma), and this was then used as an antigen solution for a mouse. The antigen solution was administered intraperitoneally to 6-week-old Balb / cc mice (Japan SLC Inc.), then administered 7 times each week, thus completing the immunization. Each spleen was excised 3 times after the final immunization, placed between two sterile glass slides, and then the spleens were crushed. The resulting product was washed with PBS (-) (Nissui) and then centrifuged at 1500 rpm for 10 minutes to remove the supernatant. This procedure was repeated 3 times so that splenocytes were obtained. The splenocytes thus obtained and the SP2 / 0 mouse myeloma cells (acquired from ATCC) were mixed in a Petition 870230039907, dated 12 / 05 / 2023, page 76 / 100 68 / 82 ratio of 10:1. The PEG solution prepared by mixing 200 μL of RPMI 1640 medium containing 10% SBF heated to 37 °C and 800 μL of PEG1500 (Boehringer) was added to the mixture, left to stand for 5 minutes for cell fusion, and then centrifuged at 1700 rpm for 5 minutes. After removing the supernatant, the cells were suspended in 150 ml of RPMI1640 medium containing 15% SBF, supplemented with a HAT solution (Gibco) (2% equivalents) (selective HAT medium), then the cell suspension was plated in fifteen 96-well plates (Nunc) at 100 μL per well. The cells were cultured for 7 days at 37 °C under 5% CO2 conditions, so that hybridomas prepared by the fusion of splenocytes and myeloma cells were obtained.

[00173] Hybridomas were selected using an antibody binding affinity marker produced by hybridomas prepared for human CAPRIN-1. The CAPRIN-1 protein solution (1 μg / ml) prepared in Example 2 was added to a 96-well plate with 100 μg per well and then incubated at 4 °C for 18 hours. Each well was washed 3 times with PBS-T, 400 μg of a 0.5% Bovine Serum Albumin (BSA) solution (Sigma) were added to each well, then the plate was incubated at room temperature for 3 hours. The solution was removed and then the wells were washed three times with 400 μg of PBS-T per well. The supernatant from the hybridoma cultures obtained above was added at a volume of 100 μL per well, and then the plates were left to stand at room temperature for 2 hours.After washing each well three times with PBS-T, an HRP-labeled anti-IgG (H+L) mouse antibody (Invitrogen) diluted 5000 times with PBS was added at 100 μL per well, and the resulting mixture was left to stand at room temperature for 1 hour. After washing the well three times with PBS-T, 100 μL of a TMB substrate solution (Thermo) was added. Petition 870230039907, dated 12 / 05 / 2023, page 77 / 100 69 / 82 added to each well, and then the plate was left to stand for 15 to 30 minutes for the staining reaction. After color development, 100 μL of 1N sulfuric acid was added to each well to stop the reaction. Then, the absorbances at 450 nm and 595 nm were measured using an absorption spectrometer. As a result, hybridomas that produce antibodies with high absorbance values ​​were selected.

[00174] The hybridomas thus selected were added to a 96-well plate at 0.5 cells per well and then cultured. After 1 week, hybridomas that formed isolated colonies in the wells were observed. These cells in the culture wells were cultured for a longer time, then the hybridomas were selected using as a marker the binding affinity of antibodies produced by the cloned hybridomas for human CAPRIN-1. The CAPRIN-1 protein solution (1 μg / ml) prepared in Example 3 was added to a 96-well plate with 100 μL per well, and then left to stand at 4 °C for 18 hours. Each well was washed three times with PBS-T, 400 μL of a 0.5% BSA solution was added to each well, then the plate was left to stand at room temperature for 3 hours. The solution was removed, and then the wells were flushed three times with 400 μL of PBS-T per well.100 μL of each culture supernatant from the hybridomas obtained above were added per well, and then the plate was left to stand at room temperature for 2 hours. After washing each well three times with PBS-T, 100 μL of an HRP-labeled anti-mouse IgG (H+L) antibody (Invitrogen) diluted 5,000 times with PBS was added per well and then left to stand for 1 hour at room temperature. After washing the well three times with PBS-T, 100 μL of a TMB substrate solution (Thermo) was added to each well, and then the plate was left to stand for 15 to 30 minutes for the staining reaction. After color development, 100 μL of sulfuric acid was added. Petition 870230039907, dated 12 / 05 / 2023, page 78 / 100 70 / 82 1N was added to each well to stop the reaction, then the absorbances at 450 nm and 595 nm were measured using an absorption spectrometer. As a result, 50 hybridoma cell lines producing monoclonal antibodies immunoreactive with human CAPRIN1 were obtained.

[00175] Next, from these monoclonal antibodies, antibodies reactive with the cell surfaces of human CAPRIN-1-expressing breast cancer cells were selected. Specifically, 106 cells of the MDA-MB-231V human breast cancer cell line were centrifuged in a 1.5 ml microcentrifuge tube, and 100 μL of culture supernatant from each of the above hybridomas was added to the tube, then the tube was left to stand on ice for 1 hour. After washing with PBS, a FITC-labeled goat anti-mouse IgG (H+L) antibody (Invitrogen) diluted 500-fold with PBS containing 0.1% SBF was added, and then left to stand on ice for 1 hour. After washing with PBS, the fluorescence intensity was measured using a FACScalibur (Becton, Dickinson & Company).Meanwhile, procedures similar to those mentioned above were performed using serum from a 6-week-old Balb / c mouse that had not been treated with antibodies and which was diluted 500 times with hybridoma culture medium, so that a control sample was obtained. As a result, three monoclonal antibodies (monoclonal antibodies #1, #2 and #3) were selected that exhibited stronger fluorescence intensity than the control, and that reacted with the cell surfaces of breast cancer cells. Example 4 Characterization of Selected Antibodies (1) Cloning of Genes from Variable Regions of the Mouse Monoclonal Antibody Anti-CAPRIN-1 Human

[00176] mRNA was extracted from each cell line of Petition 870230039907, dated 12 / 05 / 2023, page 79 / 100 71 / 82 hybridoma that produces any of the three monoclonal antibodies selected in Example 3. An RT-PCR method using primers specific for the mouse FR1-derived sequence and mouse FR4-derived sequence was performed for the same, and the genes of the variable heavy chain (VH) and variable light chain (VL) regions of all anti-CAPRIN-1 monoclonal antibodies were obtained. For sequence determination, these genes were cloned into a pCR2.1 vector (Invitrogen). (2) RT-PCR

[00177] mRNA was prepared from 106 cells of each hybridoma cell line using an mRNA micro purification kit (GE HealthCare). The mRNA thus obtained was reverse transcribed and then cDNA was synthesized using a SuperScriptII 1st strand Synthesis Kit (Invitrogen). These procedures were performed according to protocols attached to each kit.

[00178] Antibody gene amplification was performed by a PCR method using the obtained cDNA.

[00179] To obtain the gene from the VH region, a primer (SEQ ID No: 54) specific for the mouse heavy chain FR1 sequence and a primer (SEQ ID No: 55) specific for the mouse heavy chain FR4 sequence were used. Additionally, to obtain the gene from the VL region, a primer (SEQ ID No: 56) specific for the mouse light chain FR1 sequence and a primer (SEQ ID No: 57) specific for the mouse light chain FR4 sequence were used. These primers were designed with reference to Jones ST and Bending MM Bio / technology 9, 88-89 (1991). Ex-Taq (Takara Bio Inc.) was used for PCR. A cDNA sample was added to 5 μL of 10x Ex-Taq buffer, 4 μL of dNTP mixture (2.5 mM), primers (1.0 μM) (2 μL each), and 0.25 μL of Ex-Taq (5 U / μL), then... Petition 870230039907, dated 12 / 05 / 2023, p. 80 / 100 72 / 82 The total amount of the PCR mixture was adjusted with sterile water to 50 μL. PCR was performed under the following conditions: 2 minutes of treatment at 94 °C, followed by 30 cycles of 1 minute of denaturation at 94 °C, 30 seconds of annealing at 58 °C, and 1 minute of extension reaction at 72 °C. (3) Cloning

[00180] The PCR products thus obtained were subjected to agarose gel electrophoresis, and the DNA bands from the VH and VL regions were excised. The DNA fragments were purified using a “QIAquick Gel purification kit” (QIAGEN) according to the manufacturer's protocol. The purified DNA was cloned into the pCR2.1 vector using the TA cloning kit (Invitrogen). The ligated vector was transformed into competent DH5a cells (TOYOBO) according to a conventional method. 10 clones of each transformant were cultured overnight in medium (100 μg / ml ampicillin) at 37 °C, then the plasmid DNA was purified using a “Qiaspin Miniprep kit” (QIAGEN). (4) Determining the Sequence

[00181] The gene sequences of the VH and VL regions in each plasmid obtained above were analyzed with a forward M13 primer (SEQ ID NO: 58) and a reverse M13 primer (SEQ ID NO: 59) on a fluorescence sequencer (DNA Sequencer 3130XL; ABI), using a “Big Dye Terminator Cycle Sequencing Kit” Ver3.1 (ABI) according to the manufacturer's protocols. As a result, each gene sequence was determined. The sequences were identical among the 10 clones.

[00182] The gene sequences encoding the variable heavy chain regions of the monoclonal antibody obtained are presented by SEQ ID NOS: 52 and 68 and the amino acid sequences of the same are presented by SEQ ID NOS: 43 and 63, and the gene sequences encoding the variable light chain regions are presented by SEQ ID NOS: 70, 53 and 69, and the Petition 870230039907, dated 12 / 05 / 2023, page 81 / 100 73 / 82 amino acid sequences of the same are presented by SEQ IDs NOS: 47, 51 and 67. Specifically, it was revealed that monoclonal antibody #1 comprises the variable heavy chain region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 47; and antibody #2 comprises the variable heavy chain region of SEQ ID NO: 43 and the variable light chain region of SEQ ID NO: 51; and antibody #3 comprises the variable heavy chain region of SEQ ID NO: 63 and the variable light chain region of SEQ ID NO: 67. Example 5 Identification of the Caprin-1 Epitope to be Recognized by Anti-Caprin-1 Monoclonal Antibodies #1, #2 and #3

[00183] The epitope regions of CAPRIN-1 recognized by anti-CAPRIN-1 monoclonal antibodies #1, #2, and #3 (obtained in Example 3) reactive with cancer cell surfaces were identified.

[00184] 93 candidate peptides, each comprising 12 to 16 amino acids in the amino acid sequence of the human CAPRIN-1 protein, were synthesized, and then each peptide was dissolved in DMSO at a concentration of 1 mg / ml. Each peptide was dissolved in 0.1 M sodium carbonate buffer (pH 9.6) at a concentration of 30 μg / ml, added to a 96-well plate (Nunc, Product No. 436006) at 100 μL per well, then the plate was left to stand at 4 °C overnight. The solution was discarded, 10 mM ethanolamine / 0.1 M sodium carbonate buffer (pH 9.6) was added in a volume of 200 μL per well, then the resulting solution was left to stand at room temperature for 1 hour. The solution was then discarded and the plate was washed twice with PBS containing 0.5% Tween-20 (PBST), so that a plate on which each peptide was immobilized was prepared.

[00185] The cell culture supernatant containing the mouse monoclonal antibodies (#1, #2 and #3) obtained in Example 3 was Petition 870230039907, dated 12 / 05 / 2023, p. 82 / 100 74 / 82 added in a volume of 50 μL per well, then the plate was shaken at room temperature for 1 hour. The solution was removed, followed by three washes with PBST. Then, a secondary antibody solution prepared with an HRP-labeled anti-mouse IgG antibody (Invitrogen) diluted 3,000 to 4,000 times with PBST was added (50 μL each) to the mouse monoclonal antibodies. The solution was removed, followed by six washes with PBST.

[00186] The TMB (Thermo) substrate solution was added in a volume of 100 μL per well and then allowed to stand for 15 to 30 minutes for the coloration reaction. After color development, 100 μL of 1N sulfonic acid was added to each well to stop the reaction, then the absorbances at 450 nm and 595 nm were measured using an absorption spectrometer. As a result, a polypeptide comprising the amino acid sequence of SEQ ID NO: 37 was identified as a partial sequence of CAPRIN-1 recognized by both anti-CAPRIN-1 monoclonal antibodies #1 and #2.

[00187] Thus, it was revealed that the polypeptide of SEQ ID NO: 37, a partial sequence of human CAPRIN01, contains the epitope regions for the anti-CAPRIN-1 monoclonal antibodies #1, #2 and #3. Example 6 Expression of CAPRIN-1 on the Surface of Various Cancer Cells Using Anti-CAPRIN-1 Antibodies #1, #2, and #3

[00188] Next, 7 breast cancer cell lines (MDA-MB-157, T47D, MRK-nu-1, MDA-MB-231V, BT20, SK-BR-3, and DA-MB231T) in which CAPRIN-1 gene expression was observed, and the other three remaining breast cancer cell lines (MDA-MB-231c, MCF-7, and ZR75-1), 5 glioma cell lines (T98G, SNB19, U251, U87MG, and U373), 4 renal cancer cell lines (Caki-1, Caki-2, A498, and ACHN), 2 Petition 870230039907, dated 12 / 05 / 2023, page 83 / 100 75 / 82 gastric cancer cell lines (MKN28 and MKN45), 5 colorectal cancer cell lines (HT29, LoVo, Caco2, SW480, and HCT116), 3 lung cancer cell lines (A549, QG56, and PC8), 4 leukemia cell lines (AML5, Namalwa, BDCM, RPI1788), 1 cervical cancer cell line (SW756), 1 bladder cancer cell line (T24), 1 esophageal cancer cell line (KYSE180), and 1 lymphoma cell line (Ramos) were examined for CAPRIN1 protein expression on the cell surfaces of each cell line using culture supernatants containing #1, #2, and #3 obtained in Example 3. 106 cells from each cell line were centrifuged in a tube of 1.5 ml microcentrifuge. Each cell culture supernatant (100 μE) containing #1, #2, or #3 was added and then incubated on ice for 1 hour.After washing with PBS, a FITC-labeled goat anti-mouse IgG (H+L) antibody (SouthernBiotech) diluted 500 times with PBS containing 0.1% SBF was added, and the resulting solution was then left to stand on ice for 1 hour. After washing with PBS, the fluorescence intensity was measured using a FACS Calibur (Becton, Dickinson & Company). Meanwhile, a sample subjected to a reaction with only a secondary antibody was used as a negative control. As a result, the cells to which antibodies #1, #2, and #3 were added exhibited fluorescence intensity 20% or more stronger compared to the negative control. These results revealed that the CAPRIN-1 protein was expressed on the cell membrane surfaces of the above human cancer cell lines.The percentage increase in fluorescence intensity above was expressed as a percentage of the increase in mean fluorescence intensity (MFI level) in each cell type and was calculated using the following formula.

[00189] Percentage increase in average intensity of Petition 870230039907, dated 12 / 05 / 2023, page 84 / 100 76 / 82 fluorescence (percentage increase in fluorescence intensity) (%) = ((MFI level in cells that reacted with the anti-human CAPRIN-1 antibody) (MFI level of the control)) / (MFI level of the control) x 100. Example 7 Antitumor Effects (ADCC Activity and CDC Activity) of Anti-CAPRIN-1 Antibodies on Cancer Cells.

[00190] It was assessed whether or not anti-CAPRIN-1 antibodies can damage cancer cells expressing CAPRIN-1 primarily by measuring ADCC activity. The assessment was performed using a polyclonal antibody against the human CAPRIN-1 derived peptide (SEQ ID NO: 37) prepared in Example 1. 106 cells of the MDA-MB-157 human breast cancer cell line, in which CAPRIN-1 expression was confirmed, were collected in a 50 ml centrifuge tube, 100 μCi of chromium-51 was added, and then incubated at 37 °C for 2 hours. The resulting medium was then washed three times with RPMI1640 medium containing 10% fetal bovine serum. The cells were added to a 96-well V-bottom plate at a density of 103 cells per well.1 μg of the polyclonal antibody against the human CAPRIN-1 derived peptide mentioned above was added to the wells, and then lymphocytes (2 x 10⁵ each) separated from rabbit peripheral blood were added and cultured for 4 hours at 37 °C under 5% CO₂ conditions. After culture, the amount of chromium (Cr)-51 released from the damaged cancer cells in the culture supernatant was measured, so that the ADCC activity of the polyclonal antibodies against the human CAPRIN-1 derived peptide against the cancer cells was calculated. As a result, an ADCC activity of 18.1% was observed against the MDA-MB-157 cell line (see Fig. 2). On the other hand, when similar procedures were performed using a control antibody (Example 1 (5)) prepared from peripheral blood of a... Petition 870230039907, dated 12 / 05 / 2023, page 85 / 100 77 / 82 rabbits not immunized with the antigen, and when the antibody was not added, almost no activity was observed (see Fig. 2). Thus, it was revealed that the anti-CAPRIN-1 antibody can damage cancer cells that express CAPRIN-1 through ADCC activity.

[00191] In the present case, cytotoxic activity was determined as cytotoxic activity against a cancer cell line. Specifically, as described above, the result was obtained by mixing an anti-CAPRIN-1 antibody to be used in the present invention, a rabbit lymphocyte, and 103 cells of each cancer cell line that had incorporated chromium-51, culturing the cells for 4 hours, measuring the amount of chromium-51 released into the culture medium, and then calculating the cytotoxic activity against the cancer cell line using the following formula*.

[00192] * Formula: cytotoxic activity (%) = (amount of chromium-51 released from cancer cells after the addition of an anti-CAPRIN-1 antibody and rabbit lymphocytes) / (amount of chromium-51 released from target cells to which 1N hydrochloric acid was added) x 100.

[00193] Next, mouse anti-CAPRIN-1 monoclonal antibodies #1, #2, and #3 (obtained in Example 3) were evaluated for their cytotoxic activity against cancer cells. Each supernatant from the antibody-producing cell culture #1, #2, or #3 was purified using a “Hitrap ProteinA Sepharose FF” column (GE Healthcare), subjected to buffer replacement with PBS(-), and then filtered with a 0.22 μm filter (Millipore). The resulting supernatants were used as antibodies for activity measurement. 106 cells of the MDA-MB-157 human breast cancer cell line were collected in a 50 ml centrifuge tube, 100 μCi of chromium-51 was added, and then incubated at 37 °C for 2 hours. Subsequently, the resulting product was washed three times with medium Petition 870230039907, dated 12 / 05 / 2023, page 86 / 100 78 / 82 RPMI 1640 containing 10% SBF. Cells were added to a 96-well V-bottom plate at a density of 10³ cells per well for use as target cells. The purified antibodies described above (1 μg each) were added to the cells. 5 x 10⁴ mouse spleen cells isolated from the spleen of a 6-week-old BALB / C mouse (Japan SLC Inc.) according to a conventional method were additionally added and then cultured for 4 hours at 37 °C under 5% CO₂ conditions. After culture, the amount of chromium-51 released from damaged tumor cells in a culture supernatant was measured, and the cytotoxic activity of each anti-CAPRIN-1 antibody against cancer cells was calculated. As negative control samples, a sample prepared by adding PBS instead of antibodies and a sample prepared by adding an isotypic control antibody instead of antibodies were used.As a result, antibodies #1, #2, and #3 exhibited more than 25% cytotoxic activity against the MDA-MB-157 cell line. In contrast, the activity of the negative control sample prepared by adding PBS and the activity in the negative control sample prepared by adding isotypic control antibody were 1.7% and 3.0%, respectively. Similarly, antibodies #1, #2, and #3 were evaluated for their cytotoxic activities against other cancer cells, including U373 and T98G glioma cell lines, A549 and QG56 lung cancer cell lines, Caki-1 and ACHN renal cancer cell lines, SW756 cervical cancer cell line, T24 bladder cancer cell line, KYSE180 esophageal cancer cell line, MKN28 and MKN45 gastric cancer cell lines, SW480 colorectal cancer cell line, AML5 leukemia cell line, and a Ramos lymphoma cell line.As a result, antibody #1 exhibited 13.2% activity against T98G (6.4% in the isotypic control), 16.0% against U373 (4.3% in the isotypic control), and 12.0% against... Petition 870230039907, dated 12 / 05 / 2023, page 87 / 100 79 / 82 A549 (4.5% in the case of isotypic control), 12.6% against QG56 (5.3% in the case of isotypic control), 10.3% against Caki-1 (4.5% in the case of isotypic control), 9.0% against ACHN (3.8% in the case of isotypic control), 8.6% against SW756 (5.1% in the case of isotypic control), 13.0% against T24 (3.8% in the case of isotypic control), 8.9% against KYSE180 (5.7% in the case of isotypic control), 16.2% against MKN28 (4.2% in the case of isotypic control), 12.1% against MKN45 (4.6% in the case of isotypic control), 13.4% against SW480 (6.4% in the case of isotypic control), 8.9% against AML5 (4.7% in the isotypic control), and 8.1% against Ramos (2.5% in the isotypic control). Furthermore, antibodies #2 and #3 showed similar results. The above results demonstrate that the anti-CAPRIN-1 antibodies #1, #2, and #3 obtained cause damage to cancer cells expressing CAPRIN-1 through ADCC activity.The results above have shown that the mouse antiCAPRIN-1 monoclonal antibodies #1, #2, and #3 thus obtained cause damage to cancer cells expressing CAPRIN-1 through ADCC activity.

[00194] In the present case, cytotoxic activity was determined as cytotoxic activity against a cancer cell line. Specifically, as described above, the result was obtained by mixing an anti-CAPRIN-1 antibody to be used in the present invention, a mouse splenocyte, and 103 cells of each cancer cell line that had incorporated chromium-51, culturing the cells for 4 hours, measuring the amount of chromium-51 released into the culture medium, and then calculating the cytotoxic activity against the cancer cell line using the following formula*.

[00195] * Formula: cytotoxic activity (%) = (amount of chromium-51 released from cancer cells after the addition of an anti-CAPRIN-1 antibody and mouse splenocytes) / (amount of chromium-51 released from target cells to which 1N hydrochloric acid was added) x Petition 870230039907, dated 12 / 05 / 2023, page 88 / 100 80 / 82 100.

[00196] Next, monoclonal antibodies #1, #2, and #3 antiCAPRIN-1 were evaluated for their cytotoxicity activities (CDC activity) against cancer cells. Blood collected from a rabbit was added to an Eppendorf tube and left to stand at room temperature for 60 minutes, then centrifuged for 5 minutes at 3000 rpm. This prepared the serum for measuring CDC activity. 10⁵ cells of the human breast cancer cell line MDA-MB-231V were collected in a 50 ml centrifuge tube, 100 μθί of chromium 51 was added, and then incubated at 37 °C for 2 hours. The resulting product was washed three times with RPMI 1640 medium containing 10% SBF. Subsequently, the cells were suspended in RPMI medium containing the rabbit serum prepared above (50%), and then the cells were added to a 96-well V-bottom plate at a density of 103 cells per well.One pg of each of the mouse monoclonal antibodies #1 and #2 was added to the cells, and then the cells were cultured for 4 hours at 37 °C under 5% CO2 conditions. After culture, the amount of chromium 51 released from damaged tumor cells in a culture supernatant was measured, and the CDC activity of each antibody against MDA-MB-213V cells was calculated. As a result, both antibodies #1 and #2 exhibited more than 20% CDC activity. Furthermore, no cytotoxic activity was observed in the negative control group to which no antibody was added. Thus, it was revealed that antibodies #1 and #2 can damage tumor cells expressing CAPRIN-1 also through CDC activity.

[00197] In the present case, cytotoxic activity was determined as cytotoxic activity against a cancer cell line. Specifically, as described above, the result was obtained by mixing an anti-CAPRIN-1 antibody to be used in the present invention, a serum, and Petition 870230039907, dated 12 / 05 / 2023, page 89 / 100 81 / 82 103 cells from each cancer cell line that had incorporated chromium-51 were cultured for 4 hours, the amount of chromium-51 released into the culture medium was measured, and then the cytotoxic activity against the cancer cell line was calculated using the following formula*.

[00198] * Formula: cytotoxic activity (%) = (amount of chromium-51 released from cancer cells after the addition of an anti-CAPRIN-1 antibody and serum) / (amount of chromium-51 released from target cells to which 1N hydrochloric acid was added) x 100.

[00199] Next, the obtained mouse anti-CAPRIN-1 monoclonal antibodies #1 and #2 were evaluated for their in vivo antitumor effects in tumor-grafted mice. The antibodies used herein were prepared by column purification of the culture supernatant from each #1 or #2 producing cell, in the same manner as described above.

[00200] The antitumor effects of antibodies #1 and #2 were examined using tumor-bearing mice in which a CAPRIN-1-expressing mouse-derived cancer cell line was transplanted. 4T1 cells (acquired from ATCC) were transplanted subcutaneously into the dorsal region of 30 Balb / c mice (SLC Japan Inc.) at a density of 5x10⁵ cells per mouse. The tumors were allowed to grow to a size of approximately 5 mm in diameter. Each of antibodies #1 and #2 was administered intraperitoneally to 20 of the 30 tumor-bearing mice at a dose of 200 μg (200 μL) / mouse (and each antibody was administered to 10 mice). Subsequently, the same amount of antibody was administered intraperitoneally to each tumor-bearing mouse a total of 3 times within 2 days. The tumor sizes were measured daily, and the antitumor effects were examined through observation.About that,. Petition 870230039907, dated 12 / 05 / 2023, pages 90 / 100 In the 82 / 82 control group, PBS (-) was administered instead of antibodies to the remaining 10 tumor-bearing mice. Tumor size was calculated as a volume using the formula: major axis length x minor axis length x minor axis length x 0.5.

[00201] As a result of observing the antitumor effects, it was observed that the tumors almost completely regressed by day 16 after antibody administration in the examination group to which anti-CAPRIN-1 mouse monoclonal antibodies #1 and #2 were administered. On the other hand, in the control group to which PBS() was administered, the tumors showed an increase of approximately 820% by day 12. The results demonstrated that the obtained anti-human CAPRIN-1 mouse monoclonal antibodies #1 and #2 exhibit strong antitumor effects in vivo against cancer cells expressing CAPRIN-1. Industrial Applicability

[00202] The antibodies of the present invention are useful for the treatment and / or prevention of cancer.

[00203] All publications, patents and patent applications cited herein are incorporated herein by reference in full.

[00204] Listing of Sequences in Free Text SEQ ID NO: 31: primer T3 SEQ ID NO: 32: primer T7 SEQ ID NOS: 33, 34, 38, 39, and 54-59: primers SEQ ID NOS: 35 and 36: GAPDH primers Petition 870230039907, dated 12 / 05 / 2023, p. 91 / 100

Claims

1 / 2 Claims 1. ANTIBODY, which has immunological reactivity with a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 37, characterized by the antibody comprising: (i) a variable heavy chain region comprising SEQ ID NOs: 40, 41 and 42; and a variable light chain region comprising SEQ ID NOs: 44, 45, and 46; (ii) a variable heavy chain region comprising SEQ ID NOs: 40, 41, and 42; and a variable light chain region comprising SEQ ID NOs: 48, 49, and 50; or (iii) a variable heavy chain region comprising SEQ ID NOs: 60, 61 and 62; and a variable light chain region comprising SEQ ID NOs: 64, 65, and 66; and which has immunological reactivity with a CAPRIN1 protein.

2. ANTIBODY, according to claim 1, characterized in that the polypeptide is a partial polypeptide of the CAPRIN-1 protein, wherein CAPRIN-1 is represented by any of the numbered pair sequences SEQ ID NOS: 2 to 30.

3. ANTIBODY, according to any one of claims 1 to 2, characterized in that it is monoclonal.

4. ANTIBODY, according to any one of claims 1 to 2, characterized in that it is polyclonal.

5. ANTIBODY, according to any one of claims 1 to 4, characterized in that it is a human antibody, humanized antibody, chimeric antibody, single-chain antibody or bispecific antibody.

6. PHARMACEUTICAL COMPOSITION, characterized by the fact that it comprises the antibody, as defined in any of claims 1 to 5, as an active ingredient, and pharmaceutically acceptable excipients.

7. COMPOSITION, according to claim 6, characterized in that it further comprises an antitumor agent.

8. PHARMACEUTICAL COMBINATION, characterized in that it comprises the pharmaceutical composition, as defined in any one of claims 6 to 7, and a pharmaceutical composition containing an antitumor agent.

9. USE OF AN ANTIBODY, as defined in any one of claims 1 to 5, characterized in that it is for the manufacture of a medicament for the treatment and / or prevention of cancer.

10. USE OF A PHARMACEUTICAL COMPOSITION, as defined in any one of claims 6 to 7, characterized in that it is for the manufacture of a medicament for the treatment and / or prevention of cancer.

11. USE OF A PHARMACEUTICAL COMBINATION, as defined in claim 8, characterized in that it is for the manufacture of a medicament for the treatment and / or prevention of cancer.

12. USE, according to any of claims 9 to 11, characterized in that the cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, mast cell tumor, kidney cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer, or colorectal cancer. Petition 870230039907, dated 12 / 05 / 2023, pp. 93 / 100

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