Pharmaceutical composition for treatment and / or prevention of cancer

Abstract

A substance capable of reducing the expression of a CAPRIN-1 protein on the surface of a cancer cell membrane, specifically a substance capable of reducing the expression level of the CAPRIN-1 protein on the surface of a cancer cell membrane, or a substance capable of binding to the CAPRIN-1 protein on the surface of a cancer cell membrane, can be used as an active ingredient of a pharmaceutical composition for the treatment and / or prevention of cancer.

Description

Technical Field

[0001] This invention relates to pharmaceutical compositions for the treatment and / or prevention of cancer, with a substance that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes as an active ingredient. Background Technology

[0002] In cancer treatment, various molecularly targeted therapies have been explored, utilizing drugs that control the expression of proteins present in cancer cells or inhibit their function. Antibody drugs, as one type of molecularly targeted therapy, are used as therapeutic agents with high target specificity and few side effects. Examples of antibody drugs include those that specifically bind to receptors or ligands to block signal transduction; those that enhance signal transduction; and those that induce ADCC, CDC, or ADCP after binding to a target. Other molecularly targeted drugs include low-molecular-weight drugs targeting kinases and low-molecular-weight drugs targeting proteasomes. Furthermore, due to technological advancements in endowing cells with resistance to nucleases in vivo, efficient intracellular uptake has become possible, leading to the practical application of nucleic acid drugs with even higher target specificity than the aforementioned molecularly targeted drugs.

[0003] Cytoplasmic activation and proliferation-associateed protein 1 (CAPRIN-1) is an intracellular protein known to be expressed during the activation and division of normal cells in the dormant phase. It also participates in the control of mRNA transport and translation by forming intracellular stress granules with RNA. On the other hand, CAPRIN-1 protein is known to be specifically expressed on the cell membrane surface of various cancer cells, and antibodies that specifically bind to CAPRIN-1 protein expressed on the cancer cell membrane surface show promise as therapeutic agents for cancer (Patent Document 1).

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: WO2010 / 016526 Summary of the Invention

[0007] The problem that the invention aims to solve

[0008] The present invention aims to provide new medicines that are useful in the treatment and / or prevention of cancer.

[0009] Methods for solving problems

[0010] The inventors discovered that reducing the expression of CAPRIN-1 protein on the surface of cancer cell membranes can inhibit tumor formation in cancer patients. This led to the idea of ​​using a substance that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes as an effective ingredient in a drug for the treatment and / or prevention of cancer, thus completing this invention.

[0011] That is, the present invention relates to the following embodiments (1) to (14).

[0012] (1) A pharmaceutical composition for the treatment and / or prevention of cancer, wherein the active ingredient is a substance that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes.

[0013] (2) According to the pharmaceutical composition of (1), the cancer patient is a cancer patient with cancer that expresses CAPRIN-1 protein on the surface of cancer cell membrane.

[0014] (3) The pharmaceutical composition according to (1) or (2) wherein the substance is a substance that reduces the expression level of CAPRIN-1 protein on the surface of cancer cell membranes.

[0015] (4) The pharmaceutical composition according to any one of (1) to (3), wherein the substance is selected from at least one substance selected from AMPK inhibitors, Wnt agonists, Akt / PI3K inhibitors, P53 inhibitors, Rac1 inhibitors, ROS-producing factors, ErbB2 / HER2 inhibitors, GSK3β inhibitors, endocytosis inhibitors and Wnt / β-linkin inhibitors.

[0016] (5) The pharmaceutical composition of any one of (1) to (3), wherein the substance is a substance capable of inhibiting the expression of the CAPRIN-1 gene.

[0017] (6) The pharmaceutical composition according to (5) is a nucleic acid molecule.

[0018] (7) According to the pharmaceutical composition of (6), the nucleic acid molecule is a nucleic acid molecule selected from at least one of the following as a target of the CAPRIN-1 gene: interfering small RNA (siRNA), microRNA, small hairpin RNA (shRNA), guide RNA (gRNA), antisense nucleic acid, ribozyme and nucleic acid aptamer.

[0019] (8) The pharmaceutical composition according to any one of (1) to (3), wherein the substance is an enzyme capable of breaking down CAPRIN-1 protein on the surface of cancer cell membranes.

[0020] (9) In the pharmaceutical composition according to (8), the enzyme is trypsin or chymotrypsin.

[0021] (10) The pharmaceutical composition according to (1) or (2), wherein the substance is a substance that binds to CAPRIN-1 protein on the surface of cancer cell membranes.

[0022] (11) The pharmaceutical composition according to (10) wherein the substance is an antibody or an antigen-binding fragment thereof that binds to the CAPRIN-1 protein on the surface of a cancer cell membrane.

[0023] (12) The pharmaceutical composition according to any one of (1) to (11), wherein the cancer is ovarian cancer, bile duct cancer, breast cancer, kidney cancer, pancreatic cancer, colorectal cancer, melanoma, lung cancer, renal cell carcinoma, head and neck cancer, gastric cancer, biliary tract cancer, brain tumor, prostate cancer, mesothelioma, colorectal / rectal cancer, esophageal cancer, gastroesophageal junction cancer, hepatocellular carcinoma, glioblastoma, urothelial carcinoma, bladder cancer, uterine cancer, liver cancer, sarcoma, fibrosarcoma, mast cell tumor, adrenocortical carcinoma, Ewing's sarcoma, multiple myeloma, testicular cancer, thyroid cancer, basal cell carcinoma, Paget's disease, skin cancer, gastrointestinal stromal tumor (GIST), renal pelvis and ureter cancer, rare cancer, primary central nervous system lymphoma, primary testicular lymphoma, Hodgkin's lymphoma, leukemia or lymphoma.

[0024] (13) A method of treating and / or preventing cancer, including administering the pharmaceutical composition of any one of (1) to (12) to a cancer patient.

[0025] (14) The cancer patient is a cancer patient with cancer that expresses CAPRIN-1 protein on the surface of cancer cell membrane according to the cancer treatment and / or prevention method described in (13).

[0026] The effects of the invention

[0027] This invention reduces the expression of CAPRIN-1 protein on the surface of cancer cell membranes, thereby inhibiting tumor formation in cancer patients. Attached Figure Description

[0028] Figure 1This is a graph showing the changes in CAPRIN-1 protein expression on the cell membrane surface caused by inhibition of CAPRIN-1 gene expression. The upper graph represents the human cancer cell lines BT474, KKU-213, SW780, and OVCAR3, where CAPRIN-1 protein expression was confirmed on the cell membrane surface. References 1, 3, 5, and 7 indicate CAPRIN-1 protein expression on the cell membrane in various cancer cells treated with siRNA of the negative control (+control siRNA), and references 2, 4, 6, and 8 indicate CAPRIN-1 protein expression on the cell membrane in various cancer cells treated with CAPRIN-1 gene-specific siRNA (+CAPRIN-1 siRNA) (arrows indicate CAPRIN-1 protein expression on the cell membrane surface). The middle table shows the immunostaining scores of CAPRIN-1 protein in the cytoplasm and cell membrane of human cancer cells BT474, KKU-213, SW780, and OVCAR3, in which CAPRIN-1 protein expression was confirmed on the cell membrane surface. The lower figure shows the expression of CAPRIN-1 protein in various cancer cells BT474, KKU-213, SW780, and OVCAR3, in which CAPRIN-1 gene expression was confirmed on the cell membrane surface, after siRNA was used to inhibit CAPRIN-1 gene expression.

[0029] Figure 2This diagram, created from the HEC-1 and SK-MEL-5 human cancer cell lines where CAPRIN-1 protein expression was confirmed on the cell membrane surface, shows the sorting of cancer cell fractions with different CAPRIN-1 protein expression levels on the cell membrane surface. The cytoskeleton-independent colony-forming ability of each cancer cell fraction was evaluated. Figure A at the top shows the grading of cancer cell fractions with different CAPRIN-1 protein expression levels on the cell membrane surface. Reference numbers 1 and 3 represent cancer cell fractions with high CAPRIN-1 protein expression on the cell membrane surface; reference numbers 2 and 4 represent cancer cell fractions with low CAPRIN-1 protein expression on the cell membrane surface; reference numbers 5 and 7 represent cancer cell fractions with low CAPRIN-1 protein expression on the cell membrane surface after sorting (Low); and reference numbers 6 and 8 represent cancer cell fractions with high CAPRIN-1 protein expression on the cell membrane surface after sorting (High). Figure B at the bottom evaluates the cytoskeleton-independent colony-forming ability of each cancer cell fraction. Reference numbers 9 and 11 show the colony-forming images of cancer cell fractions (Low) sorted from human cancer cell line HEC-1-A in soft agar medium. Reference numbers 10 and 12 show the colony-forming images of cancer cell fractions (High) sorted from human cancer cell line SK-MEL-5 in soft agar medium. Reference number 13 shows the number of colonies formed by cancer cell fractions (Low) sorted from human cancer cell line HEC-1-A in soft agar medium. Reference number 14 shows the number of colonies formed by cancer cell fractions (High) sorted from human cancer cell line HEC-1-A in soft agar medium. Reference number 15 shows the number of colonies formed by cancer cell fractions (Low) sorted from human cancer cell line SK-MEL-5 in soft agar medium. Reference number 16 shows the number of colonies formed by cancer cell fractions (High) sorted from human cancer cell line SK-MEL-5 in soft agar medium.

[0030] Figure 3 This figure illustrates the differences in tumor-forming ability of different cancer cell fractions expressing the CAPRIN-1 protein on the cell membrane surface in mouse organisms. Reference numeral 1 represents the tumor-forming ability of the cancer cell fraction (High) sorted from human cancer cell line HEC-1-A in mouse organisms; Reference numeral 2 represents the tumor-forming ability of the cancer cell fraction (Low) sorted from human cancer cell line HEC-1-A in mouse organisms; Reference numeral 3 represents the tumor-forming ability of the cancer cell fraction (High) sorted from human cancer cell line SK-MEL-5 in mouse organisms; and Reference numeral 4 represents the tumor-forming ability of the cancer cell fraction (Low) sorted from human cancer cell line SK-MEL-5 in mouse organisms. Detailed Implementation

[0031] In this specification, "protein expression" refers to the production of protein by a gene encoding the protein, and "inhibition of protein expression" refers to the complete suppression of protein expression through certain treatments, or the reduction of protein expression compared to before the treatment.

[0032] In this specification, "inhibition of gene expression" means the complete inhibition of mRNA transcription of a gene encoding a protein by certain treatments, or the reduction of the amount of mRNA transcribed compared to before the treatment, thereby completely inhibiting protein expression or reducing protein expression compared to before the treatment.

[0033] In this specification, "treatment" refers to the treatment of cancer based on the antitumor effect of the active ingredient in the pharmaceutical composition. Furthermore, "prevention" in this specification refers not only to the prevention of cancer occurrence but also to the prevention of cancer metastasis or recurrence.

[0034] In this specification, the terms “cancer” and “tumor” refer to malignant new growths and are used interchangeably.

[0035] This invention relates to pharmaceutical compositions useful for the treatment and / or prevention of cancer, with a substance that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes as an active ingredient.

[0036] Substances that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes include substances that act inside cancer cells, directly or indirectly reducing the expression of CAPRIN-1 protein on the surface of cancer cell membranes; substances that cleave the polypeptides of CAPRIN-1 protein on the surface of cancer cell membranes; and substances that bind to CAPRIN-1 protein on the surface of cancer cell membranes, specifically substances that bind to CAPRIN-1 protein on the surface of cancer cell membranes and directly cover part or all of the CAPRIN-1 protein on the surface of cancer cell membranes. The present invention will now be described in detail.

[0037] <Decreased expression of CAPRIN-1 protein on the surface of cancer cell membrane>

[0038] As a target for substances that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes, the CAPRIN-1 gene and protein can be listed. Examples of CAPRIN-1 genes and proteins include those disclosed in WO2010 / 016526 for humans, dogs, cattle, horses, mice, rats, and chickens. Alternatively, for example, by accessing GenBank (NCBI) and using algorithms such as BLAST and FASTA (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993; Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997), the base and amino acid sequences of the CAPRIN-1 gene and protein can be obtained.

[0039] CAPRIN-1 protein expression can be detected by methods such as Western blotting using anti-CAPRIN-1 antibodies. Specific examples of anti-CAPRIN-1 antibodies include: WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2011 / 096535, WO2013 / 018886, WO2013 / 018894, and WO2013 / 01889. Anti-CAPRIN-1 antibodies described in No. 2, WO2013 / 018891, WO2013 / 018889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640, WO2013 / 147169, WO2013 / 147176 and WO2015 / 020212.

[0040] The reduction in CAPRIN-1 protein expression on the surface of cancer cell membranes, achieved by using substances that decrease the expression of CAPRIN-1 protein, can be detected using anti-CAPRIN-1 antibodies, similar to Western blotting. Specifically, after reacting the anti-CAPRIN-1 antibody with cells that have not undergone cell membrane permeation treatment, a secondary antibody labeled with arbitrary detection fluorescence is applied, followed by detection by flow cytometry. Alternatively, detection can be performed using an antibody directly labeled with arbitrary detection fluorescence on the anti-CAPRIN-1 antibody. Here, the reduction in CAPRIN-1 protein expression on the surface of cancer cell membranes refers to a state where the amount of CAPRIN-1 protein present on the surface of cancer cell membranes (hereinafter referred to as expression level) is reduced, and / or a state where part or all of the CAPRIN-1 protein present on the surface of cancer cell membranes is covered by substances that can bind to CAPRIN-1 protein on the surface of cancer cell membranes. Details will be described later, but substances that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes include low-molecular-weight compounds, substances that inhibit the expression of the CAPRIN-1 gene, and enzymes that can break down CAPRIN-1 protein on the surface of cancer cell membranes. Substances that can cover part or all of the CAPRIN-1 protein on the surface of cancer cell membranes include substances that can bind to CAPRIN-1 protein on the surface of cancer cell membranes. When evaluating using flow cytometry with an anti-CAPRIN-1 antibody that is reactive to CAPRIN-1 protein on the cell membrane surface, the expression of CAPRIN-1 protein on the cell membrane surface in untreated cells is expressed as mean fluorescence intensity and used as a 100% baseline. The mean fluorescence intensity value in cells treated with a substance that reduces the expression of CAPRIN-1 protein on the surface of cancer cell membranes can be obtained using the following formula, and the evaluation can be performed accordingly: (Inhibition of CAPRIN-1 protein expression on the cell membrane surface (%) = Mean fluorescence intensity of cells that reacted to the factor / Mean fluorescence intensity of untreated cells × 100). In this invention, "suppressed" simply means any meaningful difference. The suppression rate (%) obtained in the above evaluation is preferably 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, or 40% or less.

[0041] <Substances that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes>

[0042] In this invention, substances that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes include low molecular weight compounds, substances that can inhibit the expression of the CAPRIN-1 gene, and enzymes that can decompose CAPRIN-1 protein on the surface of cancer cell membranes.

[0043] The substances used in this invention that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes are not particularly limited as long as they can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes. Specific examples of substances that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes include low molecular weight compounds that have the function of reducing the expression of CAPRIN-1 protein on the surface of cancer cell membranes, substances that can inhibit the expression of the CAPRIN-1 gene (substances that inhibit the transcription of CAPRIN-1 gene mRNA, substances that inhibit the translation of CAPRIN-1 protein from the mRNA transcribed from the CAPRIN-1 gene, substances that decompose the mRNA transcribed from the CAPRIN-1 gene, or substances that decompose the CAPRIN-1 protein translated from the mRNA transcribed from the CAPRIN-1 gene), or hydrolytic enzymes that can decompose CAPRIN-1 protein on the surface of cancer cell membranes.

[0044] Specific examples of low-molecular-weight compounds that reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes include AMPK inhibitors, IFG-1R inhibitors, Wnt agonists, Akt / PI3K inhibitors, P53 inhibitors, Rac1 inhibitors, ROS-producing factors, ErbB2 / HER2 inhibitors, GSK3β inhibitors, IGF-1R inhibitors, endocytosis inhibitors, and Wnt / β-linkin inhibitors. Preferred examples include the AMPK inhibitor Dorsomorphin, Wnt agonist 1, the Akt / PI3K inhibitor LY294002, the P53 inhibitor Pifithrin-α, the Rac1 inhibitor Rac1 inhibitor (CAS #1177865-17-6), the ROS-producing factor Pyocyanin, the ErbB2 / HER2 inhibitor Mubritinib, and the GSK3β inhibitor GSK-3β inhibitor. VIII, endocytosis inhibitor Dynasore, Wnt / β-linkin inhibitor Wnt-C59, but not limited to these.

[0045] Nucleic acid molecules are among the preferred options for substances that can inhibit the expression of the CAPRIN-1 gene. As for nucleic acid molecules that can inhibit the expression of the CAPRIN-1 gene, there are no particular limitations as long as they can bind to the full-length or partial sequence of the CAPRIN-1 gene and inhibit its expression. Examples include nucleic acid molecules that can inhibit the transcription of the CAPRIN-1 gene, nucleic acid molecules that can disrupt the CAPRIN-1 gene, and nucleic acid molecules that can inhibit the translation of the CAPRIN-1 gene. Specifically, examples include siRNA, microRNA, or shRNA targeting the full-length or partial sequence of the CAPRIN-1 gene; guide RNA targeting the full-length or partial sequence of the CAPRIN-1 gene itself or protospacer adjacent motifs (NGC, NNGRR, NNNNRYAC, TTN, TTTR, 3'A / U / C sequences); antisense nucleic acids targeting the full-length or partial sequence of the CAPRIN-1 gene; ribozymes targeting the full-length or partial sequence of the CAPRIN-1 gene; and nucleic acid aptamers (DNA or RNA) that specifically bind to the full-length or partial sequence of the CAPRIN-1 gene. Furthermore, the aforementioned CAPRIN-1 gene not only contains the structural genes of the CAPRIN-1 protein but also its 5'-UTR and 3'-UTR, promoter region, enhancer region, and other transcriptional regulatory regions. As a specific example, the nucleic acid molecule capable of inhibiting CAPRIN-1 gene expression can be a substance capable of inhibiting transcription from CAPRIN-1 gene mRNA, a substance capable of inhibiting translation of CAPRIN-1 protein from mRNA transcribed from the CAPRIN-1 gene, a substance capable of degrading mRNA transcribed from the CAPRIN-1 gene, or a substance capable of degrading CAPRIN-1 protein translated from mRNA transcribed from the CAPRIN-1 gene. Furthermore, in the case of a nucleic acid molecule complementary to the CAPRIN-1 gene, complete complementarity to the base sequence of the CAPRIN-1 gene is preferred, but nucleic acid molecules hybridized under stringent conditions are also acceptable. The homology of the complementary sequence is preferably 90% or more, more preferably 95% or more.

[0046] The nucleic acid molecules can be designed based on the CAPRIN-1 gene sequence, with indicators such as G (guanine) and C (cytosine) content, sequence specificity, and off-target effects set, using methods known to those skilled in the art. For example, for siRNA, databases such as siDirect (http: / / sidirect2.rnai.jp) can be used to design siRNA sequences from the base sequence of the CAPRIN-1 gene, and then chemically synthesized using methods known to those skilled in the art.

[0047] The combination of the nucleic acid molecule with a pharmaceutically acceptable carrier is also a preferred option. The carrier is not particularly limited, but preferably has a morphology that enhances aggregation in cancerous tissues expressing the CAPRIN-1 gene, improves intracellular permeability, or enhances the stability of the nucleic acid molecule. For example, cationic liposomes can be used (see Yano J et al., Clinical Cancer research, 10(22), 7721, 2004). Other carriers include particles containing cationic lipids, neutral lipids, cholesterol lipids, PEG-modified lipids, lipid particles, and lipid nanoparticles. Alternatively, examples could be listed such as the amphiphilic polymers described in WO2023 / 042872, WO2023 / 032892, WO22023 / 032891, WO2016 / 066586, WO2010 / 005721, WO2016 / 187234, WO2016 / 123480, WO2006 / 107903, WO2010 / 005740, WO2017 / 003668, WO2007 / 056153, and WO2002 / 080982.

[0048] In addition, nucleic acid molecules can be chemically modified to prevent them from breaking down in organisms (see Ross J. et al., Nature, 432(7014), 155, 2004).

[0049] Alternatively, vectors capable of expressing the nucleic acid molecules can be used. Specific examples include viral vectors (e.g., adenovirus vectors) and non-viral vectors (e.g., plasmid DNA).

[0050] Specific examples of enzymes that can break down CAPRIN-1 protein on the surface of cancer cell membranes include trypsin and chymotrypsin.

[0051] In addition, substances that can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes also include: substances that can bind to CAPRIN-1 protein on the surface of cancer cell membranes to cover part or all of the CAPRIN-1 protein, or substances that can bind to CAPRIN-1 protein on the surface of cancer cell membranes to internalize CAPRIN-1 protein into the cell. These substances can be substances that can directly damage cancer cells expressing CAPRIN-1 protein on the cell membrane surface, or substances that can induce damaging activity against cancer cells. However, even substances that do not directly damage cancer cells or induce damaging activity against cancer cells can achieve the effects of the present invention. As a preferred specific example, antibodies or antigen-binding fragments thereof that can bind to CAPRIN-1 protein on the surface of cancer cell membranes (hereinafter also referred to as CAPRIN-1 expression inhibitory antibodies) can be listed. CAPRIN-1 expression inhibitory antibodies can be substances that directly damage cancer cells expressing CAPRIN-1 protein on the cell membrane surface or induce damaging activity against cancer cells, or substances that do not directly damage cancer cells or induce damaging activity against cancer cells.

[0052] The type of antibody that inhibits CAPRIN-1 expression is not particularly limited; for example, it can be any class and subtype such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b, IgG3, and IgM. Additionally, examples of antigen-binding fragments include Fab, Fv, F(ab')2, and Fab'.

[0053] CAPRIN-1 expression inhibitory antibodies can be either monoclonal or polyclonal antibodies.

[0054] The polyclonal antibody can be obtained by immunizing mice, human antibody-producing mice, rats, rabbits, chickens, etc., with natural CAPRIN-1 protein or a fusion protein with GST, or a partial peptide thereof, and then obtaining serum. The obtained serum is purified by ammonium sulfate precipitation, protein A, protein G, DEAE ion exchange column, affinity column bound to CAPRIN-1 protein or a partial peptide, etc.

[0055] The method for preparing the CAPRIN-1 protein used in the above immunization can be obtained by referring to WO2014 / 012479, or by using cells expressing the CAPRIN-1 protein, etc.

[0056] The monoclonal antibody can be obtained, for example, by immunizing mice with breast cancer cells SK-BR-3 expressing CAPRIN-1 protein, the full-length CAPRIN-1 protein, or a fragment thereof; fusing spleen cells isolated from these mice with myeloma cells; and selecting clones that produce the monoclonal antibody from the resulting fused cells (hybridoma). The antibody produced by the selected hybridoma can be obtained using the same purification method as described above for polyclonal antibodies.

[0057] The antibodies used in this invention include human antibodies, humanized antibodies, chimeric antibodies, and non-human animal antibodies.

[0058] Human antibodies can sensitize human lymphocytes infected with EB virus with proteins, protein-expressing cells or their lysates, and then fuse these sensitized lymphocytes with human-derived myeloma cells such as U266 cells. Antibodies that are immunoreactive with the full length or fragments of the CAPRIN-1 protein can be obtained from the resulting fused cells.

[0059] Humanized antibodies, also known as reshaped human antibodies, are engineered antibodies. They are constructed by transplanting the complementarity-determining region (CDR) of an antibody derived from an immunized animal into the CDR of a human antibody. Gene recombination, a well-known technique, is a common method for this. Specifically, a DNA sequence designed to link the CDR of, for example, a mouse or rabbit antibody to the framework region of a human antibody is synthesized from several oligonucleotides produced with overlapping ends using PCR. The resulting DNA is then linked to DNA encoding the constant region of the human antibody, integrated into an expression vector, and introduced into a host to produce the humanized antibody (see EP239400 and WO96 / 02576). The framework region of the human antibody linked by the CDR can be selected to allow the CDR to form a good antigen-binding site. Alternatively, amino acids in the framework region of the antibody's variable region can be replaced to allow the CDR of the reshaped human antibody to form an appropriate antigen-binding site (Sato K. et al., Cancer Research 1993, 53: 851-856). Alternatively, it can be replaced with a framework region derived from various human antibodies (see WO99 / 51743).

[0060] Antibodies are typically heteropolymeric glycoproteins containing at least two heavy chains and two light chains. An antibody consists of two identical light chains and two identical heavy chains. The heavy chain has a variable region at one end, linked to several constant regions. The light chain has a variable region at one end, also linked to several constant regions. The variable regions contain specific regions called complementarity-determining regions (CDRs), which impart binding specificity to the antibody. Relatively conserved portions of the variable regions are called framework regions (FRs). The variable regions of both the complete heavy and light chains contain four FRs, each composed of three CDRs (CDR1–CDR3).

[0061] In addition, the sequences of constant and variable regions of human heavy and light chains can be obtained from, for example, NCBI (US: GenBank, UniGene, etc.). For example, the heavy chain constant region of human IgG1 can refer to the sequence of accession number J00228, the heavy chain constant region of human IgG2 can refer to the sequence of accession number J00230, the human light chain κ constant region can refer to the sequences of accession numbers V00557, X64135, X64133, etc., and the human light chain λ constant region can refer to the sequences of accession numbers X64132, X64134, etc.

[0062] Chimeric antibodies are antibodies created by combining sequences from different animals. Examples include antibodies containing the variable regions of the heavy and light chains of mouse antibodies, and the constant regions of the variable regions of the heavy and light chains of human antibodies. Chimeric antibodies can be produced using known methods, such as by linking DNA encoding the V region of an antibody with DNA encoding the C region of a human antibody, integrating this into an expression vector, and introducing it into a host cell.

[0063] Non-human animal antibodies can be obtained by sensitizing animals with sensitizing antigens using known methods. A common method is to inject the sensitizing antigen into the peritoneal cavity, intradermal cavity, or subcutaneous cavity of animals such as mice. When injecting the sensitizing antigen, it can be mixed with various adjuvants, such as CFA (Freud's complete adjuvant), and administered to the animal multiple times. After immunizing the animal and confirming the presence of anti-CAPRIN-1 antibodies in the serum, serum is obtained and purified, as previously described, by ammonium sulfate precipitation, protein A, protein G, DEAE ion exchange columns, or affinity columns coupled with CAPRIN-1 protein or partial peptides, thereby obtaining non-human animal antibodies. Alternatively, monoclonal antibodies can be obtained from non-human animals by collecting immune cells from immunized animals and fusing them with myeloma cells. The cell fusion of immune cells and myeloma cells can be performed according to known methods (see Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46).

[0064] CAPRIN-1 expression inhibitory antibodies can also be obtained by integrating the antibody gene from a hybridoma clone into a suitable vector, introducing it into the host, and using gene recombination technology to produce recombinant antibodies (see Carl, AKBorrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

[0065] The higher the binding affinity of the CAPRIN-1 expression inhibitory antibody to the CAPRIN-1 protein on the surface of cancer cell membranes, the stronger the expected anti-tumor effect. The desired binding constant (affinity constant) Ka (kon / koff) is preferably at least 10. 7 M -1 At least 10 8 M -1 At least 5×10 8 M -1 At least 10 9 M -1 At least 5×10 9 M -1 At least 10 10 M -1 At least 5×10 10 M -1 At least 10 11 M -1 At least 5×10 11 M -1 At least 10 12 M -1 or at least 10 13 M -1 .

[0066] The ability of CAPRIN-1 expression inhibitory antibodies to bind to the CAPRIN-1 protein on the surface of cancer cell membranes can be specifically determined using binding assays such as ELISA, Western blotting, immunofluorescence, and flow cytometry.

[0067] CAPRIN-1 expression inhibitory antibodies can be chemically modified. Examples of such antibody modifiers include antibodies bound to various molecules such as polyethylene glycol (PEG). In the antibody modifiers of this invention, the bound substances are not limited. Such antibody modifiers can be obtained by chemically modifying the resulting antibody. These methods have been established in the art.

[0068] CAPRIN-1 expression inhibitory antibodies can enhance the binding affinity of anti-CAPRIN-1 antibodies to effector cells by replacing one, two, or more amino acids in the constant region of the antibody's heavy chain, or by removing fucose that binds to N-acetylglucosamine in the N-glycosidic bond glycans bound to the constant region of the heavy chain. These replacements can be individual amino acid substitutions or combinations thereof with antibodies bound to fucose.

[0069] Antibodies that replace one, two, or more amino acids in the constant region of the heavy chain can be prepared according to, for example, WO2004 / 063351, WO2011 / 120135, US Patent 8388955, WO2011 / 005481, US Patent 6737056, and WO2005 / 063351.

[0070] Antibodies containing fucose attached to N-acetylglucosamine in the N-glycosidic bond glycan chain of the heavy chain constant region, or cells that produce such antibodies, can be prepared with reference to U.S. Patent No. 6,602,684, European Patent No. 1,914,244, and U.S. Patent No. 7,579,170. Compositions of antibodies containing fucose bound to N-acetylglucosamine in the N-glycosidic bond glycan chain of the heavy chain constant region and fucose-bound antibodies, or cells that produce such compositions, can be prepared with reference to, for example, U.S. Patent No. 8,642,292.

[0071] Specific examples of CAPRIN-1 expression inhibitory antibodies can be found in WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2011 / 096535, WO2013 / 018886, WO2013 / 018894, and WO2013. Antibodies that can be produced using the following codes: / 018892, WO2013 / 018891, WO2013 / 018889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640, WO2013 / 147169, WO2013 / 147176, and WO2015 / 020212.

[0072] <Pharmaceutical Compositions>

[0073] The purpose of this pharmaceutical composition, in which the substance of the present invention capable of reducing the expression of CAPRIN-1 protein on the surface of cancer cell membranes is an active ingredient, is the treatment and / or prevention of cancer. CAPRIN-1 protein is significantly expressed on the cell membrane surface of various cancer cells. The substance of the present invention, as an active ingredient of the pharmaceutical composition capable of reducing the expression of CAPRIN-1 protein on the surface of cancer cell membranes, can reduce the tumorigenic capacity of cancer cells by decreasing the expression of CAPRIN-1 protein on the cell membrane surface of various cancer cells. The antitumor activity of the substance capable of reducing the expression of CAPRIN-1 protein on the surface of cancer cell membranes can be evaluated, as described below, by investigating the inhibition of cytoskeleton-independent colony formation capacity of cancer cells and the proportion of dead cells in vitro, and by investigating the inhibition of tumor formation or tumor proliferation in mouse organisms.

[0074] The cancers that can be targeted by the pharmaceutical compositions of the present invention are not particularly limited to any cancer that expresses CAPRIN-1 protein on the cell membrane surface, but are preferably ovarian cancer, bile duct cancer, breast cancer, kidney cancer, pancreatic cancer, colorectal cancer, melanoma (including postoperative melanoma), lung cancer (including non-small cell lung cancer and small cell lung cancer), renal cell carcinoma, head and neck cancer, gastric cancer, biliary tract cancer, brain tumors, prostate cancer, mesothelioma (including malignant pleural mesothelioma), colorectal cancer (e.g., colorectal cancer with MSI-high), esophageal cancer, esophagogastric junction cancer, hepatocellular carcinoma, glioblastoma, urothelial carcinoma, bladder cancer, and uterine cancer. This includes cervical cancer, endometrial cancer, liver cancer, sarcomas (including osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, cutaneous angiosarcoma, soft tissue sarcoma, head and neck sarcoma, cardiac sarcoma, uterine sarcoma, uterine carcinosarcoma, endometrial intramural sarcoma, uterine leiomyosarcoma, undifferentiated endometrial sarcoma, retroperitoneal sarcoma, pediatric sarcoma, alveolar soft tissue sarcoma, clear cell sarcoma, dermatofibrosarcoma protuberans, epithelioid sarcoma), mast cell tumor, adrenocortical carcinoma, Ewing's sarcoma, multiple myeloma, testicular cancer, thyroid cancer, basal cell carcinoma, Paget's disease. Special diseases, skin cancer, gastrointestinal stromal tumors (GIST), renal pelvis and ureter cancer, rare cancers (including eye tumors, orbital sarcomas, eyelid tumors, intracranial lymphoma, adnexal lymphoma, optic nerve tumors, choroidal malignant melanoma, retinoblastoma, lacrimal gland cancer, adenoid cystic carcinoma, auditory organ cancer, oral cancer, male breast cancer, special types of breast cancer, malignant phyllodes tumors of the breast, malignant pericardial mesothelioma, malignant pleural mesothelioma, thymoma, thymic carcinoma, SMARC4 defect tumors of the chest, cancers originating from neuroendocrine cells (including NEC, NET, pulmonary neuroendocrine tumors, digestive tract endocrine tumors), malignant peritoneal mesothelioma, Anal cancer, squamous cell carcinoma of the anal canal, small bowel cancer, duodenal cancer, jejunal cancer, ileal cancer, pancreatic neuroendocrine tumors, pheochromocytoma, paraganglioma, malignant testicular mesothelioma, vaginal cancer, urachal cancer, peritoneal cancer, sweat gland cancer, sebaceous gland cancer, skin appendage cancer, Merkel cell carcinoma, acanthoma, sarcoma of the trunk, ligamentous tumors, germ cell tumors, internal auditory canal cancer, external auditory canal cancer, gingival cancer (including mandibular gingival cancer), gastrinoma, divergent ligamentous tumors, tongue cancer, urachal cancer, uveal malignant melanoma, primary central nervous system lymphoma, primary testicular lymphoma, Hodgkin lymphoma, leukemia or lymphoma. Furthermore, these cancers can be primary cancer, cancer of unknown primary origin, metastatic cancer, metastatic or recurrent cancer, postoperative cancer, or unresectable cancer. In addition, melanoma is often used synonymously with malignant melanoma or malignant melanoma.

[0075] The cancer patient (subject) to whom the pharmaceutical composition of the present invention is administered may be a cancer patient confirmed to have cancer, a patient suspected of having cancer, but preferably a cancer patient with cancer expressing CAPRIN-1 protein on the surface of cancer cell membranes. The term "cancer patient with cancer expressing CAPRIN-1 protein on the surface of cancer cell membranes" here includes not only cancer patients confirmed to have the cancer at the time of administration of the pharmaceutical composition of the present invention, but also cancer patients previously confirmed to have the cancer, and patients to whom the pharmaceutical composition of the present invention is administered for the prevention of recurrence of the cancer. Whether a patient has cancer expressing CAPRIN-1 protein on the surface of cancer cell membranes can be confirmed, for example, by detecting the CAPRIN-1 protein expressed on the surface of cancer cell membranes in cancer cells / cancer tissue isolated from the cancer patient using, for example, the method disclosed in WO2010 / 016527. Furthermore, as a biological classification of the cancer patient (subject), mammals such as humans, pets, livestock, and racing animals are preferred, and humans are more preferred.

[0076] The pharmaceutical compositions of the present invention can be formulated using methods known to those skilled in the art. The pharmaceuticals of the present invention can be used non-orally in the form of sterile solutions or suspensions in water or pharmaceutically acceptable liquids other than water. In the pharmaceuticals of the present invention, each formulation or pharmaceutical composition may also be formulated by appropriately combining its active ingredient (the component that inhibits the expression of CAPRIN-1 protein on the surface of cancer cell membranes) with, for example, a pharmacologically acceptable carrier, medium, or additive, specifically sterile water, physiological saline, isotonic solution, buffer (buffer solution, etc.), vegetable oil, oily liquid, antioxidant, solubilizer, emulsifier, suspending agent, surfactant, stabilizer, fragrance, excipient, binder, etc., preferably by mixing with them in a unit dosage manner as generally considered necessary for pharmaceutical implementation. The amount of active ingredient in these formulations is such that an appropriate dosage within the indicated range is obtained.

[0077] Sterile compositions for injection can be formulated using a medium such as distilled water for injection, following standard pharmaceutical preparation practices. As an aqueous solution for injection, it can be mixed, for example, with physiological saline, isotonic solutions containing D-sorbitol, D-mannose, D-mannitol, other excipients, monohydric alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80(TM), HCO-60), benzyl benzoate, benzyl alcohol, and other solubilizers, oily solutions (e.g., sesame oil, soybean oil), buffers (e.g., phosphate buffer, sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol, phenol), and antioxidants. The prepared injection solution is typically filled into appropriate ampoules.

[0078] The administration can be oral or non-oral, with non-oral administration being preferred. Specifically, examples include injectable dosage forms, nasal dosage forms, pulmonary dosage forms, and percutaneous dosage forms. Examples of injectable dosage forms include systemic or local administration via intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intratumoral injection. Examples of percutaneous dosage forms include dosage forms referred to as ointments or topical medications. Topical medications include solid dosage forms, liquids, sprays, ointments, creams, and gels.

[0079] Furthermore, an appropriate administration method can be selected based on the patient's age, weight, sex, symptoms, etc. The dosage of the pharmaceutical composition containing at least one of the active ingredients of the present invention is determined by the amount of each active ingredient; for example, it can be selected from 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, for example, the dosage of each active ingredient can be selected from 0.001 to 100000 mg per patient, or for example, 1 mg to 30 mg per kg of body weight per patient, but is not necessarily limited to these values. The dosage and administration method vary depending on the patient's weight, age, sex, symptoms, etc., and can be appropriately selected by those skilled in the art.

[0080] <Treatment and / or prevention of cancer>

[0081] By administering the pharmaceutical composition of the present invention to the cancer patient (subject) according to the aforementioned method, it is possible to treat and / or prevent cancer in the cancer patient. The cancer patient (subject) who is the object of treatment and / or prevention may be a cancer patient confirmed to have cancer, a patient suspected of having cancer, but preferably a cancer patient with cancer expressing CAPRIN-1 protein on the surface of cancer cell membranes. Furthermore, the pharmaceutical composition of the present invention may be administered to the cancer patient (subject) simultaneously or separately with other antitumor agents (known antitumor agents, etc.).

[0082] Example

[0083] The present invention will now be described in detail with reference to specific embodiments, but the scope of the present invention is not limited to these specific embodiments.

[0084] (Example 1) Expression of CAPRIN-1 gene in cancer cells

[0085] The expression of the CAPRIN-1 gene in various cell lines was investigated using RT-PCR (Reverse Transcription-PCR). The reverse transcription reaction was performed as follows: 50–100 mg of each tissue and 5–10 × 10⁶ mg of each cell line were used. 6Total RNA was extracted from cells using TRIZOL reagent (Invitrogen) according to the accompanying protocol. cDNA was synthesized from this total RNA using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) according to the accompanying protocol. PCR reactions were performed using the obtained gene-specific primers (sense: 5'-AAGGTTTGAATGGAGTGC-3' (Sequence No. 1) and antisense: 5'-TGCTCCTTTTCACCACTG-3' (Sequence No. 2)) as follows: 0.25 μl of the sample prepared by reverse transcription, 2 μM of each of the above primers, 0.2 mM of each dNTP, and 0.65 U of ExTaq polymerase (Takarazuchi Co., Ltd.) were added to the reagents and accompanying buffer to a total volume of 25 μl. The reaction was performed 30 times using a Thermal Cycler (BIO RAD Co., Ltd.) at 94°C / 30 sec, 60°C / 30 sec, and 72°C / 30 sec. Specifically, the aforementioned gene-specific primers amplified the base sequence of the canine CAPRIN-1 gene from position 206 to 632 starting from the 5' end, and the base sequence of the human CAPRIN-1 gene from position 698 to 1124 starting from the 5' end. For comparison, GAPDH-specific primers (sense: 5'-GGGCTGCTTTTAACTCTG-3' (Sequence No. 3) and antisense: 5'-TGCTCCTTTTCACCACTG-3' (Sequence No. 4)) were also used.

[0086] The results showed that CAPRIN-1 gene expression was detected in multiple cancer cell lines. Specifically, the following cancer cells were selected from the American Type Culture Collection (ATCC): human breast cancer cells BT-474 (HTB-20), MCF7 (HTB-22), T-47D (HTB-133), SK-BR-3 (HTB-30), HCC70 (CRL-2315), BT-20 (HTB-19), MDA-MB-231 (CRM-HTB-26), MDA-MB-453 (HTB-131), MDA-MB-468 (HTB-132), human bladder cancer cells SW780 (CRL-2169), T24 (HTB-4), HT-1197 (CRL-1473), RT4 (HTB-2), TCCSUP (HTB-5), HT-1376 (CRL-1472), and human pancreatic cancer cells Panc10.05 (CRL-2547), PANC1 (CRL-1469), and SU.86.86 (CRL-1837), human colorectal cancer cells HT-29 (HTB-38), HCT-116 (CCL-247), COLO 205 (CCL-222), DLD-1 (CCL-221), LoVo (CCl-229), human gastric cancer cells NCI-N87 (CRL-5822), human prostate cancer cells 22Rv1 (CRL-2505), human lung cancer cells A549 (CCL-185), NCI-H2228 (CRL-5935), human kidney cancer cells ACHN (CRL-1611), Caki-1 (HTB-46), Caki-2 (HTB-47), human liver cancer cells Hep3B (HB-8064), human head and neck cancer cells FaDu (HTB-43), human ovarian cancer cells TOV-21G (CRL-11730), SKOV3 (HTB-77), OVCAR3 (HTB-161), OV90 (C Human uterine cancer cells HEC-1-A (HTB-112), human prostate cancer cells PC-3 (JCRB9110), DU-145 (HTB-81), human melanoma Malme-3M (HTB-64), SK-MEL-5 (HTB-70), A-375 (CRL-1619), G-361 (CRL-1424), SK-MEL-28 (HTB-72), human brain tumor cells U-87MG (HTB-14), human lymphoma Ramos (CRL-1596), human fibrosarcoma cells HT-1080 (CCL-121), mouse breast cancer cells 4T1 (CRL-2539), mouse kidney cancer cells Renca (CRL-2947), can be obtained from Japanese The following human gallbladder cancer cells were obtained from the Collection of Research Bioresources (JCRB): KKU-213 (JCRB1557), HuCCA-1 (JCRB1657), HuCCT-1 (JCRB0425), KKU-100 (JCRB1568), MRK-nu-1 (JCRB0628), HepG2 (JCRB1054), MCAS (JCRB0240), OVISE (JCRB1043), MIA PaCa-2 (JCRB0070), and IM95 (JCRB1075).CAPRIN-1 gene expression was confirmed in several cancer cell lines, including MKN1 (JCRB0252), MKN74 (JCRB0255), NUGC-2 (JCRB0921), NUGC-3 (JCRB0822), NUGC-4 (JCRB0834), human gallbladder cancer cells TGBC14TKB (RCB1186) obtained from RIKEN Cell Bank, human esophageal cancer cells OE33, and human leukemia cells OCI-AML5 (ACC247). These results confirm that the CAPRIN-1 gene is expressed in various cancer cells.

[0087] (Example 2) Expression of CAPRIN-1 protein in cancer cells

[0088] The expression of CAPRIN-1 protein in the cancer cells used in Example 1 was confirmed by Western blotting. Cell lysates were prepared by dispersing the lysate in RIPA buffer (Nacalai tesque, 16488-34) containing a protease inhibitor (Nacalai tesque, 25955-24) for protein extraction. These lysates were mixed with a sample buffer solution containing DTT, and fractions of different molecular weights were separated in an acrylamide gel and then transferred to a PVDF membrane. Anti-GPIP137 antibodies (Invitrogen, 703658), used as anti-CAPRIN-1 antibodies, were reacted with the transferred PVDF membrane using a prescribed method. The membrane was blocked with TBS-T solution containing 5% skim milk, and then reacted with HRP-labeled anti-rabbit IgG antibodies. After the secondary antibody reaction, a Western Lightning Plus-ECL reaction was performed, and the results were detected using a FUSION SOLO.6S.EDGE (VilberLourmat) imaging system. The results confirmed the expression of CAPRIN-1 protein in the cancer cells described in Example 1.

[0089] (Example 3) Expression of CAPRIN-1 protein on the surface of cancer cell membranes

[0090] The expression of CAPRIN-1 protein on the cell membrane surface of cancer cells from which CAPRIN-1 gene expression was confirmed in Example 1 was verified by flow cytometry. 2 × 10⁻⁶ cells were suspended in Dublin PBS(-) (hereinafter, 1% FCS-PBS(-)) containing 1% fetal bovine serum. 5 One cancer cell was reacted with an antibody against CAPRIN-1 at a concentration of 1–10 μg / mL and reacted on ice (or at 4°C) for 1 hour.

[0091] The anti-CAPRIN-1 antibody used numbers WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2011 / 096535, WO2013 / 018886, WO2013 / 018894, and WO2013 / 018892. Anti-CAPRIN-1 antibodies described in WO2013 / 018891, WO2013 / 018889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640, WO2013 / 147169, WO2013 / 147176 and WO2015 / 020212.

[0092] The negative control used cells that reacted at the same concentration with the same type of antibody that did not show a reaction to CAPRIN-1 protein.

[0093] After the antibody reaction, the cells were washed with 1% FCS-PBS(-), and the fluorescently labeled anti-IgG secondary antibody (FITCGoat anti-rabbit IgG (BD Biosciences, 554020), Alexa Fluor 488 anti-rabbit IgG(H+L) (Invitrogen, A21206), Alexa Fluor 647 anti-rabbit IgG (BioLegend, 406414), Alexa Fluor 488 F(ab')2 anti-mouse IgG(H+L) (Invitrogen, A11017) or Alexa Fluor488 anti-human IgG(H+L) (Invitrogen, A11013)) was reacted with the antibody against CAPRIN-1 at a concentration of 1–10 μg / mL and incubated on ice (or at 4°C) for 1 hour. After reaction with fluorescently labeled secondary antibody, cells were washed with 1% FCS-PBS(-), and reacted with 0.1 μg / mL PI (Propidium Iodide) solution (DOJINDO, 341-07881) or Fixed Viability Stain 450 (BD Horizon) for 15 min. Cells were then washed with 1% FCS-PBS(-), and the scattered light intensity and fluorescence intensity were measured by flow cytometry according to standard methods. The results showed that cells reacted with anti-CAPRIN-1 antibody exhibited stronger fluorescence intensity compared to the negative control. This confirmed that CAPRIN-1 protein is expressed on the cell surface of various human cancer cells.

[0094] (Example 4) Changes in CAPRIN-1 protein expression on the cell membrane surface caused by inhibition of CAPRIN-1 gene expression.

[0095] For the cancer cell lines BT-474, KKU-213, SW780, and OVCAR3, in which CAPRIN-1 protein expression on the cell membrane surface was confirmed in Example 3, Stealth RNAi siRNA for CAPRIN-1 (Invitrogen, HSS106231 and HSS106233) and Stealth RNAi siRNA Negative Control (Invitrogen, 12935300) were transfected with Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, 13778150) according to conventional methods.

[0096] The changes in CAPRIN-1 protein expression on the cell membrane surface when CAPRIN-1 gene expression was suppressed were detected using an anti-CAPRIN-1 antibody and immunostaining according to standard methods. The results confirmed that the expression of CAPRIN-1 protein on the cell membrane surface was reduced by suppressing CAPRIN-1 gene expression in all cancer cells. Figure 1 ).

[0097] (Example 5) Expression profile of CAPRIN-1 protein on cell membrane surface; differences in cytoskeleton-independent colony-forming ability among different cancer cell fractions.

[0098] From cancer cells where CAPRIN-1 protein expression on the cell membrane surface was confirmed in Example 3, two fractions of cancer cell lines HEC-1-A, SK-MEL-5, and Malme-3M, which are cancer cells with different CAPRIN-1 protein expression profiles on the cell membrane, were separated using a cell sorting system (FACS Aria Fusion, BD Biosciences) to separate cancer cell fractions with low CAPRIN-1 protein expression on the cell membrane surface (Low; fraction 1) and cancer cell fractions with high CAPRIN-1 protein expression on the cell membrane surface (High; fraction 2).

[0099] Fraction 1 and Fraction 2 were seeded with the same number of cells in 96-well cells culture plates and incubated for 3 days at 37°C and 5% CO2 in complete medium containing 10% fetal bovine serum. Cell proliferation was then evaluated using the CellTiter-GloLuminescent Cell Viability Assay (Promega, G7575). No significant difference in cell proliferation ability was confirmed between Fraction 1 and Fraction 2 when cultured in standard culture flasks. Next, the cytoskeleton-independent colony-forming ability of the isolated Fraction 1 and Fraction 2 cells was evaluated using a soft agar colony-forming assay. Specifically, 10 cells from each Fraction 1 and Fraction 2 were seeded... 4 Cells were suspended in DMEM culture gel containing 0.4% soft agar (Difco, 214220) and 10% fetal bovine serum, and added to 35 mm diameter culture dishes. The cells were incubated at 37°C and 5% CO2 for 2–4 weeks. After incubation, the number of colonies formed in the gel was counted. Results showed that in all HEC-1-A, SK-MEL-5, and Malme-3M cancer cells, fraction 1 showed a significantly lower colony formation number compared to fraction 2. Figure 2 ).

[0100] These results indicate that the expression of CAPRIN-1 protein on the surface of cancer cell membranes becomes low, thereby reducing the ability of cancer cells to form cytoskeleton-independent colonies.

[0101] (Example 6) Expression profile of CAPRIN-1 protein on cell membrane surface; Differences in tumorigenesis ability of different cancer cell fractions in mouse organisms.

[0102] The cancer cell fractions isolated in Example 5, exhibiting low CAPRIN-1 protein expression on their cell membranes (Low; Fraction 1) and high CAPRIN-1 protein expression on their cell membranes (High; Fraction 2), were cultured in complete culture medium containing 10% fetal bovine serum in conventional culture flasks for 4 days to 1 week. Then, they were resuspended at 1–2.5 × 10⁻⁶ μL in 100 μL of DMEM medium containing 25% Matrigel-Based Membrane Matrix (Corning, 354234). 5 In fractions 1 and 2, the same number of cells were subcutaneously transplanted into NOD-SCID (NOD.CB17-Prkdcscid / NcrCrl) mice under anesthesia. After transplantation, the size of the tumor in the cancer-bearing mice was measured over time using calipers. The tumor volume was calculated using the formula: (length of the major axis of the tumor) × (length of the minor axis of the tumor) according to standard methods. 2 ×0.5 was calculated for each individual, and the mean of the treatment group was also calculated. The results showed that tumor growth in mice transplanted in grade 2 was significantly faster than that in mice transplanted in grade 1. Figure 3 ).

[0103] These results indicate that reducing the expression of CAPRIN-1 protein on the surface of cancer cell membranes can significantly inhibit tumor formation in vivo.

[0104] (Example 7) Changes in the cytoskeleton-independent colony-forming ability of cancer cells caused by enzymatic treatment that breaks down CAPRIN-1 protein on the surface of cancer cell membranes.

[0105] Because the results of Examples 5 and 6 clarified that the ability to form tumors in vitro and in vivo is significantly reduced by decreasing the expression of CAPRIN-1 protein on the surface of cancer cell membranes, thus achieving an anti-tumor effect, in this example, the difference in cytoskeleton-independent colony-forming ability when cancer cells are treated with trypsin, one of the hydrolytic enzymes, to reduce the expression of CAPRIN-1 protein on the cell membrane surface was compared with that of cancer cells that were not treated with trypsin.

[0106] SK-MEL-5 and OVCAR3 cancer cells, which were confirmed to express CAPRIN-1 protein on their cell membrane surface in Example 3, were cultured in standard culture flasks at 37°C and 5% CO2 using complete culture medium containing 10% fetal bovine serum. The cultured cancer cell lines SK-MEL-5 and OVCAR3 were recovered from the flasks using a cell scraper. A portion of the recovered cells was treated for 5 minutes with a solution containing trypsin (Thermo Biotech, Trypsin / EDTA (0.05%)) as a digestive enzyme. The ratio of live cells to dead cells stained with trypsin was then measured and calculated using the trypsin exclusion method according to standard procedures, and the cell density of live cells was calculated. Using the cancer cells SK-MEL-5 and OVCAR3 treated with trypsin as described above, and SK-MEL-5 and OVCAR3 recovered with a cell scraper but not treated with trypsin, the expression of CAPRIN-1 protein on the cell membrane surface was confirmed by flow cytometry, similar to Example 3. The results confirmed that CAPRIN-1 protein was expressed on the cell membrane surface in the untreated cancer cell lines recovered by cell scraping. Conversely, it was confirmed that CAPRIN-1 protein expression was reduced on the cell membrane surface in cancer cell lines treated with trypsin.

[0107] The cytoskeleton-independent colony-forming ability of SK-MEL-5 and OVCAR3 expressed on the cell membrane surface using the CAPRIN-1 protein prepared above, and SK-MEL-5 and OVCAR3 with reduced CAPRIN-1 protein expression on the cell membrane surface, was evaluated by soft agar colony formation assay. Ten cells of each were used... 4 Cells were suspended in DMEM culture gel containing 0.4% soft agar (Difco, 214220) and 10% fetal bovine serum, and added to 35 mm diameter culture dishes. The cells were incubated at 37°C and 5% CO2 for 2–4 weeks. After incubation, the number of colonies formed in the gel was counted. The results showed that the expression of CAPRIN-1 protein on the cell membrane surface was reduced in SK-MEL-5 and OVCAR3, resulting in significantly lower colony numbers compared to SK-MEL-5 and OVCAR3 where CAPRIN-1 protein expression was lower on the cell membrane surface.

[0108] These results indicate that by enzymatically breaking down CAPRIN-1 protein on the surface of cancer cell membranes, the expression of CAPRIN-1 protein on the surface of cancer cell membranes is reduced, thereby decreasing the ability of cancer cells to form cytoskeleton-independent colonies.

[0109] (Example 8) Changes in the cytoskeleton-independent colony-forming ability of cancer cells induced by treatment with anti-CAPRIN-1 expression inhibitory antibody.

[0110] In this embodiment, the cytoskeleton-independent colony-forming ability was investigated by treating cancer cells with a rabbit-derived anti-CAPRIN-1 polyclonal antibody, which is one of the CAPRIN-1 expression inhibitory antibodies, thereby reducing the expression of CAPRIN-1 protein on the surface of cancer cells.

[0111] Use of rabbit-derived anti-CAPRIN-1 polyclonal antibodies: Refer to WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2011 / 096535, WO2013 / 018886, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, WO2 Antibodies identified in serial numbers 013 / 018889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640, WO2013 / 147169, WO2013 / 147176, and WO2015 / 020212 were obtained by purifying peripheral blood from rabbits immunized with recombinant human CAPRIN-1 protein using standard methods via a Protein A column, replacing the solvent with DuPont PBS (-). A negative control was obtained by purifying peripheral blood from unimmunized rabbits using the same Protein A column method, replacing the solvent with PBS (-).

[0112] SK-MEL-5 cells, which were cancer cells whose CAPRIN-1 protein expression on the cell membrane surface was confirmed in Example 3, were cultured in conventional culture flasks using complete culture medium containing 10% fetal bovine serum at 37°C and 5% CO2. The cultured SK-MEL-5 cells were recovered from the flasks using a cell scraper. A portion of the recovered cells was reacted in a solution containing a rabbit-derived anti-CAPRIN-1 polyclonal antibody prepared at 0.1 mg / mL at 4°C for 1 hour. As a negative control, the cytoskeleton-independent colony-forming ability was evaluated using a soft agar colony formation assay, using the product of reacting the negative control antibody with SK-MEL-5 cells prepared in the same manner as described above. Ten cells of each... 4Cells were suspended in DMEM medium gel containing 0.4% soft agar (Difco, 214220) and 10% fetal bovine serum, and added to 35 mm diameter culture dishes. The cells were incubated at 37°C and 5% CO2 for 2–4 weeks. After incubation, the number of colonies formed in the gel was counted. The results showed that SK-MEL-5 cells treated with rabbit-derived anti-CAPRIN-1 polyclonal antibody had significantly lower colony formation numbers compared to SK-MEL-5 cells treated with the negative control antibody.

[0113] These results indicate that by using anti-CAPRIN-1 antibodies to reduce the CAPRIN-1 protein on the surface of cancer cell membranes, the ability of cancer cells to form cytoskeleton-independent colonies is reduced.

[0114] (Example 9) Decreased expression of CAPRIN-1 protein on the surface of cancer cell membranes induced by low molecular weight compounds

[0115] The following inhibitors were used: Dorsomorphin (AMPK inhibitor, final concentration: 0.1 mM), Wnt agonist 1 (final concentration: 20 μM), Akt / PI3K inhibitor LY294002 (final concentration: 10 μM), P53 inhibitor Pifithrin-α (final concentration: 30 μM), Rac1 inhibitor Rac1 inhibitor (CAS # 1177865-17-6) (final concentration: 250 nM), ROS-producing factor Pyocyanin (final concentration: 20 μM), ErbB2 / HER2 inhibitor Mubritinib (final concentration: 1 μM), and GSK-3β inhibitor GSK-3β inhibitor. VIII (final concentration 10 μM), the endocytosis inhibitor Dynasore (final concentration 100 μM), and the Wnt / β-linkin inhibitor Wnt-C59 (final concentration 5 μM) were added to the wells of a standard 6-well plate containing complete culture medium containing 10% fetal bovine serum. HCT-116 cancer cells, whose cell membrane expression of CAPRIN-1 protein was confirmed, were cultured at 37°C and 5% CO2 for 1–3 days. After culture, HCT-116 cells treated with various low-molecular-weight compounds and HCT-116 cells in a negative control group (containing an equal volume of the various low-molecular-weight compounds in DMSO) were recovered by cell scraping. The expression of CAPRIN-1 protein on the cancer cell membrane surface was confirmed in both groups by flow cytometry as described in Example 3. The results confirmed that, in all groups with various low-molecular-weight compounds, the expression of CAPRIN-1 protein on the cancer cell membrane surface was significantly reduced compared to the negative control.

[0116] These results demonstrate that the aforementioned low-molecular-weight compounds can reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes.

[0117] (Example 10) Decreased expression of CAPRIN-1 protein on the surface of cancer cell membrane induced by AMPK inhibitor and its anti-tumor effect

[0118] Dorsomorphin, an AMPK inhibitor confirmed in Example 9 to reduce the expression of CAPRIN-1 protein on the surface of cancer cell membranes, was found to have antitumor effects. HCT-116 cancer cells with confirmed CAPRIN-1 protein expression on their cell membranes were cultured in standard culture flasks using complete medium containing 10% fetal bovine serum, followed by mixing with RPMI-1640 medium containing 20% ​​Matrigel Basement Membrane Matrix (Corning, 354234). 1–2 × 10⁶ mg / L of this medium was administered to each nude mouse. 7 Cancer cells were transplanted subcutaneously under anesthesia. After transplantation, the size of the tumor in the cancer-bearing mice was measured over time using calipers. The tumor volume was calculated using the formula: (length of the major axis of the tumor) × (length of the minor axis of the tumor). 2 ×0.5 was calculated for each individual, and the average value of the treatment group was also calculated. After transplantation of the cancer cells, the tumor size reached 100–200 mm. 3 Dorsomorphin was administered twice weekly at a dose of 10 mg / kg via the tail vein of the above-mentioned cancer-bearing mice. The evaluation results showed that, with the tumor size of untreated cancer-bearing mice being 100%, the tumor size of cancer-bearing mice treated with Dorsomorphin was less than 70%.

Claims

1. A pharmaceutical composition for the treatment and / or prevention of cancer, wherein the active ingredient is a substance capable of reducing the expression of CAPRIN-1 protein on the surface of cancer cell membranes.

2. The pharmaceutical composition according to claim 1, wherein the cancer patient is a cancer patient with cancer in which CAPRIN-1 protein is expressed on the surface of cancer cell membranes.

3. The pharmaceutical composition according to claim 1 or 2, wherein the substance is a substance that reduces the expression level of CAPRIN-1 protein on the surface of cancer cell membranes.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the substance is selected from at least one substance selected from AMPK inhibitors, Wnt agonists, Akt / PI3K inhibitors, P53 inhibitors, Rac1 inhibitors, ROS-producing factors, ErbB2 / HER2 inhibitors, GSK3β inhibitors, endocytosis inhibitors, and Wnt / β-linkin inhibitors.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the substance is a substance capable of inhibiting the expression of the CAPRIN-1 gene.

6. The pharmaceutical composition according to claim 5, wherein the substance is a nucleic acid molecule.

7. The pharmaceutical composition according to claim 6, wherein the nucleic acid molecule is a nucleic acid molecule selected from at least one of the following: interfering small RNA (siRNA), microRNA, small hairpin RNA (shRNA), guide RNA (gRNA), antisense nucleic acid, ribozyme, and nucleic acid aptamer, with CAPRIN-1 gene as the target.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein the substance is an enzyme capable of breaking down CAPRIN-1 protein on the surface of cancer cell membranes.

9. The pharmaceutical composition according to claim 8, wherein the enzyme is trypsin or chymotrypsin.

10. The pharmaceutical composition according to claim 1 or 2, wherein the substance is a substance that binds to the CAPRIN-1 protein on the surface of cancer cell membranes.

11. The pharmaceutical composition according to claim 10, wherein the substance is an antibody or an antigen-binding fragment thereof that binds to the CAPRIN-1 protein on the surface of a cancer cell membrane.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the cancer is ovarian cancer, bile duct cancer, breast cancer, kidney cancer, pancreatic cancer, colorectal cancer, melanoma, lung cancer, renal cell carcinoma, head and neck cancer, gastric cancer, biliary tract cancer, brain tumor, prostate cancer, mesothelioma, colorectal / rectal cancer, esophageal cancer, esophagogastric junction cancer, hepatocellular carcinoma, glioblastoma, urothelial carcinoma, bladder cancer, uterine cancer, liver cancer, sarcoma, fibrosarcoma, mast cell tumor, adrenocortical carcinoma, Ewing's sarcoma, multiple myeloma, testicular cancer, thyroid cancer, basal cell carcinoma, Paget's disease, skin cancer, gastrointestinal stromal tumor (GIST), renal pelvis and ureter cancer, rare cancer, primary central nervous system lymphoma, primary testicular lymphoma, Hodgkin's lymphoma, leukemia, or lymphoma.

13. A method of treating and / or preventing cancer, comprising administering the pharmaceutical composition of any one of claims 1 to 12 to a cancer patient.

14. The method for treating and / or preventing cancer according to claim 13, wherein the cancer patient is a cancer patient suffering from cancer in which CAPRIN-1 protein is expressed on the surface of the cancer cell membrane.

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