# Patent

## Title
Method for detecting cancer via measurement of caprin-1 expression level

## Abstract

This invention provides: a method for detecting a cancer, comprising measuring the expression of a polypeptide having binding reactivity through antigen-antibody reaction with an antibody against CAPRIN-1 having the amino acid sequence shown in any even-numbered SEQ ID NO of SEQ ID NOs: 2 to 30 in the Sequence Listing in a biological sample; a method for detecting a cancer which involves determining the presence and the amount of CAPRIN-1 in a sample of a cancer patient in order to determine the administration of a CAPRIN-1-targeting drug to the cancer patient; and a drug and a kit for the diagnosis of a cancer, comprising an anti-CAPRIN-1 antibody.

## Background
TECHNICAL FIELD
The present invention relates to a method for detecting a cancer with CAPRIN-1 as a tumor marker.
BACKGROUND ART
Cancer is the leading cause of death. This disease is currently treated principally by surgical therapy in combination with radiation therapy and/or chemotherapy. Owing to previous advances in medical technology, cancer is now a disease highly curable if early detected, depending on its type. Therefore, there is a demand for a method for detecting a cancer which places neither physical nor economic burdens on cancer patients and can be achieved by convenient tests.
Recently, methods for assaying tumor products such as tumor markers have been widely available. The tumor products refer to, for example, tumor-related antigens, enzymes, particular proteins, metabolites, oncogenes, oncogene products, and tumor suppressor genes. Carcinoembryonic antigen CEA, glycoprotein CA19-9, prostate-specific antigen PSA, calcitonin (peptide hormone produced in the thyroid gland), and the like are exploited as tumor markers in cancer diagnosis for some cancers. For many types of cancers, however, tumor markers useful in cancer diagnosis have not yet been found. In addition, a large majority of currently known tumor markers is present only in very small amounts (of the order of pg/mL) in body fluids and requires highly sensitive assay methods or special techniques for detecting these markers. Under such circumstances, it can be expected that doors will be opened for diagnostic use for various types of cancers if a novel cancer testing approach capable of highly sensitively detecting various types of cancers by convenient operation can be provided.
Meanwhile, in spite of recent development of novel surgical techniques or discovery of novel anticancer agents, the existing cancer treatment has an insufficiently improved outcome. This is because an effective cancer diagnosis technique has not been established for many cancers, except for some cancers. Inability to detect these cancers early is partly responsible for this situation.
With recent advances in molecular biology or cancer immunology, antibodies specifically reacting with cancer, molecular targeting drugs for cancer antigens related to malignant transformation or cancer exacerbation, and the like have been identified, raising expectations on specific cancer therapy targeting cancer antigens.
Among others, a plurality of antibody drugs for cancer treatment targeting antigenic proteins on cancer cells have been launched and used in the cancer treatment. These antibody drugs have received attention because of their certain efficacy as cancer-specific therapeutic agents. A large majority of antigenic proteins targeted by the drugs, however, are also expressed in normal cells. As a result of administering the antibodies, cancer cells as well as normal cells expressing the antigens are therefore damaged, resulting in undesired adverse reactions. In addition, the effects of cancer treatment differ very largely among individuals due to various factors of the individual cancer patients. For example, surgery, chemotherapy, or radiation therapy largely varies in the treatment and prognosis depending on the stages of cancers. Different persons are known to have distinctive sensitivities to the same therapeutic drug for cancers. This indicates that a certain drug is effective for some patients but ineffective for others due to the diversity of individuals.
Thus, as for some therapeutic drugs, their administration to cancer patients is determined by measuring in advance the expression of disease-related genes or proteins in the patients and evaluating whether a particular drug is effective for a patient expressing a particular gene or protein. Specifically, the presence of a cancer antigen in a sample, for example, serum or tissue, derived from a cancer patient is tested in clinical practice by use of a detection method for assaying a disease-related gene or protein of a certain kind of cancer. Then, the administration of a cancer antigen-specific therapeutic drug is determined. For example, cancer tissues from a large bowel cancer patient are evaluated by an immunohistochemical staining EGFR detection method “EGFR pharm (Dako)” to predict the effectiveness of Erbitux® (cetuximab) for the large bowel cancer. Then, the administration of Erbitux is determined. Further, cancer tissues from a breast cancer patient are evaluated by an immunohistochemical staining Her2 detection method “HercepTest” to predict the effectiveness of Herceptin® (trastuzumab) for the breast cancer. Then, the application of Herceptin is determined.
Incidentally, companion animals have been raised recently as family members and often have lifestyles similar to those of their owners. For this reason, from the occurrence of cancers in companion animals, it can reportedly be predicted that their owners have the high risk of developing cancers in the future.
Dogs, typical companion animals, are known to age 7 times more quickly than humans. Reportedly, the number of dogs currently raised is approximately 6.7 million in Japan and approximately 17.64 million in the USA. Rabies shots as well as combined vaccines such as quintuple, septuple, or octuple combination shots are generally available, leading to decreased rates of highly lethal infections including canine parvovirus infection, canine distemper infection, canine parainfluenza virus infection (kennel cough), canine adenovirus type 2 infection (kennel cough), canine infectious hepatitis, canine coronavirus infection, and leptospirosis. An average dog life-span has therefore been increased, and 7-year-old or older dogs account for 35.5% of the total number of pet dogs. The causes of death such as cancer, hypertension, and heart disease are ever increasing in dogs, as in humans. In the USA, approximately 4 million dogs are yearly diagnosed with cancers. Also in Japan, approximately 1.6 million dogs allegedly have some potential tumor. Checkup examination, however, is not very common in companion animals, unlike humans. This leads to the late detection of disease. In most cases, their owners notice pets' symptoms for the first time after tumors have already become large, and then visit animal hospitals. If such large tumors are malignant, even surgical therapy (e.g., surgical operation) or medication using anticancer agents or the like is very often too late to cure the tumors. Tumors confirmed by veterinarians to be malignant are generally treated with anticancer agents without surgery. Even in the case of performing surgery, it is required to secure surgical margins or to take stringent measures for surgery such as measures against the spread of blood or cells during surgery. Desirably, treatment with anticancer agents is initiated immediately after surgery, and a follow-up is also performed at short intervals. Thus, the medication using therapeutic drugs for cancers is also essential for the cancer-affected companion animals. A detection method, if any, for assaying a disease-related gene or protein of a certain kind of cancer permits more effective treatment than ever and is advantageous both for owners and for veterinarians.
Cytoplasmic- and proliferation-associated protein 1 (CAPRIN-1) is an intracellular protein known to be expressed upon activation or cell division of resting normal cells and to form cytoplasmic stress granules with intracellular RNAs to participate in the regulation of transport and translation of mRNAs. Meanwhile, it has been found that: CAPRIN-1 is highly expressed on the membrane surface of breast cancer cells; and an antibody against CAPRIN-1 exerts strong antitumor effects on breast cancer cells (Patent Literature 1). According to another report, the expression of CAPRIN-1 in a patient-derived sample can be measured using an antibody binding to CAPRIN-1 expressed on cell surface to thereby detect a cancer and to evaluate the grade of the cancer (Patent Literature 2). Specifically, the report states that a plasma membrane protein CAPRIN-1 may serve as a target for cancer treatment or the like. As mentioned above, due to the diversity of cancer patients, it is required to test the presence of CAPRIN-1 in a cancer patient-derived sample for determining the administration of a CAPRIN-1-targeting therapeutic drug, for example, an antibody. Nonetheless, there exists no report on a method for detecting CAPRIN-1 for the application of such a specific therapeutic drug, or there exists no reagent for detecting a cancer using a cancer patient-derived sample.
CITATION LIST
Patent Literature


    Patent Literature 1: WO2010/016526
    Patent Literature 2: WO2010/016527


SUMMARY OF INVENTION
Technical Problem
An object of the present invention is to provide a cancer detection approach useful in the diagnosis of a cancer. Another object of the present invention is to provide a method for detecting a cancer which involves determining the presence and the amount of CAPRIN-1 in a sample of a cancer patient in order to determine the administration of a CAPRIN-1-targeting drug to the cancer patient, and a drug and a kit for the diagnosis of a cancer.
Solution to Problem
As a result of conducting diligent studies, the present inventors have obtained cDNAs encoding proteins binding to antibodies present in cancer-bearing organism-derived serum by SEREX using a dog testis-derived cDNA library and the serum of cancer-bearing dogs, and prepared dog CAPRIN-1s having the amino acid sequences shown in SEQ ID NOs: 6, 8, 10, 12, and 14 on the basis of the cDNAs. The present inventors have also prepared human CAPRIN-1s having the amino acid sequences shown in SEQ ID NOs: 2 and 4 on the basis of human homologous genes of the obtained genes. Consequently, the present inventors have found that: genes encoding these proteins are specifically expressed in dog and human testis, respectively, and in malignant cancer cells (see Example 1 mentioned later); and monoclonal antibodies prepared using, as antigens, recombinant polypeptides prepared on the basis of the amino acid sequences of these proteins can bind to CAPRIN-1 in various cancer tissues and damage cancer cells having CAPRIN-1 on their surface. As a result, the present inventors have gained the finding that CAPRIN-1 can be used as a target for cancer treatment. The present inventors have further found that CAPRIN-1 can be specifically detected from cancer patient-derived samples by use of the monoclonal antibodies mentioned above. Specifically, the present invention provides a method for detecting a cancer, comprising measuring the expression of CAPRIN-1 using a predetermined anti-CAPRIN-1 antibody for application to a sample separated from an organism. In addition, the present invention has established a method for detecting CAPRIN-1 in a cancer patient-derived sample and evaluating the expression level thereof by an immunological assay method using any of the monoclonal antibodies mentioned above, for example, by ELISA for cancer patient-derived serum using a predetermined anti-CAPRIN-1 monoclonal antibody or an immunohistochemical staining method for cancer tissues. The present inventors have also found that as a result of evaluating a cancer-derived sample by this method, the applicability of a CAPRIN-1-targeting drug to a patient is indicated if the expression of CAPRIN-1 and a high abundance thereof are found in the patient. On the basis of these findings, the present invention has been completed.
The present invention provides a method for detecting a cancer which is applied to a sample separated from an organism, the method comprising detecting CAPRIN-1 in the sample and measuring the amount thereof. The present invention also provides a diagnosis method comprising measuring the expression level of CAPRIN-1 in a tissue before administration of a CAPRIN-1-targeting drug to a patient to thereby predict the effectiveness thereof and reveal the applicability of a therapeutic drug against CAPRIN-1 (e.g., whether a CAPRIN-1-targeting drug, for example, an antibody, can be applied to the cancer patient). The present invention further provides a drug or kit for the diagnosis of a cancer, comprising an antibody capable of antigen-antibody reaction with CAPRIN-1, or an antigen-binding fragment thereof.
Specifically, the present invention has the following aspects:
(1) A method for detecting a cancer, comprising measuring the expression level of CAPRIN-1 in a biological sample through antigen-antibody reaction using an antibody having immunological reactivity with a polypeptide having the amino acid sequence shown in SEQ ID NO: 66, or an antigen-binding fragment thereof.
(2) The method for detecting a cancer according to (1), wherein the CAPRIN-1 to be measured is (a) a polypeptide having the amino acid sequence shown in any even-numbered SEQ ID NO of SEQ ID NOs: 2 to 30 in the Sequence Listing, or (b) a polypeptide having 85% or higher sequence identity to the polypeptide having the amino acid sequence shown in any even-numbered SEQ ID NO of SEQ ID NOs: 2 to 30 in the Sequence Listing.
(3) The method for detecting a cancer according to (1) or (2), wherein the biological sample is derived from a human, a dog, or a cat.
(4) The method for detecting a cancer according to any of (1) to (3), wherein the biological sample is derived from a dog, and the CAPRIN-1 to be measured has the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, or 14.
(5) The method for detecting a cancer according to any of (1) to (3), wherein the biological sample is derived from a human, and the CAPRIN-1 to be measured has the amino acid sequence shown in SEQ ID NO: 2 or 4.
(6) The method for detecting a cancer according to any of (1) to (5), wherein a measured CAPRIN-1 expression level which is higher than that of a healthy individual, indicates the presence of the cancer targeted by the antibody as a therapeutic drug for the cancer.
(7) The method for detecting a cancer according to any of (1) to (6), wherein the measurement of the expression level of CAPRIN-1 is carried out using an immunological assay method.
(8) The method for detecting a cancer according to (7), wherein the immunological assay method is ELISA and/or an immunohistochemical staining method.
(9) The method for detecting a cancer according to any of (1) to (8), wherein the sample is a body fluid, a tissue, or a cell.
(10) The method for detecting a cancer according to any of (1) to (9), wherein the cancer is at least one cancer selected from the group consisting of breast cancer, brain tumor, esophagus cancer, stomach cancer, lung cancer, liver cancer, kidney cancer, thyroid gland cancer, spleen cancer, pancreas cancer, large bowel cancer, skin cancer, ovary cancer, uterus cancer, prostate cancer, bladder cancer, testis cancer, osteosarcoma, and fibrosarcoma.
(11) The method for detecting a cancer according to any of (1) to (10), wherein the antibody or the antigen-binding fragment thereof is a monoclonal antibody having a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 70 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 71, or an antigen-binding fragment thereof.
(12) A drug or kit for diagnosis of a cancer, comprising an antibody having immunological reactivity with a polypeptide having the amino acid sequence shown in SEQ ID NO: 66, or an antigen-binding fragment thereof.
(13) The drug or kit for diagnosis of a cancer according to (12), wherein the antibody or the antigen-binding fragment thereof is a monoclonal antibody having a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 70 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 71, or an antigen-binding fragment thereof.
(14) A method for selecting an individual-specific therapeutic drug for a cancer, comprising: measuring the expression level of CAPRIN-1 in a biological sample using an antibody having immunological reactivity with a polypeptide having the amino acid sequence shown in SEQ ID NO: 66, or an antigen-binding fragment thereof; and, if the expression level is statistically significantly higher than that of a healthy individual, determining selecting a CAPRIN-1-targeting drug as a therapeutic drug for a cancer suitable for administration to the individual from which the biological sample is derived.
(15) The method for selecting an individual-specific therapeutic drug for a cancer according to (14), wherein the CAPRIN-1-targeting drug is an antibody having immunological reactivity with CAPRIN-1, or an antigen-binding fragment thereof.
The present application claims the priority from Japanese Patent Application No. 2012-160763, the disclosed content of which is incorporated herein in its entirety.
Advantageous Effects of Invention
The present invention provides a novel method for detecting a cancer, comprising measuring the expression of CAPRIN-1 in a sample separated from a cancer patient. As specifically shown in Examples mentioned later, antibodies prepared using, as antigens, recombinant polypeptides prepared on the basis of the amino acid sequence of CAPRIN-1 (also referred to as Caprin-1 or CAPRIN-1 protein) specifically react with CAPRIN-1 in the body fluids (e.g., serum) or tissues of cancer patients. As also described later in Examples, CAPRIN-1 itself is specifically expressed at high levels in various cancer tissues. The presence and the amount of CAPRIN-1 in a sample separated from a cancer patient can therefore be measured to thereby detect a cancer. In addition, the presence or absence of sensitivity to a CAPRIN-1-targeting drug such as a CAPRIN-1-targeting therapeutic drug, for example, an antibody drug, can be determined in advance to thereby select a patient to which this drug is applicable. Specifically, the expression and the amount of CAPRIN-1 can be measured in advance by the application of the present invention to a cancer patient to thereby provide more efficient treatment using an antibody against CAPRIN-1.



BRIEF DESCRIPTION OF DRAWING
FIG. 1 is a diagram showing the expression patterns of a CAPRIN-1-encoding gene in normal tissues and tumor cell lines. Reference number 1 depicts the expression patterns of the gene encoding the CAPRIN-1 protein. Reference number 2 depicts the expression patterns of the GAPDH gene. The uppermost panel shows the results for dog normal tissues. The left middle panel shows the results for dog breast cancer tissues. The right middle panel shows the results for human breast cancer cell lines. The lowermost panel shows the results for various human cancer cell lines.



DESCRIPTION OF EMBODIMENTS
The method for detecting a cancer according to the present invention comprises measuring the amount (expression level) of CAPRIN-1 (CAPRIN-1 protein) in a sample separated from an organism (biological sample). The measurement of the expression level of CAPRIN-1 in a sample of a cancer patient can be carried out by use of, for example, an immunological assay method which involves detecting CAPRIN-1 using an antibody against CAPRIN-1 (anti-CAPRIN-1 antibody). Various immunological assay methods applicable to the measurement of the expression level of CAPRIN-1 are well known in the art. Examples thereof include immunohistochemical analysis, Western blot analysis, immunoprecipitation, molecular binding assay, ELISA, and biochemical enzyme activity assay. The results of measuring the expression level of CAPRIN-1 by such an assay method can also indicate, for example, the presence of CAPRIN-1 in the sample, the ratio of cells expressing CAPRIN-1, the distribution of expression sites in tissues, and expression intensity on a site basis. In this context, the “expression level” used herein includes the intracellular accumulation level and abundance of the protein.
The results of measuring the expression level of CAPRIN-1 in the sample can be classified into scores shown in Examples. A higher score indicates that CAPRIN-1 is contained in a larger amount in the biological sample (e.g., cancer tissue or cancer serum) of a cancer patient. In the present invention, the term “measurement” or “assay” encompasses all of detection and qualitative, quantitative, and semiquantitative approaches.
The amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, or 14 is the amino acid sequence of dog CAPRIN-1. The dog CAPRIN-1 having this amino acid sequence has been found by SEREX using a dog testis-derived cDNA library and serum derived from cancer-bearing dogs, and identified from the cDNA library as a polypeptide binding to an antibody specifically present in the serum derived from cancer-bearing dogs (see Example 1). CAPRIN-1 itself having the amino acid sequence of SEQ ID NO: 6, 8, 10, 12, or 14 can also be assayed as an antigen in dog tissues by the method mentioned above to thereby diagnose the presence or absence of sensitivity to a CAPRIN-1-targeting drug (see Examples).
In this context, the phrase “having an (the) amino acid sequence” used herein means that amino acid residues are arranged in the order presented in predetermined amino acid sequence information. Thus, for example, the “polypeptide having the amino acid sequence shown in SEQ ID NO: 2” means a polypeptide of 709 amino acid residues in size in which the amino acid residues are linked according to the amino acid sequence Met Pro Ser Ala . . . (snip) . . . Gln Gln Val Asn as shown in SEQ ID NO: 2. Also, for example, the “polypeptide having the amino acid sequence shown in SEQ ID NO: 2” is abbreviated to the “polypeptide of SEQ ID NO: 2”. The same holds true for the phrase “having a (the) nucleotide sequence”. The term “having” in the phrase “having an (the) amino acid sequence” and “having a (the) nucleotide sequence” may be replaced with the term “consisting of”.
The “polypeptide” used herein refers to a molecule that is formed through the peptide bonds of a plurality of amino acids, and encompasses not only a polypeptide molecule constituted by a large number of amino acids but a low-molecular-weight molecule having a small number of amino acids (oligopeptide or peptide) and a full-length protein. The polypeptide according to the present invention also encompasses the full-length proteins of CAPRIN-1 having the amino acid sequences shown in even-numbered SEQ ID NOs of SEQ ID NOs: 2 to 30.
In the method of the present invention, additional mammalian CAPRIN-1 other than the dog CAPRIN-1 of SEQ ID NO: 6, 8, 10, 12, or 14 can also be measured. In the present specification, such non-dog mammalian CAPRIN-1 is also referred to as a “homolog” of dog CAPRIN-1. The term “CAPRIN-1” encompasses CAPRIN-1 derived from not only dogs but other mammals. Examples of the additional mammalian CAPRIN-1 that may be measured in the method of the present invention include, but not limited to, human CAPRIN-1 and cat CAPRIN-1.
As specifically described below in Examples, mRNA encoding human CAPRIN-1 is significantly expressed at high levels in human testis and cancer cells, as with the dog CAPRIN-1 of SEQ ID NO: 6, 8, 10, 12, or 14. An anti-human CAPRIN-1 antibody, however, is not detected in the bodies of healthy humans. An anti-cat CAPRIN-1 antibody is not detected in the bodies of healthy cats, but is detected only in cancer-bearing cats. Thus, the applicability of a CAPRIN-1-targeting drug to a non-dog mammal can also be determined by the measurement of the expression level of CAPRIN-1 derived from the non-dog mammal.
The nucleotide sequence encoding human CAPRIN-1 and the amino acid sequence thereof are shown in SEQ ID NO: 1 or 3 and SEQ ID NO: 2 or 4, respectively, in the Sequence Listing. The sequence identity of human CAPRIN-1 to dog CAPRIN-1 is 94% for the nucleotide sequence and 98% for the amino acid sequence. Since the amino acid sequence identity of CAPRIN-1 is as very high as 98% even between dogs and humans, which are genetically distantly related mammals from each other, many non-human and non-dog mammalian CAPRIN-1 proteins have high (approximately 85% or higher) sequence identity to the human or dog CAPRIN-1. The CAPRIN-1 whose expression level is to be measured in the method of the present invention may be, but not particularly limited to, a protein having preferably 85% or higher, more preferably 95% or higher sequence identity to the amino acid sequence of the dog or human CAPRIN-1 shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14.
An antigenic substance, such as a protein, which has a complicated structure with a large molecular weight, usually contains a plurality of epitopes differing in structure on its molecule. Thus, plural types of antibodies that respectively recognize and bind to a plurality of epitopes on such an antigenic substance are produced in vivo. In other words, antibodies produced in vivo against the antigenic substance (e.g., protein) are polyclonal antibodies, which are mixtures of plural types of antibodies. The antibodies found by the present inventors to be specifically present in serum derived from cancer-affected organisms and to specifically bind to recombinant CAPRIN-1 through antigen-antibody reaction are also polyclonal antibodies. The term “polyclonal antibody” used in the present invention refers to an antibody that is found in serum derived from an organism containing an antigenic substance in the body, and has been induced in the organism against the antigenic substance.
Specific examples of a preferred polypeptide for use as an antigen for obtaining an anti-CAPRIN-1 antibody include polypeptides of even-numbered SEQ ID NOs of SEQ ID NOs: 2 to 30 and fragments thereof. Particularly, an anti-CAPRIN-1 antibody that is obtained using, as an antigen, the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 or a fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 66 containing a preferred epitope and specifically binds to (i.e., has immunological reactivity with) a polypeptide having the amino acid sequence shown in SEQ ID NO: 66 can be preferably used in the method of the present invention.
The nucleotide sequences of polynucleotides encoding polypeptides consisting of the amino acid sequences shown in even-numbered SEQ ID NOs of SEQ ID NOs: 2 to 30 (i.e., SEQ ID NOs: 2, 4, 6, . . . 28, and 30) are shown in odd-numbered SEQ ID NOs of SEQ ID NOs: 1 to 29 (i.e., SEQ ID NOs: 1, 3, 5, . . . 27, and 29), respectively.
It is widely known to those skilled in the art that even a protein derived from a protein antigen by the substitution, deletion, addition, or insertion of a small number of amino acid residues in the amino acid sequence of the protein may generally have almost the same antigenicity as that of the original protein. Thus, a polypeptide having a sequence derived from the amino acid sequence of CAPRIN-1 by the substitution, deletion, addition and/or insertion of a small number of (preferably 1 or several) amino acid residues can also be used in the production of an anti-CAPRIN-1 antibody, as with the polypeptides consisting of the amino acid sequences shown in even-numbered SEQ ID NOs of SEQ ID NOs: 2 to 30, as long as the polypeptide has 80% or higher or 85% or higher, preferably 90% or higher, more preferably 95% or higher, further preferably 98% or higher sequence identity to the original sequence and specifically binds to a polyclonal antibody against CAPRIN-1 through antigen-antibody reaction (hereinafter, this polypeptide is also referred to as a “specifically-reactive modified polypeptide” for the sake of convenience). Preferably, the specifically-reactive modified polypeptide has an amino acid sequence derived from the amino acid sequence of CAPRIN-1 by the substitution, deletion, addition, and/or insertion of 1 or several amino acid residues. The term “several” used herein refers to an integer of 2 to 10, preferably an integer of 2 to 6, more preferably an integer of 2 to 4. The “sequence identity” used herein for the amino acid sequence refers to the percentage of a value determined by dividing the number of matched amino acid residues by the total number of amino acid residues in the best-matching alignments of the amino acid residues in two amino acid sequences to be compared. For the alignments, if necessary, one or both of these two sequences to be compared can be gapped. Such sequence alignments can be carried out using a well known program, for example, BLAST, FASTA, or CLUSTAL W (Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A., 87: 2264-2268, 1993; and Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997).
Twenty types of amino acids constituting a natural protein can be divided according to similar properties into the following groups: neutral amino acids having a low polar side chain (Gly, Ile, Val, Leu, Ala, Met, and Pro); neutral amino acids having a hydrophilic side chain (Asn, Gln, Thr, Ser, Tyr, and Cys); acidic amino acids (Asp and Glu); basic amino acids (Arg, Lys, and His); and aromatic amino acids (Phe, Tyr, Trp, and His). It is known that substitution within each of these groups, i.e., conservative substitution, does not change the properties of the polypeptide in most cases. Thus, in the case of substituting the amino acid residues of CAPRIN-1, a member in each of these groups can be substituted by another member in the same group so that the binding activity against the appropriate antibody is likely to be maintained. In the present invention, however, the modified form may have non-conservative substitution as long as the modified form is provided with immunity-inducing activity equivalent to or substantially equivalent to that of the unmodified form.
The polypeptide used in the present invention can be synthesized according to chemical synthesis methods, for example, Fmoc (fluorenylmethyloxycarbonyl) and tBoc (t-butyloxycarbonyl) methods (Seikagaku Jikken Koza (Biochemical Experimentation Course in English) 1, the Japanese Biochemical Society ed., Protein Chemistry IV, Chemical Modification and Peptide Synthesis, Tokyo Kagaku Dojin Co., Ltd. (Japan), 1981). Also, the polypeptide can be synthesized by routine methods using various commercially available peptide synthesizers. Alternatively, the polypeptide can be easily prepared using genetic engineering approaches known in the art (Sambrook et al., Molecular Cloning, the 2nd edition, Current Protocols in Molecular Biology (1989), Cold Spring Harbor Laboratory Press; Ausubel et al., Short Protocols in Molecular Biology, the 3rd edition, A compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons; etc.). For example, RNA, is extracted from a tissue expressing a gene encoding the human CAPRIN-1 of SEQ ID NO: 2 or a homolog thereof. From this RNA, cDNA of the gene is prepared by RT-PCR. The full-length cDNA or a desired partial fragment thereof is incorporated into expression vectors, which can then be transferred to host cells to obtain the polypeptide of interest. The nucleotide sequences of cDNAs encoding the dog CAPRIN-1 proteins of SEQ ID NOs: 6, 8, 10, 12, and 14 are shown in SEQ ID NOs: 5, 7, 9, 11, and 13, respectively. The nucleotide sequences of cDNAs encoding the human CAPRIN-1 proteins of SEQ ID NOs: 2 and 4 as human homologs thereof are shown in SEQ ID NOs: 1 and 3, respectively. Primers for use in RT-PCR can therefore be easily designed with reference to these nucleotide sequences. As mentioned later, a gene encoding non-human mammalian CAPRIN-1 can be amplified with primers designed with reference to the nucleotide sequence of any odd-numbered SEQ ID NO of SEQ ID NOs: 5 to 29. Thus, cDNA encoding, for example, cat CAPRIN-1, can also be easily prepared by the same approach as above. The RNA extraction, RT-PCR, the incorporation of cDNA into vectors, and the transfer of the vectors to host cells can be carried out, for example, by well known methods as described below. Also, the vectors or host cells used are well known, and various products are commercially available.
The host cells may be any cell capable of expressing the above polypeptide. Examples of prokaryotic cells include E. coli. Examples of eukaryotic cells include: cultured mammalian cells such as monkey kidney cells COS1, Chinese hamster ovary cells CHO, a human embryonic kidney cell line HEK293, and mouse embryonic skin cell line NIH3T3; and budding yeast, fission yeast cells, silkworm cells, and Xenopus egg cells.
In the case of using prokaryotic cells as the host cells, the expression vectors used have an origin that permits replication in the prokaryotic cells, a promoter, a ribosomal binding site, a multicloning site, a terminator, a drug resistance gene, an auxotrophic complementary gene, etc. Examples of expression vectors for E. coli can include pUC series, pBluescript II, pET expression systems, and pGEX expression systems. DNA encoding the above polypeptide can be incorporated into such expression vectors, with which prokaryotic host cells are then transformed, followed by the culture of the obtained transformants so that the polypeptide encoded by the DNA is expressed in the prokaryotic host cells. In this respect, the polypeptide may be expressed as a fusion protein with an additional protein. In this context, the DNA encoding the above polypeptide can be obtained, for example, by the preparation of cDNA by RT-PCR as mentioned above. Alternatively, the DNA may be synthesized by routine methods using commercially available nucleic acid synthesizers as mentioned later. The nucleotide sequences of cDNAs of genes encoding the CAPRIN-1 proteins of SEQ ID NOs: 2 and 4 are shown in SEQ ID NOs: 1 and 3, respectively, in the Sequence Listing.
In the case of using eukaryotic cells as the host cells, expression vectors for eukaryotic cells having a promoter, a splicing region, a poly(A) addition site, etc. are used as the expression vectors. Examples of such expression vectors can include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, and pYES2 vectors. In the same way as above, the DNA encoding the above polypeptide used in the present invention can be incorporated into such expression vectors, with which eukaryotic host cells are then transformed, followed by the culture of the obtained transformants so that the polypeptide encoded by the DNA is expressed in the eukaryotic host cells. In the case of using expression vectors such as pIND/V5-His, pFLAG-CMV-2, pEGFP-N1, or pEGFP-C1, the polypeptide may be expressed as various fusion proteins tagged with His tag (e.g., (His)6 to (His)10), FLAG tag, myc tag, HA tag, GFP, or the like.
The expression vectors can be transferred to the host cells using well known methods such as electroporation, a calcium phosphate method, a liposome method, a DEAE dextran method, microinjection, viral infection, lipofection, and binding with cell-penetrating peptides.
The polypeptide of interest can be isolated and purified from the host cells by a combination of separation operations known in the art. Examples thereof include treatment with a denaturant (e.g., urea) or a surfactant, ultrasonication, enzymatic digestion, salting-out, solvent fractionation and precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing electrophoresis, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse-phase chromatography.
The polypeptides obtained by these methods also include their forms of fusion proteins with other arbitrary proteins. Examples thereof can include fusion proteins with glutathione-S-transferase (GST) or His tag. Such polypeptides in the form of fusion proteins are also encompassed by the specifically reactive added polypeptide mentioned above. The polypeptides expressed in transformed cells may undergo various intracellular modifications after translation. Such posttranslationally modified polypeptides may be used as long as these polypeptides have binding activity against the polyclonal antibody against CAPRIN-1. Examples of such posttranslational modifications can include N-terminal methionine elimination, N-terminal acetylation, glycosylation, intracellular protease-mediated limited degradation, myristoylation, isoprenylation, and phosphorylation.
The CAPRIN-1 as mentioned above or a fragment thereof can be used as an antigen to prepare an anti-CAPRIN-1 antibody. The anti-CAPRIN-1 antibody used in the present invention may be a polyclonal antibody or may be a monoclonal antibody. A monoclonal antibody is more preferred.
In the method of the present invention, an anti-CAPRIN-1 antibody specifically binding to (having immunological reactivity with) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 66, among the anti-CAPRIN-1 antibodies obtained as mentioned above, can be preferably used in analysis such as the measurement of the expression level of CAPRIN-1. Such an anti-CAPRIN-1 antibody can bind to human or dog CAPRIN-1 (e.g., a polypeptide having the amino acid sequence shown in any even-numbered SEQ ID NO of SEQ ID NOs: 2 to 30) or a homolog thereof (e.g., a polypeptide having 85% or higher sequence identity to the polypeptide having the amino acid sequence shown in any even-numbered SEQ ID NO of SEQ ID NOs: 2 to 30) as a target.
The anti-CAPRIN-1 antibody having immunological reactivity with a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 66 can be obtained as a polyclonal antibody by: immunizing an animal with the above CAPRIN-1 or a fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 66; and screening the produced polyclonal antibodies for the immunological reactivity with the polypeptide of SEQ ID NO: 66. Alternatively, the anti-CAPRIN-1 antibody having immunological reactivity with a polypeptide comprising the amino acid sequence SEQ ID NO: 66 can also be obtained as a monoclonal antibody by: immunizing an animal with the above CAPRIN-1 or the fragment thereof; preparing monoclonal antibody-producing hybridomas using its immunocytes such as spleen cells; and further screening for an antibody having immunological reactivity with the polypeptide of SEQ ID NO: 66.
The animal to be immunized can be any non-human animal having spleen cells or the like that permit preparation of hybridoma cells. Examples thereof include mice, rats, hamsters, rabbits, and chickens. A mouse can be used more preferably.
The immunization method involves immunizing the animal with, for example, CAPRIN-1 or a fragment thereof conjugated with a carrier protein such as keyhole limpet hemocyanin (KLH), casein, or serum albumin as an immunogen together with an adjuvant to thereby induce an antibody against CAPRIN-1. More specifically, the above CAPRIN-1 or fragment thereof is subcutaneously or intraperitoneally administered several times together with an adjuvant to, for example, a 4- to 10-week-old mouse. After confirmation of an elevated antibody titer in blood, only CAPRIN-1 or a fragment thereof is intravenously or intraperitoneally administered to the mouse for a boost. At day 3 to 10, blood, ascites, or spleen cells can be collected. In this case, serum obtained from the collected blood, or the ascites contains polyclonal antibodies including anti-CAPRIN-1 antibodies. The obtained polyclonal antibodies can be screened by routine methods such as affinity chromatography for the binding to the polypeptide of SEQ ID NO: 66 to select an antibody having immunological reactivity with the polypeptide of SEQ ID NO: 66.
Examples of the adjuvant can include complete Freund's adjuvants, incomplete Freund's adjuvants, mixtures of aluminum hydroxide gel and pertussis vaccine, MPL+TDM adjuvant (Sigma-Aldrich Corp.), Titer Max Gold (Vaxel Inc.), and GERBU adjuvant (GERBU Biotechnik GmbH).
The antibody titer in blood can be measured by: collecting blood from the eye-ground venous plexus or tail vein of the immunized animal; and examining the presence or absence of the CAPRIN-1-reactive antibody in the obtained blood by an immunological assay method.
The spleen cells can be collected 3 to 10 days after boosting from the immunized animal which had been found to have an elevated antibody titer in blood and then boosted, and fused with myeloma cells to prepare hybridoma cells capable of growing autonomously. These hybridomas can be screened for hybridoma cells producing antibodies having the specificity of interest to prepare monoclonal antibodies in large amounts.
For the cell fusion, for example, SP2/0, P3-X63Ag8-U1 (P3-U1), P3-X63-Ag8653 (653), P3-X63-Ag8 (X63), or P3/NS1/1-Ag4-1 (NS1) can be used as the myeloma cells. These cell lines are available from, for example, ATCC (American Type Culture Collection), ECACC (European Collection of Cell Cultures), or Riken BioResource Center.
The cell fusion of the spleen cells with the myeloma cells can be carried out by: washing the cells of both lines; then mixing the myeloma cells and the spleen cells at a ratio of 1:1 to 10; and adding thereto polyethylene glycol or polyvinyl alcohol having an average molecular weight of 1000 to 6000 as a fusion promoter or using a commercially available cell fusion apparatus based on electrical stimulation (e.g., electroporation).
After the completion of the treatment for cell fusion, the fused cells are washed by suspension in a medium and cloned by a limiting dilution method or by a colony formation method in a methylcellulose medium. In this context, examples of the limiting dilution method can include a method which involves, for example, diluting the cells to 103 to 107 cells/mL and then inoculating the dilution at 102 to 106 cells/well to a 96-well microplate for cell culture, followed by the culture of the cells.
The culture medium for the hybridoma cell cloning is preferably supplemented with a HAT supplement in order to selectively obtain only the fused cells of interest. More specifically, the hybridoma cells of interest can be obtained and cloned according to the methods described in Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, 1988) or Selected Methods in Cellular Immunology (W.H. Freeman and Company, 1980).
The screen for the hybridoma cells producing the antibody having immunological reactivity with the polypeptide of SEQ ID NO: 66 can be performed for as follows: for example, CAPRIN-1 or a fragment thereof is immobilized onto a carrier, to which the culture supernatant (containing anti-CAPRIN-1 antibodies produced by the hybridoma cells) of each hybridoma cell line is then added. After reaction under conditions of 4 to 37° C. for a time long enough to form an antibody/antigen complex, a secondary antibody labeled with, for example, an enzyme, a dye, or a radioisotope is contacted with the formed antibody/antigen complex and reacted under conditions of 4 to 37° C. for a time long enough to form an antibody/antigen/secondary antibody complex. The presence or absence of the formed antibody/antigen/secondary antibody complex is further detected using a signal from the enzyme, dye, or radioisotope label on the secondary antibody as an indicator. An anti-CAPRIN-1 antibody confirmed to form the complex can be selected as the antibody of interest so that hybridoma cells producing this antibody of interest are screened for.
The hybridoma cells thus selected are used to condition a serum-free medium and monoclonal antibodies can be prepared from the resulting culture supernatant. For the large-scale preparation of monoclonal antibodies, for example, 0.5 mL of pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered to a 6- to 8-week-old nude mouse or SCID mouse. After rearing for 2 weeks, the hybridoma cells are intraperitoneally administered thereto at a dose of 5×106 to 2×107 cells/mouse. Monoclonal antibodies can be prepared from ascites generated by rearing for 10 to 21 days.
The thus-obtained anti-CAPRIN-1 antibody having immunological reactivity with the polypeptide having the amino acid sequence shown in SEQ ID NO: 66, or an antigen-binding fragment thereof can be used in the present invention. The antigen-binding fragment of the antibody means any antibody fragment that retains the ability to bind to the antigen. Examples thereof include Fv, scFv, Fab, Fab′, and F(ab)2. The anti-CAPRIN-1 antibody or the antigen-binding fragment may be conjugated with a metal such as manganese or iron.
In the method of the present invention, CAPRIN-1 that may be contained in the sample obtained from an organism (biological sample) is assayed. As mentioned above, cancer cells have been found to have a significantly high expression level (accumulation level) of CAPRIN-1 as an antigen. CAPRIN-1 itself can be assayed in cancer cells or cancer tissues to thereby determine the applicability of a CAPRIN-1-targeting drug to the patient having a high expression level of CAPRIN-1. This is as specifically described below in Examples.
The polypeptide in the biological sample can be easily assayed, as mentioned above, by a well known immunological assay method based on antigen-antibody reaction using the anti-CAPRIN-1 antibody or the antigen-binding fragment thereof. As mentioned above, not only the dog CAPRIN-1 of SEQ ID NO: 6 but its homologs in other mammals, for example, non-dog mammalian CAPRIN-1 (e.g., human CAPRIN-1 of SEQ ID NO: 2 or 4 or cat CAPRIN-1), can be assayed using an antibody capable of antigen-antibody reaction with, for example, the dog CAPRIN-1 of SEQ ID NO: 6, or an antigen-binding fragment thereof, due to cross-reactivity of the antibodies.
The organism from which the biological sample is derived or to which the method of the present invention is applied is a mammal and is preferably a human, a dog, or a cat.
Examples of the biological sample that is subjected to the method of the present invention typically include, but not limited to, body fluids, tissues, and cells. The “body fluid” used herein refers to a biological sample in a liquid state. Examples thereof include blood (including serum, plasma, and interstitial fluid), lymph, ascites, pleural effusion, spinal fluid, sputum, lacrimal fluid, nasal discharge, saliva, urine, vaginal fluid, and semen. The body fluid may additionally include, for example, peritoneal washings with saline. The body fluid used as the biological sample in the present invention is preferably serum, plasma, ascites, or pleural effusion.
For example, the expression level of CAPRIN-1 in the biological sample is measured using the anti-CAPRIN-1 antibody. If the expression level is higher (preferably, statistically significantly higher) than that of a healthy individual, this biological sample is indicated to contain cancer cells or cancer tissues. In the present invention, the “healthy individual” refers to a cancer-unaffected, normal individual of the same organism species as the test subject.
According to one embodiment, the anti-CAPRIN-1 antibody may be immunohistochemically tested for its reactivity with CAPRIN-1 in a tissue sample by an immunological assay method well known to those skilled in the art using a paraformaldehyde- or acetone-fixed frozen section or paraformaldehyde-fixed paraffin-embedded section of a tissue such as a tissue obtained from a patient during surgical operation or from an animal carrying a xenograft tissue inoculated with a cell line expressing CAPRIN-1 either spontaneously or after transfection.
The expression level (accumulation level or abundance) of CAPRIN-1 in the sample thus immunohistochemically stained can be quantitatively determined by numerical scoring based on staining patterns. Two or more scores are preferably set. In the most preferred aspect, the staining patterns are classified into 4 scores. For example, CAPRIN-1 expressed on the surface of cancer cells in a tissue sample is stained by a usual immunohistochemical staining method, and the amount thereof is given any of 4 scores reflecting its staining pattern. In such a case, each score is set as follows.

    
    
        Score 0 (without CAPRIN-1 overexpression): Positive staining of the cell membrane is not observed or is observed in less than 10% of the cancer cells.
        Score 1 (without CAPRIN-1 overexpression): Faint, almost unperceivable staining of the cell membrane is observed in 10% or more of the cancer cells, and these cancer cells are partially stained only at their cell membranes.
        Score 2 (with CAPRIN-1 overexpression): Weak to moderate complete positive staining of the cell membrane is observed in 10% or more of the cancer cells, or strong complete positive staining of the cell membrane is observed in 10% or more and 30% or less of the cancer cells.
        Score 3 (with CAPRIN-1 overexpression): Strong complete positive staining of the cell membrane is observed in 30% or more of the cancer cells.
    
    


This score system is specified by American Society of Clinical Oncology (USA) and approved by The Japanese Society of Pathology (Japan). A similar scoring system is also exploited in “HercepTest” for quantitatively determining the abundance of a cancer antigen Her2 in samples of patients. The quantification of Her2 is specified by the ASCO/CAP Her2 testing guidelines. In Japan, a guideline for Her2 testing including this scoring system is also specified by the pathological committee for trastuzumab.
The ratio of stained cancer cells after immunohistochemical staining as indicated in each score can be determined by: counting at least 500 cells in the field of view using a light microscope with sensitivity increased to 4 times, 10 times, or 20 times; measuring cells that exhibit stain images of their cell membranes as described in each score; and making a trial calculation according to the following expression.

The number of positive cells/The total number of cells(approximately 500 cells)×100

In these scoring criteria, biological samples with scores 2 and 3 can be determined to contain CAPRIN-1-expressing cancer tissues.
For the immunohistochemical staining, the antigen-antibody reaction of the anti-CAPRIN-1 antibody can be visualized by various methods. For example, the anti-CAPRIN-1 antibody is reacted with a secondary antibody labeled with an enzyme such as horseradish peroxidase or alkaline phosphatase, and the reaction (e.g., color reaction, chemiluminescence, or chemical fluorescence) of the enzyme can be induced to thereby visualize the binding of the anti-CAPRIN-1 antibody to CAPRIN-1. A fluorescent label, a radioisotope label, a biotin label, or the like can be used in the labeling of the secondary antibody.
CAPRIN-1 has been found to be a plasma membrane protein that is expressed on the surface of cancer cells. Since organisms contain many proteolytic enzymes, the extracellular region of CAPRIN-1 expressed on cancer cells in the body of a cancer patient is separated from the cancer cells upon degradation. The extracellular region thus separated is therefore present in larger amounts in the outside of the cells, compared with the intracellular region of CAPRIN-1. Accordingly, CAPRIN-1 present not only in cancer tissues but in body fluids or cell populations derived from cancer-affected individuals (e.g., cancer tissues fixed on slide glass or the serum of cancer patients) can be detected by the detection of CAPRIN-1 using an anti-CAPRIN-1 antibody or an antigen-binding fragment thereof capable of binding more strongly to the extracellular region of CAPRIN-1 present on the surface of cancer cells. Thus, in the present invention, an anti-CAPRIN-1 antibody binding to a portion expressed on the surface of cancer cells (CAPRIN-1 extracellular region) in the CAPRIN-1 protein is preferably used. Examples of the partial peptide of CAPRIN-1 recognized by such an antibody include partial peptides consisting of a sequence in extracellular regions in the amino acid sequences shown in even-numbered SEQ ID NOs of SEQ ID NOs: 2 to 30 in the Sequence Listing except for SEQ ID NOs: 6 and 18. Such sequence in these extracellular regions corresponds to a sequence of 7 or more consecutive amino acids in the region of amino acid residues (aa) 50 to 98 or amino acid residues (aa) 233 to 344 based on SEQ ID NO: 2 as a reference. Specifically, such a preferred anti-CAPRIN-1 antibody binds to a partial peptide of CAPRIN-1 comprising, for example, a sequence in the amino acid sequences of SEQ ID NOs: 43, 61, and 62 located in the extracellular region of CAPRIN-1 expressed on cancer cells. Also, an anti-CAPRIN-1 antibody particularly preferably used binds to a peptide comprising an amino acid sequence having 80% or higher, preferably 85% or higher, more preferably 90% or higher, further preferably 95% or higher sequence identity to any of these amino acid sequences. The anti-CAPRIN-1 antibody used in the method of the present invention, which specifically binds to (has immunological reactivity with) the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 66, can bind to the extracellular region of CAPRIN-1. Thus, CAPRIN-1 can be detected with high sensitivity by use of the method of the present invention. The anti-CAPRIN-1 antibody specifically binding to (having immunological reactivity with) the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 66 is further preferably an antibody (preferably a monoclonal antibody) having a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 70 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 71, or an antigen-binding fragment thereof.
The cancer to be detected by the method of the present invention is a cancer overexpressing CAPRIN-1 and examples thereof include, but not limited to, breast cancer, brain tumor, esophagus cancer, stomach cancer, lung cancer, liver cancer, kidney cancer, thyroid gland cancer, spleen cancer, pancreas cancer, large bowel cancer, skin cancer, ovary cancer, uterus cancer (uterine cervix cancer and uterine body cancer), prostate cancer, bladder cancer, testis cancer, and osteosarcoma. Other examples thereof can include, but not limited to, squamous cell cancer of the head and neck, melanoma, various types of adenocarcinomas, hepatocellular cancer, basal cell cancer, acanthoma-like gingival tumor, tumor mass in the oral cavity, perianal gland cancer, tumor mass of the anal sac, anal sac apocrine adenocarcinoma, Sertoli cell carcinoma, vaginal vestibule cancer, sebaceous cancer, sebaceous epithelioma, sebaceous adenoma, sweat gland cancer, adenocarcinoma in the nasal cavity, adenocarcinoma of the nose, bronchial adenocarcinoma, ducal cancer, mammary gland cancer, mammary complex carcinoma, malignant mixed tumor of the mammary gland, intraductal papillary adenocarcinoma, fibrosarcoma, hemangiopericytoma, chondrosarcoma, soft tissue sarcoma, histiocytic sarcoma, myxosarcoma, primitive sarcoma, lung cancer, mastocytoma, cutaneous leiomyoma, intraperitoneal leiomyoma, leiomyoma, chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, lymphoma of the digestive organ, small/medium cell lymphoma, adrenal medullary tumor, granulosa cell tumor, and pheochromocytoma.
In the method of the present invention, if the measured CAPRIN-1 expression level is higher (preferably, statistically significantly higher) than that of a healthy individual, the presence of a cancer that can be specifically bound by the anti-CAPRIN-1 antibody used in the measurement (i.e., a cancer that can be targeted by the antibody as a therapeutic drug for the cancer) in the organism (individual) from which the biological sample is derived, is indicated. Based on this, the expression level of CAPRIN-1 in a cancer patient-derived biological sample can be measured by the method of the present invention and compared with that of a healthy individual to determine whether a CAPRIN-1-targeting drug is applicable to the cancer in the patient (e.g., whether the cancer in the patient can be targeted by the antibody as a therapeutic drug for the cancer).
Thus, the present invention enables an identification of a cancer patient that can be expected to get therapeutic effects by the administration of a CAPRIN-1-targeting drug including a CAPRIN-1-targeting antibody, and thus the provision of more effective cancer treatment.
According to one embodiment, the present invention relates to a method for selecting an individual-specific therapeutic drug for a cancer, comprising: measuring the expression level of CAPRIN-1 in a biological sample using an antibody having immunological reactivity with a polypeptide having the amino acid sequence shown in SEQ ID NO: 66; and, if the expression level is higher (preferably, statistically significantly higher) than that of a healthy individual, selecting a CAPRIN-1-targeting drug, preferably an antibody having immunological reactivity with CAPRIN-1 or an antigen-binding fragment thereof, as a therapeutic drug for a cancer suitable for administration to the individual from which the biological sample is derived. This selection of the individual-specific therapeutic drug for a cancer realizes so-called tailor-made medicine, which offers cancer therapy optimized for an individual patient.
The term “statistically significantly” used herein means that statistically treated quantitative difference between the two is a significant difference. Specifically, examples thereof include the case where a significance level is smaller than 5%, 1%, or 0.1%. The method of verification is not particularly limited as long as the method is known in the art and is capable of determining the presence or absence of significance. For example, a Student's t test or multiple comparison test method can be used.
The present invention also provides a drug or kit for the diagnosis of a cancer, comprising, as a reagent, an anti-CAPRIN-1 antibody (particularly, an anti-CAPRIN-1 antibody having immunological reactivity with a polypeptide having the amino acid sequence shown in SEQ ID NO: 66) or an antigen-binding fragment thereof for use in the measurement of the expression of CAPRIN-1 according to the present invention. In this case, the drug or kit for the diagnosis of a cancer may further comprise, for example, various additives useful in the stabilization or the like of the antibody or the antigen-binding fragment. The anti-CAPRIN-1 antibody or the antigen-binding fragment may be conjugated with a metal such as manganese or iron. Such a metal-conjugated antibody or antigen-binding fragment, when administered to the body, accumulates to a site containing a larger amount of the antigenic protein. Accordingly, the presence of cancer cells producing the antigenic protein can be detected by the MRI measurement or the like of the metal.
EXAMPLES
Hereinafter, the present invention will be described more specifically with reference to Examples. However, the scope of the present invention is not intended to be limited by these Examples.
Example 1
Analysis of CAPRIN-1 Expression in Each Tissue
The expression of the CAPRIN-1 gene in dog and human normal tissues and various cancer tissues and cancer cell lines was examined by RT-PCR according to Example 1(4) of WO2010/016526. As a result, its strong expression was observed in the testis among the normal tissues of the healthy dog. Also, the expression was observed in dog breast cancer (FIG. 1) and adenocarcinoma tissues. Further, the expression of the gene was also confirmed in human tissues. As a result, the expression was confirmed only in the testis among the normal tissues, as with the dog CAPRIN-1 gene, but was detected in many types of cancer cell lines including 8 human breast cancer cell lines (ZR75-1, MCF7, T47D, SK-BR-3, MDA-MB-157, BT-20, MDA-MB-231V, and MRK-nu-1) as well as a brain tumor cell line, a leukemia-derived cell line, a lung cancer cell line, and an esophagus cancer cell line (FIG. 1). These results demonstrated that CAPRIN-1 is not expressed in normal tissues except for the testis, but is expressed in many cancer cells including breast cancer cells.
Example 2
Preparation of Antibody Against CAPRIN-1
(1) Preparation of Mouse Anti-Human CAPRIN-1 Monoclonal Antibody
100 μg of human CAPRIN-1 having the amino acid sequence of SEQ ID NO: 2 as prepared in Example 3 of WO2010/016526 was mixed with an equal amount of MPL+TDM adjuvant (Sigma-Aldrich Corp.). This mixture was used as an antigen solution per mouse. This antigen solution was intraperitoneally administered to each 6-week-old Balb/c mouse (Japan SLC, Inc.). Then, 3 boosters were performed every 1 week. Three days after the final immunization, the spleen of the mouse was excised and ground between two sterilized glass slides. Procedures of washing with PBS(-) (Nissui Pharmaceutical Co., Ltd.) and removing the supernatant by centrifugation at 1500 rpm for 10 minutes were repeated three times to obtain spleen cells. The obtained spleen cells were mixed with mouse myeloma cells SP2/0 (purchased from ATCC) at a ratio of 10:1. 200 μL of an RPMI1640 medium containing 10% FBS was heated to 37° C. and mixed with 800 μL of PEG1500 (Boehringer Ingelheim GmbH), and the PEG solution thus prepared was added to the cell mixture, which was then left standing for 5 minutes for cell fusion. After removal of the supernatant by centrifugation at 1700 rpm for 5 minutes, the cells were suspended in 150 ml of an RPMI1640 medium containing 15% FBS supplemented with 2% equivalent of a HAT solution (Life Technologies, Inc./Gibco) (HAT selective medium). This suspension was inoculated to fifteen 96-well plates (Nunc) at 100 μL/well. The spleen cells and the myeloma cells were fused by culture under conditions of at 37° C. for 7 days in 5% CO2 to obtain hybridomas.
The prepared hybridomas were screened for the binding affinity of antibodies produced by the hybridomas against CAPRIN-1 as an indicator. The 1 μg/ml CAPRIN-1 protein solution was added to a 96-well plate at 100 μL/well and left standing at 4° C. for 18 hours. Each well was washed three times with PBS-T. Then, a 0.5% bovine serum albumin (BSA) solution (Sigma-Aldrich Corp.) was added thereto at 400 μL/well and left standing at room temperature for 3 hours. The solution in each well was discarded, and each well was washed three times with 400 μL of PBS-T. Then, the culture supernatant of each hybridoma obtained above was added thereto at 100 μL/well and left standing at room temperature for 2 hours. Each well was washed three times with PBS-T. Then, HRP-labeled anti-mouse IgG (H+L) antibodies (Life Technologies, Inc.) diluted 5000-fold with PBS were added thereto at 100 μL/well and left standing at room temperature for 1 hour. Each well was washed three times with PBS-T. Then, a TMB substrate solution (Thermo Fisher Scientific Inc.) was added thereto at 100 μL/well and left standing for 15 to 30 minutes to cause color reaction. After the color development, the reaction was terminated by the addition of 1 N sulfuric acid at 100 μL/well. The absorbance was measured at 450 nm and 595 nm using an absorption spectrometer. As a result, several hybridomas producing antibodies having high absorbance were selected as candidate lines of the hybridoma of interest.
The selected hybridomas were added to a 96-well plate at a density of 0.5 cells/well and cultured in the plate. One week later, hybridomas forming single colonies in the wells were observed. The cells in these wells were further cultured, and the cloned hybridomas were screened for the binding affinity of antibodies produced by the hybridomas against CAPRIN-1 as an indicator. The 1 μg/ml CAPRIN-1 protein solution was added to a 96-well plate at 100 μL/well and left standing at 4° C. for 18 hours. Each well was washed three times with PBS-T. Then, a 0.5% BSA solution was added thereto at 400 μL/well and left standing at room temperature for 3 hours. The solution in each well was discarded, and each well was washed three times with 400 μL of PBS-T. Then, the culture supernatant of each hybridoma obtained above was added thereto at 100 μL/well and left standing at room temperature for 2 hours. Each well was washed three times with PBS-T. Then, HRP-labeled anti-mouse IgG (H+L) antibodies (Life Technologies, Inc.) diluted 5000-fold with PBS were added thereto at 100 μL/well and left standing at room temperature for 1 hour. Each well was washed three times with PBS-T. Then, a TMB substrate solution (Thermo Fisher Scientific Inc.) was added thereto at 100 μL/well and left standing for 15 to 30 minutes to cause color reaction. After the color development, the reaction was terminated by the addition of 1 N sulfuric acid at 100 μL/well. The absorbance was measured at 450 nm and 595 nm using an absorption spectrometer. As a result, a plurality of hybridoma lines producing monoclonal antibodies reactive with CAPRIN-1 were obtained. The culture supernatants of these hybridomas were purified using a protein G carrier and 150 types of monoclonal antibodies binding to CAPRIN-1 were obtained.
Next, these monoclonal antibodies were screened for the reactivity with the surface of breast cancer cells expressing CAPRIN-1. Specifically, 106 cells of a human breast cancer cell line MDA-MB-231V were centrifuged in a 1.5-ml microcentrifuge tube. 100 μL of the supernatant of each hybridoma obtained above was added thereto and left standing for 1 hour on ice. After washing with PBS, FITC-labeled goat anti-mouse IgG antibodies (Life Technologies, Inc.) diluted 500-fold with PBS containing 0.1% fetal bovine serum were added thereto and left standing for 1 hour on ice. After washing with PBS, the fluorescence intensity was measured using FACSCalibur (Becton, Dickinson and Company). On the other hand, the same operation as above was performed with the addition of a medium instead of the antibodies as a control. As a result, 10 monoclonal antibodies (#1 to #10) having stronger fluorescence intensity than that of the control, i.e., reactive with the surface of breast cancer cells, were selected. The respective sequences of the heavy chain and light chain variable regions of these monoclonal antibodies are shown in SEQ ID NOs: 44 to 60. The monoclonal antibody #1 comprises the heavy chain variable region of SEQ ID NO: 44 and the light chain variable region of SEQ ID NO: 45; the monoclonal antibody #2 comprises the heavy chain variable region of SEQ ID NO: 44 and the light chain variable region of SEQ ID NO: 46; the monoclonal antibody #3 comprises the heavy chain variable region of SEQ ID NO: 44 and the light chain variable region of SEQ ID NO: 47; the monoclonal antibody #4 comprises the heavy chain variable region of SEQ ID NO: 44 and the light chain variable region of SEQ ID NO: 48; the monoclonal antibody #5 comprises the heavy chain variable region of SEQ ID NO: 49 and the light chain variable region of SEQ ID NO: 50; the monoclonal antibody #6 comprises the heavy chain variable region of SEQ ID NO: 51 and the light chain variable region of SEQ ID NO: 52; the monoclonal antibody #7 comprises the heavy chain variable region of SEQ ID NO: 53 and the light chain variable region of SEQ ID NO: 54; the monoclonal antibody #8 comprises the heavy chain variable region of SEQ ID NO: 55 and the light chain variable region of SEQ ID NO: 56; the monoclonal antibody #9 comprises the heavy chain variable region of SEQ ID NO: 57 and the light chain variable region of SEQ ID NO: 58; and the monoclonal antibody #10 comprises the heavy chain variable region of SEQ ID NO: 59 and the light chain variable region of SEQ ID NO: 60.
(2) Identification of Peptide in CAPRIN-1 Bound by Mouse Anti-CAPRIN-1 Antibody Reactive with Cancer Cell Surface
The cancer cell surface-reactive mouse anti-CAPRIN-1 monoclonal antibodies #1 to #10 obtained above were used to identify partial sequences in CAPRIN-1 recognized thereby.
First, DTT (Fluka) was added at a final concentration of 10 mM to 100 μL of a 1 μg/μL protein solution of recombinant CAPRIN-1 dissolved in PBS, and reacted at 95° C. for 5 minutes to reduce disulfide bonds in the CAPRIN-1 proteins. Next, 20 mM (final concentration) iodoacetamide (Wako Pure Chemical Industries, Ltd.) was added thereto, followed by the alkylation reaction of thiol groups at 37° C. for 30 minutes under shading conditions. 50 μg each of the mouse anti-CAPRIN-1 monoclonal antibodies #1 to #10 was added to 40 μg of the obtained reduced alkylated CAPRIN-1 proteins. The total amount of each mixture was adjusted to 1 mL with a 20 mM phosphate buffer (pH 7.0). The resulting mixture was reacted overnight at 4° C. while mixed by stirring.
Next, trypsin (Promega K.K.) was added at a final concentration of 0.2 μg to each reaction mixture and reacted at 37° C. for 1 hour, 2 hours, 4 hours, or 12 hours. Then, the reaction mixture was mixed with protein A-glass beads (GE Healthcare Bio-Sciences Ltd.) blocked with PBS containing 1% BSA (Sigma-Aldrich Corp.) and washed with PBS in advance, 1 mM calcium carbonate, and NP-40 buffer (20 mM phosphate buffer (pH 7.4), 5 mM EDTA, 150 mM NaCl, 1% NP-40) and reacted for 30 minutes.
Each reaction solution was washed with a 25 mM ammonium carbonate buffer (pH 8.0), followed by the elution of antigen-antibody complexes using 100 μL of 0.1% formic acid. The eluate was analyzed by LC-MS using Q-TOF Premier (Waters-MicroMass). This analysis followed the protocol attached to the instrument.
As a result, the polypeptide of SEQ ID NO: 61 was identified as a partial CAPRIN-1 sequence recognized by all of the mouse anti-human CAPRIN-1 monoclonal antibodies #1 to #10. In the polypeptide of SEQ ID NO: 61, the peptide of SEQ ID NO: 62 was further identified as a partial sequence recognized by the monoclonal antibodies #1 to #4, #5 to #7, and #9. As a partial sequence peptide, the peptide of SEQ ID NO: 63 was further found to be recognized by the monoclonal antibody #10.
(3) Preparation of Chicken Anti-Human CAPRIN-1 Monoclonal Antibody
300 μg of human CAPRIN-1 having the amino acid sequence of SEQ ID NO: 2 as prepared in Example 3 of WO2010/016526 was mixed with an equal amount of a complete Freund's adjuvant. This mixture was used as an antigen solution per chicken. The antigen solution was intraperitoneally administered to 7-week-old chickens. Then, 7 boosters were performed every 4 weeks to complete immunization. Four days after the final shot, the spleen of each chicken was excised and ground between two sterilized glass slides. Procedures of washing with PBS(-) (Nissui Pharmaceutical Co., Ltd.) and removing the supernatant by centrifugation at 1500 rpm for 10 minutes were repeated three times to obtain spleen cells. The obtained spleen cells were mixed with light chain-deficient chicken myeloma cells established from chickens by transformation using avian reticuloendotheliosis virus, at a ratio of 5:1. 200 μL of an IMDM medium containing 10% FBS was heated to 37° C. and mixed with 800 μL of PEG1500 (Boehringer Ingelheim GmbH), and the PEG solution thus prepared was added to the cell mixture, which was then left standing for 5 minutes for cell fusion. After removal of the supernatant by centrifugation at 1700 rpm for 5 minutes, the cells were suspended in 300 ml of an IMDM medium containing 10% FBS supplemented with 2% equivalent of a HAT solution (Gibco) (HAT selective medium). This suspension was inoculated to thirty 96-well plates (Nunc) at 100 μL/well. The spleen cells and the chicken myeloma cells were fused by culture at 37° C. for 7 days in 5% CO2 to obtain hybridomas.
The prepared hybridomas were screened for the binding affinity of antibodies produced by the hybridomas against CAPRIN-1 proteins as an indicator. The 1 μg/ml CAPRIN-1 protein solution was added to a 96-well plate at 100 μL/well and left standing at 4° C. for 18 hours. Each well was washed three times with PBS-T. Then, a 0.5% bovine serum albumin (BSA) solution (Sigma-Aldrich Corp.) was added thereto at 400 μL/well and left standing at room temperature for 3 hours. The solution in each well was discarded, and each well was washed three times with 400 μL of PBS-T. Then, the culture supernatant of each hybridoma obtained above was added thereto at 100 μL/well and left standing at room temperature for 2 hours. Each well was washed three times with PBS-T. Then, HRP-labeled anti-chicken IgY antibodies (Sigma-Aldrich Corp.) diluted 5000-fold with PBS were added thereto at 100 μL/well and left standing at room temperature for 1 hour. Each well was washed three times with PBS-T. Then, a TMB substrate solution (Thermo Fisher Scientific Inc.) was added thereto at 100 μL/well and left standing for 15 to 30 minutes to cause color reaction. After the color development, the reaction was terminated by the addition of 1 N sulfuric acid at 100 μL/well. The absorbance was measured at 450 nm and 595 nm using an absorption spectrometer. As a result, several hybridomas producing antibodies having high absorbance were selected as candidate lines of the hybridoma of interest.
The selected hybridomas were added to a 96-well plate at a density of 0.5 cells/well and cultured in the plate. One week later, hybridomas forming single colonies in the wells were observed. The cells in these wells were further cultured, and the cloned hybridomas were screened for the binding affinity of antibodies produced by the hybridomas against CAPRIN-1 proteins as an indicator. The 1 μg/ml CAPRIN-1 protein solution was added to a 96-well plate at 100 μL/well and left standing at 4° C. for 18 hours. Each well was washed three times with PBS-T. Then, a 0.5% BSA solution was added thereto at 400 μL/well and left standing at room temperature for 3 hours. The solution in each well was discarded, and each well was washed three times with 400 μL of PBS-T. Then, the culture supernatant of each hybridoma obtained above was added thereto at 100 μL/well and left standing at room temperature for 2 hours. Each well was washed three times with PBS-T. Then, HRP-labeled anti-chicken IgY antibodies (Sigma-Aldrich Corp.) diluted 5000-fold with PBS were added thereto at 100 μL/well and left standing at room temperature for 1 hour. Each well was washed three times with PBS-T. Then, a TMB substrate solution (Thermo Fisher Scientific Inc.) was added thereto at 100 μL/well and left standing for 15 to 30 minutes to cause color reaction. After the color development, the reaction was terminated by the addition of 1 N sulfuric acid at 100 μL/well. The absorbance was measured at 450 nm and 595 nm using an absorption spectrometer. As a result, a plurality of hybridoma lines producing monoclonal antibodies reactive with CAPRIN-1 proteins were obtained as candidate lines of the hybridoma of interest.
Next, these monoclonal antibodies were screened for the reactivity with the surface of breast cancer cells expressing CAPRIN-1. Specifically, 5×105 cells of a human breast cancer cell line MDA-MB-231V were centrifuged in a 1.5-ml microcentrifuge tube. 100 μL of the culture supernatant of each hybridoma obtained above was added thereto and left standing for 1 hour on ice. After washing with PBS, FITC-labeled goat anti-chicken IgG (H+L) antibodies (SouthernBiotech) diluted 30-fold with PBS containing 0.1% FBS were added thereto and left standing for 1 hour on ice. After washing with PBS, the fluorescence intensity was measured using FACSCalibur (Becton, Dickinson and Company). On the other hand, the same operation as above was performed using a medium for hybridoma culture to prepare a control sample. As a result, 1 monoclonal antibody (chicken anti-human CAPRIN-1 monoclonal antibody #11) having stronger fluorescence intensity than that of the control, i.e., reactive with the surface of breast cancer cells expressing CAPRIN-1, was selected.
(4) Preparation of Mouse-chicken Chimeric Recombinant Antibody
The gene amplification fragment of the heavy chain variable region (SEQ ID NO: 64) of the chicken anti-human CAPRIN-1 monoclonal antibody #11 obtained in the preceding paragraph (3) was treated at both ends with restriction enzymes, then purified, and inserted according to a routine method into a pcDNA4/myc-His vector (Life Technologies, Inc.) into which a chicken antibody-derived leader sequence and a mouse IgG1 H chain constant region have been inserted. Also, the gene amplification fragment of the light chain variable region (SEQ ID NO: 65) of the chicken anti-human CAPRIN-1 monoclonal antibody #11 was treated at both ends with restriction enzymes, then purified, and inserted according to a routine method into a pcDNA3.1/myc-His vector (Life Technologies, Inc.) into which a chicken antibody-derived leader sequence and a mouse IgG1 L chain constant region have been inserted.
Next, the recombinant vector having the gene insert of the heavy chain variable region of the chicken anti-human CAPRIN-1 monoclonal antibody #11 and the recombinant vector having the gene insert of the light chain variable region of the chicken anti-human CAPRIN-1 monoclonal antibody #11 were introduced into CHO-K1 cells (obtained from Riken Cell Bank). Specifically, 2×105 CHO-K1 cells were cultured in 1 ml of Ham's F12 medium (Life Technologies, Inc.) containing 10% FBS per well of a 12-well culture plate, and washed with PBS(-). Then, 1 ml of fresh Ham's F12 medium containing 10% FBS per well was added thereto. The vectors (250 ng each) dissolved in 30 μL of OptiMEM (Life Technologies, Inc.) was mixed with 30 μL of Polyfect transfection reagent (Qiagen N.V.), and this mixture was added to each well. The CHO-K1 cells cotransfected with the recombinant vectors were cultured in a Ham's F12 medium containing 10% FBS supplemented with 200 μg/ml Zeocin (Life Technologies, Inc.) and 200 μg/ml Geneticin (Roche Diagnostics K.K.) and then inoculated to a 96-well plate at a density of 0.5 cells/well to prepare a cell line stably producing a mouse-chicken chimeric anti-human CAPRIN-1 monoclonal antibody #12 having the variable regions of the chicken anti-human CAPRIN-1 monoclonal antibody #11 and the constant regions of mouse IgG1. The prepared cell line was cultured for 5 days in a 150-cm2 flask at a density of 5×10≡ cells/ml using 30 ml of a serum-free OptiCHO medium (Life Technologies, Inc.) to obtain a culture supernatant containing #12.
(5) Identification of CAPRIN-1 Epitope Recognized by Mouse-chicken Chimeric Anti-human CAPRIN-1 Monoclonal Antibody #12
The cancer cell surface-reactive mouse-chicken chimeric anti-human CAPRIN-1 monoclonal antibody #12 obtained in the paragraph (4) was used to identify a CAPRIN-1 epitope region recognized thereby. 100 μg of recombinant CAPRIN-1 proteins was dissolved in a protein inhibitor-free dissolving buffer and reacted with the mouse-chicken chimeric anti-human CAPRIN-1 monoclonal antibody #12. To this solution, a digestive enzyme trypsin or chymotrypsin was added, followed by digestion reaction at a suitable temperature. After the reaction, a protein G Sepharose carrier was added thereto, then reacted, and precipitated by centrifugation operation. After removal of the supernatant, the carrier was washed with a dissolving buffer and PBS and dissolved in 0.1% formic acid, and the supernatant was recovered. The recovered supernatant sample was applied to a reverse-phase column (HLB Extraction Cartridge (Waters-OASIS)) to obtain an antibody-free sample solution. The obtained sample was subjected to reverse-phase liquid chromatography (Chromatography Nanosytem (KYA Technologies Corp.)) to recover a solution containing only peptides. The solution was introduced to a tandem-type mass spectrometer Quadrupole-TOF Mass Spectrometer (Waters-MicroMass) and analyzed by MS/MS to detect the peptides contained in the sample. As a result, a peptide consisting of the amino acid sequence of SEQ ID NO: 66 was identified as a partial CAPRIN-1 sequence recognized by the mouse-chicken chimeric anti-human CAPRIN-1 monoclonal antibody #12. The chicken anti-CAPRIN-1 monoclonal antibody #11 has the same heavy chain and light chain variable regions as those of the mouse-chicken chimeric anti-human CAPRIN-1 monoclonal antibody #12 and as such, recognizes this peptide consisting of the amino acid sequence of SEQ ID NO: 66 as a partial CAPRIN-1 sequence.
(6) Preparation of Human-chicken Chimeric Anti-human CAPRIN-1 Antibody
The gene amplification fragment of the heavy chain variable region (SEQ ID NO: 64) of the chicken anti-human CAPRIN-1 monoclonal antibody #11 obtained in the preceding paragraph (3) was treated at both ends with restriction enzymes, then purified, and inserted according to a routine method into a pcDNA4/myc-His vector (Life Technologies, Inc.) into which a chicken antibody-derived leader sequence comprising SEQ ID NO: 67 and a human IgG1 H chain constant region comprising SEQ ID NO: 68 have been inserted. Also, the gene amplification fragment of the light chain variable region (SEQ ID NO: 65) of the chicken anti-human CAPRIN-1 monoclonal antibody #11 was treated at both ends with restriction enzymes, then purified, and inserted according to a routine method into a pcDNA3.1/myc-His vector (Life Technologies, Inc.) into which a chicken antibody-derived leader sequence comprising SEQ ID NO: 68 and a human IgG1 L chain constant region comprising SEQ ID NO: 69 have been inserted.
Next, the recombinant vector having the gene insert of the heavy chain variable region of the chicken monoclonal antibody #11 and the recombinant vector having the gene insert of the light chain variable region of the chicken monoclonal antibody #11 were introduced into CHO-K1 cells (obtained from Riken Cell Bank). Specifically, 2×105 CHO-K1 cells were cultured in 1 ml of Ham's F12 medium (Life Technologies, Inc.) containing 10% FBS per well of a 12-well culture plate, and washed with PBS(-). Then, 1 ml of fresh Ham's F12 medium containing 10% FBS per well was added thereto. The vectors (250 ng each) dissolved in 30 μL of OptiMEM (Life Technologies, Inc.) was mixed with 30 μL of Polyfect transfection reagent (Qiagen N.V.), and this mixture was added to each well. The CHO-K1 cells cotransfected with the recombinant vectors were cultured in a Ham's F12 medium containing 10% FBS supplemented with 200 μg/ml Zeocin (Life Technologies, Inc.) and 200 μg/ml Geneticin (Roche Diagnostics K.K.) and then inoculated to a 96-well plate at a density of 0.5 cells/well to prepare a cell line stably producing a human-chicken chimeric anti-human CAPRIN-1 antibody #13 having the variable regions of the chicken anti-human CAPRIN-1 monoclonal antibody #11 and the constant regions of human IgG1. The prepared cell line was cultured for 5 days in a 150-cm2 flask at a density of 5×105 cells/ml using 30 ml of a serum-free OptiCHO medium (Life Technologies, Inc.) to obtain a culture supernatant containing the antibody #13.
(7) Preparation of Mouse Anti-human CAPRIN-1 Monoclonal Antibody #14
In the same way as in the paragraph (1), a fusion protein of the amino acid sequence of SEQ ID NO: 66 identified in the paragraph (5) and a carrier protein KLH (keyhole limpet hemocyanin) was mixed as an immunogen with an equal amount of an adjuvant TiterMax Gold® (CytRx Corp.), and this mixture was subcutaneously administered at a dose of 20 μg/shot to each mouse at 7-day intervals. After administration with four shots in total, spleen cells were obtained from the mouse 3 days after the final immunization and fused with mouse myeloma cells in the same way as in the paragraph (1) to prepare hybridomas. Then, antibodies were screened using, as an indicator, the reactivity of each antibody contained in the culture supernatants of the prepared hybridomas with a 1 μg/ml CAPRIN-1 protein solutions prepared in Example 3 of WO2010/016526 or a fusion protein of the amino acid sequence of SEQ ID NO: 66 used as an immunogen and a carrier protein KLH. The 1 μg/ml CAPRIN-1 protein solution prepared in Example 3 of WO2010/016526 and the fusion protein (30 μg/ml) of the amino acid sequence of SEQ ID NO: 66 and a carrier protein KLH were separately added at 100 μL/well to 96-well plates and left standing at 4° C. for 18 hours. Each well was washed with PBS-T. Then, a Block Ace (DS Pharma Biomedical Co., Ltd.) solution was added thereto at 400 μL/well and left standing at room temperature for 3 hours. The solution in each well was removed, and each well was washed with PBS-T. Then, the culture supernatant of each hybridoma obtained above was added thereto at 100 μL/well and left standing at room temperature for 2 hours. Each well was washed with PBS-T. Then, HRP-labeled anti-mouse IgG (H+L) antibodies (Life Technologies, Inc.) diluted 5000-fold with PBS were added thereto at 100 μL/well and left standing at room temperature for 1 hour. Each well was washed with PBS-T. Then, a TMB substrate solution (Thermo Fisher Scientific Inc.) was added thereto at 100 μL/well and left standing for 5 to 30 minutes to cause color reaction. After the color development, the reaction was terminated by the addition of 1 N sulfuric acid at 100 μL/well. The absorbance was measured at 450 nm and 595 nm using an absorption spectrometer. As a result, hybridomas producing antibodies having high absorbance were selected.
The selected hybridomas were added to a 96-well plate at a density of 0.3 cells/well and cultured in the plate. One week later, hybridomas forming single colonies in the wells were observed. The cells in these wells were further cultured, and the cloned hybridomas were screened in the same way as above with the binding affinity of antibodies produced by the hybridomas to the amino acid sequence of SEQ ID NO: 66 as a partial CAPRIN-1 sequence as an indicator to obtain hybridomas producing antibodies against the amino acid of SEQ ID NO: 66.
Monoclonal antibodies produced by the obtained hybridomas were screened for the reactivity with the surface of breast cancer cells expressing CAPRIN-1. Specifically, 106 cells of a human breast cancer cell line MDA-MB-231 were centrifuged in a 1.5-ml microcentrifuge tube. 100 μL of the culture supernatant of each hybridoma obtained above was added thereto and left standing for 1 hour on ice. After washing with PBS, FITC-labeled goat anti-mouse IgG antibodies (Life Technologies, Inc.) diluted 500-fold with PBS containing 0.1% FBS were added thereto and left standing for 1 hour on ice. After washing with PBS, the fluorescence intensity was measured using FACSCalibur (Becton, Dickinson and Company). On the other hand, the same operation as above was performed using instead of the antibodies a sample of the serum of each untreated 6-week-old Balb/c mouse diluted 500-fold with a medium for hybridoma culture, or using secondary antibodies alone for reaction as a negative control. As a result, a mouse anti-human CAPRIN-1 monoclonal antibody #14 having stronger fluorescence intensity than that of the negative control, i.e., reactive with the surface of breast cancer cells, was obtained. The monoclonal antibody #14 comprises the heavy chain variable region of SEQ ID NO: 70 and the light chain variable region of SEQ ID NO: 71.
The obtained mouse anti-human CAPRIN-1 monoclonal antibody #14 was examined for its specific reaction with the amino acid sequence of SEQ ID NO: 66 that is a partial CAPRIN-1 sequence used as an immunogen. 30 μg/ml of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 in a 0.1 M aqueous sodium carbonate solution and 30 μg/ml of a polypeptide consisting of a partial CAPRIN-1 sequence free from the amino acid sequence of SEQ ID NO: 66 in a 0.1 M aqueous sodium carbonate solution were separately added to 96-well plates Immobilizer Amino for ELISA (Nunc) at a concentration of 100 μg/ml and reacted all night and all day at 4° C. to bind the peptides to the wells. A 0.1 M aqueous sodium carbonate solution containing 10 mM ethanolamine was added to the resulting peptide-bound well and left standing at room temperature for 1 hour. The solution in each well was removed, and each well was then washed with PBS-T. Then, a Block Ace solution was added thereto at 400 μL/well and left standing at room temperature for 3 hours. The solution in each well was removed, and each well was washed with PBS-T. Then, the culture supernatant containing the mouse monoclonal antibody #14 was added thereto at 50 μL/well and reacted at room temperature for 1 hour. Then, each well was washed with PBS-T, and HRP-labeled anti-mouse IgG (H+L) antibodies (Life Technologies, Inc.) diluted 5000-fold with a Block Ace solution were added thereto at 50 μL/well and left standing at room temperature for 1 hour. Each well was fully washed with PBS-T. Then, a TMB substrate solution (Thermo Fisher Scientific Inc.) was added thereto at 100 μL/well and left standing for 5 to 30 minutes to cause color reaction. After the color development, the reaction was terminated by the addition of 1 N sulfuric acid at 100 μL/well. The absorbance was measured at 450 nm and 595 nm using an absorption spectrometer. As a result, the mouse monoclonal antibody #14 did not react with the partial CAPRIN-1 sequence free from the amino acid sequence of SEQ ID NO: 66, but specifically reacted only with the amino acid sequence of SEQ ID NO: 66. Thus, the polypeptide of SEQ ID NO: 66 was confirmed to contain an epitope region recognized by the mouse anti-human CAPRIN-1 antibody #14.
Example 3
Analysis of CAPRIN-1 Protein Expression on Cancer Cell
Next, 8 human breast cancer cell lines (ZR75-1, MCF7, T47D, SK-BR-3, MDA-MB-157, BT-20, MDA-MB-231V, and MRK-nu-1) confirmed to have a high rate of CAPRIN-1 gene expression were examined for their expression of CAPRIN-1 proteins on the cell surface. 5×105 cells of each human breast cancer cell line were centrifuged in a 1.5-ml microcentrifuge tube. 2 μg (5 μl) of the mouse-chicken anti-human CAPRIN-1 monoclonal antibody (#12) prepared in Example 2(4) was added thereto, further mixed by the addition of 95 μl of PBS containing 0.1% fetal bovine serum, and left standing for 1 hour on ice. After washing with PBS, the cells were mixed by the addition of 2 μl of Alexa 488-labeled goat anti-mouse IgG antibodies (Life Technologies, Inc.) and 98 μl PBS containing 0.1% fetal bovine serum (FBS) and left standing for 30 hours on ice. After washing with PBS, the fluorescence intensity was measured using FACSCalibur (Becton, Dickinson and Company). On the other hand, the same operation as above was performed using mouse IgG1 instead of the mouse-chicken anti-human CAPRIN-1 monoclonal antibody (#12), as a control. As a result, the cancer cell lines supplemented with the mouse-chicken anti-human CAPRIN-1 monoclonal antibody (#12) all exhibited fluorescence intensity at least 35% stronger than that of the control. This demonstrated that CAPRIN-1 proteins are expressed on the cell membrane surface of the human cancer cell lines. The above rate of enhancement in fluorescence intensity was indicated by the rate of increase in mean fluorescence intensity (MFI) in each cell line and calculated according to the following expression.

Rate of increase in mean fluorescence intensity(Rate of enhancement in fluorescence intensity)(%)=((MFI of cells reacted with the anti-CAPRIN-1 antibody)−(Control MFI))/(Control MFI)×100

Also, the fluorescence intensity was measured for 3 kidney cancer cell lines (Caki-1, Caki-2, and A498), a bladder cancer cell line (T24), an ovary cancer cell line (SKOV3), a lung cancer cell line (QG56), a prostate cancer cell line (PC3), a uterine cervix cancer cell line (Hela), a fibrosarcoma cell line (HT1080), 2 brain tumor cell lines (T98G and U87MG), a gastric cancer cell line (MNK28), a large intestinal cancer cell line (Lovo), and pancreatic cancer cell lines (Capan-2, MIAPaCa-2, Panc-1, and BxPC-3) using the same approach as above. As a result, all the cancer cell lines had fluorescence intensity at least 35% stronger than that of the control.
As with the results obtained above, the CAPRIN-1 expression on cancer cell surface was also confirmed using the human-chicken chimeric anti-human CAPRIN-1 monoclonal antibody (#13) obtained in Example 2(6) or the mouse anti-human CAPRIN-1 monoclonal antibody (#14) obtained in Example 2(7).
Example 4
Selection of Optimum Antibody for CAPRIN-1 Detection
(1) Selection of Antibody Using Human Breast Cancer Tissue
31 breast cancer tissue samples of a paraffin-embedded human breast cancer tissue array (Medical & Biological Laboratories Co., Ltd.) were used in immunohistochemical staining. The human breast cancer tissue array was treated at 60° C. for 3 hours and then placed in a staining bottle filled with xylene, and procedures of replacing xylene with a fresh one every 5 minutes were performed three times. Subsequently, the same operation as in xylene was performed using ethanol and PBS-T. The human breast cancer tissue array was placed in a staining bottle filled with a 10 mM citrate buffer solution (pH 6.0) containing 0.05% Tween 20, treated at 125° C. for 5 minutes, and then left standing at room temperature for 40 minutes or longer. Excess water around a section was wiped off with a Kimwipe. The section on a glass slide was encircled with a Dako pen (Dako), and an appropriate amount of Peroxidase Block (Dako) was added dropwise thereto. The glass slide was left standing at room temperature for 5 minutes and then placed in a staining bottle filled with PBS-T, and procedures of replacing PBS-T with a fresh one every 5 minutes were performed three times. A PBS-T solution containing 10% FBS was applied thereto as a blocking solution, and the glass slide was left standing at room temperature for 1 hour in a moist chamber. Next, 10 μg/ml of the mouse anti-human CAPRIN-1 monoclonal antibody #8 or #14 prepared in Example 2 in a PBS-T solution containing 5% FBS was applied thereto. The glass slide was left standing overnight at 4° C. in a moist chamber. After washing with PBS-T for 10 minutes three times, an appropriate amount of Peroxidase Labelled Polymer Conjugated (Dako) was added dropwise thereto, and the glass slide was left standing at room temperature for 30 minutes in a moist chamber. After washing with PBS-T for 10 minutes three times, a DAB staining solution (Dako) was applied thereto, and the glass slide was left standing at room temperature for approximately 10 minutes. Then, the staining solution was discarded, and the glass slide was washed with PBS-T for 10 minutes three times. After rinsing with distilled water, the glass slide was placed in 70%, 80%, 90%, 95%, and 100% ethanol solutions in this order for 1 minute per solution, and finally left standing overnight in xylene. The glass slide was taken out and the section was embedded in Glycergel Mounting Medium (Dako), followed by observation. The expression level of CAPRIN-1 in the tissues was evaluated according to the criteria given below. A slide that exhibited positive results was selected, and its CAPRIN-1 stain image was observed. First, the CAPRIN-1 stain image of cancer cells in the tissues, the intensity of positive staining, and the ratio of positive cells were observed by use of a ×4 objective lens of a light microscope. Next, the objective lens was changed to a ×10 or ×20 lens, and examination was made on whether the positive results were localized to the cell membrane or the cytoplasm. The detection results were evaluated in this way and classified into scores 0 to 3: The details of the scores are as follows.

    
    
        Score 0 (without CAPRIN-1 overexpression): Positive staining of the cell membrane is not observed or is observed in less than 10% of the cancer cells.
        Score 1 (without CAPRIN-1 overexpression): Faint, almost unperceivable staining of the cell membrane is observed in 10% or more of the cancer cells, and these cancer cells are partially stained only at their cell membranes.
        Score 2 (with CAPRIN-1 overexpression): Weak to moderate complete positive staining of the cell membrane is observed in 10% or more of the cancer cells, or strong complete positive staining of the cell membrane is observed in 10% or more and 30% or less of the cancer cells.
        Score 3 (with CAPRIN-1 overexpression): Strong complete positive staining of the cell membrane is observed in 30% or more of the cancer cells.
    
    


A cancer tissue was determined to be CAPRIN-1-positive, if its assay results were given score 2 or 3.
As a result, the expression of CAPRIN-1 in the breast cancer tissues was successfully confirmed using any of the antibodies. The results of immunohistochemical staining using the antibody #8 exhibited score 2 for 14 samples and score 3 for 1 sample, and therefore the number of CAPRIN-1-positive samples was 15 samples. The result of immunohistochemical staining using the antibody #14 exhibited score 2 for 18 samples and score 3 for 8 samples, and therefore the number of CAPRIN-1-positive samples was 26 samples. Thus, the antibody #14 was selected for the detection of CAPRIN-1 using human cancer tissues.
(2) Detection of CAPRIN-1 on Various Human Normal Tissues by Immunohistochemical Staining Method Using Antibody #14
A human normal tissue array (US Biomax, Inc.) (including brain, thyroid gland, lung, spleen, kidney, esophagus, stomach, large bowel, pancreas, muscle, skin, salivary gland, ovary, uterus, mammary gland, placenta, bone marrow, testis, and prostate tissues) was used in immunohistochemical staining. Excess water around a section was wiped off with a Kimwipe. The section on a glass slide was encircled with a Dako pen (Dako), and an appropriate amount of Peroxidase Block (Dako) was added dropwise thereto. The glass slide was left standing at room temperature for 5 minutes and then placed in a staining bottle filled with PBS-T, and procedures of replacing PBS-T with a fresh one every 5 minutes were performed three times. A PBS-T solution containing 10% FBS was applied thereto as a blocking solution, and the glass slide was left standing at room temperature for 1 hour in a moist chamber. Next, 10 μg/ml of the mouse anti-human CAPRIN-1 monoclonal antibody #14 prepared in Example 2 in a PBS-T solution containing 5% FBS was applied thereto. The glass slide was left standing overnight at 4° C. in a moist chamber. After washing with PBS-T for 10 minutes three times, an appropriate amount of Peroxidase Labelled Polymer Conjugated (Dako) was added dropwise thereto, and the glass slide was left standing at room temperature for 30 minutes in a moist chamber. After washing with PBS-T for 10 minutes three times, a DAB staining solution (Dako) was applied thereto, and the glass slide was left standing at room temperature for approximately 10 minutes. Then, the staining solution was discarded, and the glass slide was washed with PBS-T for 10 minutes three times. After rinsing with distilled water, the glass slide was placed in 70%, 80%, 90%, 95%, and 100% ethanol solutions in this order for 1 minute per solution, and finally left standing overnight in xylene. The glass slide was taken out and the section was embedded in Glycergel Mounting Medium (Dako), followed by observation.
The expression level of CAPRIN-1 in the tissues was evaluated according to the criteria given below. A slide that exhibited positive results was selected, and its CAPRIN-1 stain image was observed. First, the CAPRIN-1 stain image of cancer cells in the tissues, the intensity of positive staining, and the ratio of positive cells were observed by use of a ×4 objective lens of a light microscope. Next, the objective lens was changed to a ×10 or ×20 lens, and examination was made on whether the positive results were localized to the cell membrane or the cytoplasm. The detection results were evaluated in this way and classified into scores 0 to 3. The details of the scores are as follows.

    
    
        Score 0 (without CAPRIN-1 overexpression): Positive staining of the cell membrane is not observed or is observed in less than 10% of the cancer cells.
        Score 1 (without CAPRIN-1 overexpression): Faint, almost unperceivable staining of the cell membrane is observed in 10% or more of the cancer cells, and these cancer cells are partially stained only at their cell membranes.
        Score 2 (with CAPRIN-1 overexpression): Weak to moderate complete positive staining of the cell membrane is observed in 10% or more of the cancer cells, or strong complete positive staining of the cell membrane is observed in 10% or more and 30% or less of the cancer cells.
        Score 3 (with CAPRIN-1 overexpression): Strong complete positive staining of the cell membrane is observed in 30% or more of the cancer cells. A cancer tissue with score 2 or 3 was determined to be CAPRIN-1-positive.
    
    


The uterus and prostate tissues were given score 1, whereas the other tissues were all given score 0. Thus, the expression of CAPRIN-1 was not observed in the human normal tissues.
(3) Detection of CAPRIN-1 Protein on Various Human Cancer Tissues by Immunohistochemical Staining Method Using Mouse Anti-Human CAPRIN-1 Antibody #14
Various cancer tissues of a paraffin-embedded human cancer tissue array (US Biomax, Inc.) were used in immunohistochemical staining. The human cancer tissue array was treated at 60° C. for 3 hours and then placed in a staining bottle filled with xylene, and procedures of replacing xylene with a fresh one every 5 minutes were performed three times. Subsequently, the same operation as in xylene was performed using ethanol and PBS-T. The human cancer tissue array was placed in a staining bottle filled with a 10 mM citrate buffer solution (pH 6.0) containing 0.05% Tween 20, treated at 125° C. for 5 minutes, and then left standing at room temperature for 40 minutes or longer. Excess water around a section was wiped off with a Kimwipe. The section on a glass slide was encircled with a Dako pen (Dako), and an appropriate amount of Peroxidase Block (Dako) was added dropwise thereto. The glass slide was left standing at room temperature for 5 minutes and then placed in a staining bottle filled with PBS-T, and procedures of replacing PBS-T with a fresh one every 5 minutes were performed three times. A PBS-T solution containing 10% FBS was applied thereto as a blocking solution, and the glass slide was left standing at room temperature for 1 hour in a moist chamber. Next, 10 μg/ml of the mouse anti-human CAPRIN-1 monoclonal antibody #14 prepared in Example 2 in a PBS-T solution containing 5% FBS was applied thereto. The glass slide was left standing overnight at 4° C. in a moist chamber. After washing with PBS-T for 10 minutes three times, an appropriate amount of Peroxidase Labelled Polymer Conjugated (Dako) was added dropwise thereto, and the glass slide was left standing at room temperature for 30 minutes in a moist chamber. After washing with PBS-T for 10 minutes three times, a DAB staining solution (Dako) was applied thereto, and the glass slide was left standing at room temperature for approximately 10 minutes. Then, the staining solution was discarded, and the glass slide was washed with PBS-T for 10 minutes three times. The glass slide was rinsed with distilled water and placed in 70%, 80%, 90%, 95%, and 100% ethanol solutions in this order for 1 minute per solution, and finally left standing overnight in xylene. The glass slide was taken out and the section was embedded in Glycergel Mounting Medium (Dako), followed by observation.
The expression level of CAPRIN-1 in the tissues was evaluated according to the criteria given below. A slide that exhibited positive results was selected, and its CAPRIN-1 protein stain image was observed. First, the CAPRIN-1 stain image of cancer cells in the tissues, the intensity of positive staining, and the ratio of positive cells were observed by use of a ×4 objective lens of a light microscope. Next, the objective lens was changed to a ×10 or ×20 lens, and examination was made on whether the positive results were localized to the cell membrane or the cytoplasm. The detection results were evaluated in this way and classified into scores 0 to 3. The details of the scores are as follows.

    
    
        Score 0 (without CAPRIN-1 overexpression): Positive staining of the cell membrane is not observed or is observed in less than 10% of the cancer cells.
        Score 1 (without CAPRIN-1 overexpression): Faint, almost unperceivable staining of the cell membrane is observed in 10% or more of the cancer cells, and these cancer cells are partially stained only at their cell membranes.
        Score 2 (with CAPRIN-1 overexpression): Weak to moderate complete positive staining of the cell membrane is observed in 10% or more of the cancer cells, or strong complete positive staining of the cell membrane is observed in 10% or more and 30% or less of the cancer cells.
        Score 3 (with CAPRIN-1 overexpression): Strong complete positive staining of the cell membrane is observed in 30% or more of the cancer cells.
    
    


A cancer tissue was determined to be CAPRIN-1-positive, if its assay results were given score 2 or 3.
As a result, CAPRIN-1 was shown to be positive in 16 out of 22 brain tumor tissue samples (64%), 19 out of 32 lung cancer tissue samples (59%), 18 out of 21 uterus cancer tissue samples (86%), 10 out of 16 esophagus cancer tissue samples (63%), 27 out of 30 kidney cancer tissue samples (90%), 14 out of 17 liver cancer tissue samples (82%), 11 out of 15 thyroid gland cancer tissue samples (73%), 10 out of 14 stomach cancer tissue samples (71%), 17 out of 19 pancreas cancer tissue samples (89%), 13 out of 13 prostate cancer tissue samples (100%), 12 out of 14 bladder cancer tissue samples (86%), 11 out of 14 large bowel cancer tissue samples (79%), 24 out of 30 skin cancer tissue samples (80%), and 16 out of 21 breast cancer tissue samples (76%).
(4) Detection of CAPRIN-1 Protein on Dog Breast Cancer Tissue by Immunohistochemical Staining Method Using Mouse Anti-Human CAPRIN-1 Antibody #14
100 frozen breast cancer tissue samples of dogs pathologically diagnosed as malignant breast cancer were used in immunohistochemical staining. Each frozen dog breast cancer tissue was sliced into 10 to 20 μm sections using Cryostat (Leica Biosystems), mounted on a glass slide, and dried in air, together with the glass slide, for 30 minutes using a hair dryer to prepare a glass slide with a tissue slice mounted thereon. Next, the glass slide was placed in a staining bottle filled with PBS-T (saline containing 0.05% Tween 20), and procedures of replacing PBS-T with a fresh one every 5 minutes were performed three times. Excess water around a section was wiped off with a Kimwipe. The section on the glass slide was encircled with a Dako pen (Dako). Then, a PBS-T solution containing 10% fetal bovine serum was applied thereto as a blocking solution, and the glass slide was left standing at room temperature for 1 hour in a moist chamber. Next, 10 μg/ml of the mouse anti-human CAPRIN-1 monoclonal antibody #8 or #14 prepared in Example 2 in a blocking solution, and this solution was applied thereto. The glass slide was left standing overnight at 4° C. in a moist chamber. After washing with PBS-T for 10 minutes three times, MOM biotin-labeled anti-IgG antibodies (Vectastain) diluted 250-fold with a blocking solution were applied thereto, and the glass slide was left standing at room temperature for 1 hour in a moist chamber. After washing with PBS-T for 10 minutes three times, avidin-biotin ABC reagent (Vectastain) was applied thereto, and the glass slide was left standing at room temperature for 5 minutes in a moist chamber. After washing with PBS-T for 10 minutes three times, a DAB staining solution (10 mg of DAB+10 μL of 30% H2O2/50 ml of 0.05 M Tris-HCl (pH 7.6)) was applied thereto, and the glass slide was left standing at room temperature for 30 minutes in a moist chamber. The glass slide was rinsed with distilled water. A hematoxylin reagent (Dako) was applied thereto, and the glass slide was left standing at room temperature for 1 minute and then rinsed with distilled water. The glass slide was placed in 70%, 80%, 90%, 95%, and 100% ethanol solutions in this order for 1 minute per solution, and then left standing overnight in xylene. The glass slide was taken out and the section was embedded in Glycergel Mounting Medium (Dako), followed by observation. The expression level of CAPRIN-1 in the tissues was evaluated according to the criteria given below. A slide that exhibited positive results was selected, and its CAPRIN-1 stain image was observed. First, the CAPRIN-1 stain image of cancer cells in the tissues, the intensity of positive staining, and the ratio of positive cells were observed by use of a ×4 objective lens of a light microscope. Next, the objective lens was changed to a ×10 or ×20 lens, and examination was made on whether the positive results were localized to the cell membrane or the cytoplasm. The detection results were evaluated in this way and classified into scores 0 to 3. The details of the scores are as follows.

    
    
        Score 0 (without CAPRIN-1 overexpression): Positive staining of the cell membrane is not observed or is observed in less than 10% of the cancer cells.
        Score 1 (without CAPRIN-1 overexpression): Faint, almost unperceivable staining of the cell membrane is observed in 10% or more of the cancer cells, and these cancer cells are partially stained only at their cell membranes.
        Score 2 (with CAPRIN-1 overexpression): Weak to moderate complete positive staining of the cell membrane is observed in 10% or more of the cancer cells, or strong complete positive staining of the cell membrane is observed in 10% or more and 30% or less of the cancer cells.
        Score 3 (with CAPRIN-1 overexpression): Strong complete positive staining of the cell membrane is observed in 30% or more of the cancer cells.
    
    


A cancer-bearing dog tissue was determined to be CAPRIN-1-positive and to be expected to get effective therapeutic effects by the administration of a CAPRIN-1-targeting drug, if its assay results were given score 2 or 3.
As a result, the expression of CAPRIN-1 in the dog breast cancer tissues was successfully shown using any of the antibodies. Specifically, the results of immunohistochemical staining using the antibody #8 exhibited score 2 for 69 samples and score 3 for 11 samples, and thus the number of CAPRIN-1-positive samples was 80 samples (80%). The result of immunohistochemical staining using the antibody #14 exhibited score 2 for 46 samples and score 3 for 36 samples, and thus the number of CAPRIN-1-positive samples was 82 samples (82%).
(5) Detection of CAPRIN-1 on Cat Breast Cancer Tissue by Immunohistochemical Staining Method Using Mouse Anti-human CAPRIN-1 Antibody #14
30 frozen breast cancer tissue samples of cats pathologically diagnosed as malignant breast cancer were used in immunohistochemical staining. Each frozen cat cancer tissue was sliced into 10 to 20 μm sections using Cryostat (Leica Biosystems), mounted on a glass slide, and dried in air, together with the glass slide, for 30 minutes using a hair dryer to prepare a glass slide with a tissue slice mounted thereon. Next, the glass slide was placed in a staining bottle filled with PBS-T (saline containing 0.05% Tween 20), and procedures of replacing PBS-T with a fresh one every 5 minutes were performed three times. Excess water around a section was wiped off with a Kimwipe. The section on the glass slide was encircled with a Dako pen (Dako). Then, a PBS-T solution containing 10% fetal bovine serum was applied thereto as a blocking solution, and the glass slide was left standing at room temperature for 1 hour in a moist chamber. Next, 10 μg/ml of the mouse anti-human CAPRIN-1 monoclonal antibody #8 or #14 prepared in Example 2 in a blocking solution was applied thereto. The glass slide was left standing overnight at 4° C. in a moist chamber. After washing with PBS-T for 10 minutes three times, MOM biotin-labeled anti-IgG antibodies (Vectastain) diluted 250-fold with a blocking solution were applied thereto, and the glass slide was left standing at room temperature for 1 hour in a moist chamber. After washing with PBS-T for 10 minutes three times, avidin-biotin ABC reagent (Vectastain) was applied thereto, and the glass slide was left standing at room temperature for 5 minutes in a moist chamber. After washing with PBS-T for 10 minutes three times, a DAB staining solution (10 mg of DAB+10 μl, of 30% H2O2/50 ml of 0.05 M Tris-HCl (pH 7.6)) was applied thereto, and the glass slide was left standing at room temperature for 30 minutes in a moist chamber. The glass slide was rinsed with distilled water. A hematoxylin reagent (Dako) was applied thereto, and the glass slide was left standing at room temperature for 1 minute and then rinsed with distilled water. The glass slide was placed in 70%, 80%, 90%, 95%, and 100% ethanol solutions in this order for 1 minute per solution, and then left standing overnight in xylene. The glass slide was taken out and the section was embedded in Glycergel Mounting Medium (Dako), followed by observation. The expression level of CAPRIN-1 in the tissues was evaluated according to the criteria given below. A slide that exhibited positive results was selected, and its CAPRIN-1 stain image was observed. First, the CAPRIN-1 stain image of cancer cells in the tissues, the intensity of positive staining, and the ratio of positive cells were observed by use of a ×4 objective lens of a light microscope. Next, the objective lens was changed to a ×10 or ×20 lens, and examination was made on whether the positive results were localized to the cell membrane or the cytoplasm. The detection results were evaluated in this way and classified into scores 0 to 3. The details of the scores are as follows.

    
    
        Score 0 (without CAPRIN-1 overexpression): Positive staining of the cell membrane is not observed or is observed in less than 10% of the cancer cells.
        Score 1 (without CAPRIN-1 overexpression): Faint, almost unperceivable staining of the cell membrane is observed in 10% or more of the cancer cells, and these cancer cells are partially stained only at their cell membranes.
        Score 2 (with CAPRIN-1 overexpression): Weak to moderate complete positive staining of the cell membrane is observed in 10% or more of the cancer cells, or strong complete positive staining of the cell membrane is observed in 10% or more and 30% or less of the cancer cells.
        Score 3 (with CAPRIN-1 overexpression): Strong complete positive staining of the cell membrane is observed in 30% or more of the cancer cells.
    
    


A cancer-bearing cat tissue was determined to be CAPRIN-1-positive and to expected to get effective therapeutic effects by the administration of a CAPRIN-1-targeting drug, if its assay results were given score 2 or 3.
As a result, the expression of CAPRIN-1 in the cat breast cancer tissues was successfully shown using any of the antibodies. Specifically, the results of immunohistochemical staining using the antibody #8 exhibited score 2 for 20 samples and score 3 for 4 samples, and thus the number of CAPRIN-1-positive samples was 24 samples (80%). The result of immunohistochemical staining using the antibody #14 exhibited score 2 for 18 samples and score 3 for 9 samples, and thus the number of CAPRIN-1-positive samples was 27 samples (90%).
Example 5
Correlation of CAPRIN-1 Expression Evaluated Using Cancer Sample with Antitumor Effect of Antibody Against CAPRIN-1-I
(1) Detection of CAPRIN-1 by Immunohistochemical Staining Method Using Cancer Tissue Derived from Cancer-Bearing Mouse in which Mouse Cancer Cells were Transplanted
Two mouse-derived cancer cell lines (B16F10 and EMT-6) were subcutaneously transplanted (each for 5 mice) into the dorsal regions of 26 Balb/c mice (Japan SLC, Inc.) and grown until the size of tumor became approximately 7 mm in diameter. Three subjects were selected from each of these two mouse groups respectively having the 2 types of transplanted cancer cells. A tumor mass was excised from each mouse, cut open in PBS, and perfusion-fixed overnight in a 0.1 M phosphate buffer solution (pH 7.4) containing 4% paraformaldehyde (PFA). The perfusate was discarded. The tissue surface of each organ was rinsed with PBS. A PBS solution containing 10% sucrose was added to a 50-ml centrifuge tube, each cancer tissue was then placed therein and shaken at 4° C. for 2 hours using a rotor. The solution was replaced with a PBS solution containing 20% sucrose, and the sample was left standing at 4° C. until the cancer tissue was precipitated. Then, the solution was replaced with a PBS solution containing 30% sucrose, and the sample was left standing at 4° C. until the cancer tissue was precipitated. The cancer tissue was taken out, and desired portions were cut off with a surgical knife. Next, OCT compound (Tissue Tek) was poured onto the tissue surface and spread over the surface. Then, the tissue was placed on Cryomold. The Cryomold was placed on dry ice to quickly freeze the tissue, then sliced into 10 to 20 μm sections using Cryostat (Leica Biosystems), mounted on a glass slide, and dried in air, together with the glass slide, for 30 minutes using a hair dryer to prepare a glass slide with a tissue slice mounted thereon. On the next day, the glass slide was washed with PBS(-) three times. PBS(-) containing 5% goat serum was applied thereto as a blocking solution, and the glass slide was left standing at room temperature for 1 hour in a moist chamber. Next, 10 μg/ml of the mouse anti-human CAPRIN-1 monoclonal antibody #8 or #14 prepared in Example 2 in a PBS(-) solution was applied thereto. The glass slide was left standing overnight at 4° C. in a moist chamber. After washing with PBS(-) for 5 minutes five times, an appropriate amount of Peroxidase Labelled Polymer Conjugated (Dako) was added dropwise thereto, and the glass slide was left standing at room temperature for 30 minutes in a moist chamber. After washing with PBS-T for 5 minutes six times, a DAB staining solution (Dako) was applied thereto, and the glass slide was left standing at room temperature for approximately 10 minutes. Then, the staining solution was discarded, and the glass slide was washed with PBS(-) for 5 minutes three times. Then, the section on the glass slide was embedded in Glycergel Mounting Medium (Dako), followed by observation. As a result of scoring as described in Example 4, the results of immunohistochemical staining using the antibody #8 exhibited score 1 both for the melanoma-derived cells B16F10 and for the breast cancer-derived cells EMT-6. Thus, CAPRIN-1 expression was not detected. On the other hand, the results of immunohistochemical staining using the antibody #14 exhibited score 1 for the cancer cells B16F10, but exhibited score 3 for the cancer cells EMT-6.
(2) Antitumor Effect of Antibody Against CAPRIN-1
The human-chicken chimeric anti-human CAPRIN-1 monoclonal antibody #13 was studied for its antitumor effect using the cancer-bearing mice prepared in the preceding paragraph (1). Of the cancer-bearing mice in which each cancer cell line (B16F10 or EMT-6) was transplanted, 5 cancer-bearing mice in each group underwent the intraperitoneal administration of the antibody #13 at a dose of 200 μg (200 μL) per mouse. Then, the antibody was intraperitoneally administered at the same dose as above to each cancer-bearing mouse a total of 3 times for 2 days. The size of tumor was measured every day, and the antitumor effect of the antibody #13 was observed (study group). On the other hand, PBS(-) was administered instead of the antibody to the remaining 5 cancer-bearing mice, which were in turn used as a control group.
As a result of observing the antitumor effect, the tumor volumes of the cancer cell B16F10-transplanted mice in the study group receiving the antibody #13 were increased to approximately 150%, 200%, 370%, and 630% at days 4, 6, 8, and 11, respectively, with the tumor volume at the start of antibody administration defined as 100%. On the other hand, the tumor volumes of the cancer cell EMT-6-transplanted mice in the study group were reduced to 51% at day 4, approximately 31% at day 6, and 9% at day 8 with the tumor volume at the start of antibody administration defined as 100%, and their tumors were almost completely regressed by days 10 to 14. The tumor volumes of both tumor-transplanted mice in the control group receiving PBS(-) were increased to approximately 230%, 290%, 470%, and 800% at days 4, 6, 8, and 11, respectively.
From the results mentioned above, the results of measuring the expression of CAPRIN-1 using the antibody #8 were not shown to correlate with cancer therapeutic effects based on the antitumor activity of the antibody, whereas the results of measuring the expression of CAPRIN-1 using the antibody #14 were shown to correlate with cancer therapeutic effects based on the antitumor activity of the antibody. Specifically, the results of measuring the expression level of CAPRIN-1 using the antibody #14 exhibited score 3 for the EMT-6 transplant-derived cancer tissues, which indicates CAPRIN-1 overexpression, and pharmacological effects based on the antitumor activity of the administered antibody were shown. On the other hand, the results of measuring the expression level of CAPRIN-1 using the antibody #14 exhibited score 1 for the transplanted B16F10-derived cancer tissues, which indicates that the expression of CAPRIN-1 was not observed. In addition, the antibody #13 having antitumor activity did not produce pharmacological effects when administered to the cancer-bearing mice in which the cancer cells B16F10 were transplanted.
These results indicated that a cancer or an individual determined to have a high expression level of CAPRIN-1 in a cancer tissue by detection of CAPRIN-1 in the cancer tissue using the antibody #14 of the present invention specifically binding to CAPRIN-1, can get high therapeutic effects by administering the anti-CAPRIN-1 antibody according to the present invention, based on the antitumor effect of the antibody.
Example 6
Correlation of CAPRIN-1 Expression Evaluated Using Cancer Sample with Antitumor Effect of Antibody Against CAPRIN-1-II
(1) Detection of CAPRIN-1 by Immunohistochemical Staining Method Using Cancer Tissue Derived from Cancer-Bearing Mouse in which Mouse Cancer Cells were Transplanted
Two mouse-derived cancer cell lines (B16 and CT26) were subcutaneously transplanted (each for 5 mice) into the dorsal regions of 26 Balb/c mice (Japan SLC, Inc.) and grown until the size of tumor became approximately 7 mm in diameter. Three subjects were selected from each of these two mouse groups respectively having the 2 types of transplanted cancer cells. A tumor mass was excised from each mouse, cut open in PBS, and perfusion-fixed overnight in a 0.1 M phosphate buffer solution (pH 7.4) containing 4% paraformaldehyde (PFA). The perfusate was discarded. The tissue surface of each organ was rinsed with PBS. A PBS solution containing 10% sucrose was added to a 50-ml centrifuge tube, each cancer tissue was then placed therein and shaken at 4° C. for 2 hours using a rotor. The solution was replaced with a PBS solution containing 20% sucrose, and the sample was left standing at 4° C. until the cancer tissue was precipitated. Then, the solution was replaced with a PBS solution containing 30% sucrose, and the sample was left standing at 4° C. until the cancer tissue was precipitated. The cancer tissue was taken out, and desired portions were cut off with a surgical knife. Next, OCT compound (Tissue Tek) was poured onto the tissue surface and spread over the surface. Then, the tissue was placed on Cryomold. The Cryomold was placed on dry ice to quickly freeze the tissue, then sliced into 10 to 20 μm sections using Cryostat (Leica Biosystems), mounted on a glass slide, and dried in air, together with the glass slide, for 30 minutes using a hair dryer to prepare a glass slide with a tissue slice mounted thereon. On the next day, the glass slide was washed with PBS(-) three times. PBS(-) containing 5% goat serum was applied thereto as a blocking solution, and the glass slide was left standing at room temperature for 1 hour in a moist chamber. Next, 10 μg/ml of the mouse anti-human CAPRIN-1 monoclonal antibody #8 or #14 prepared in Example 2 in a PBS(-) solution was applied thereto. The glass slide was left standing overnight at 4° C. in a moist chamber. After washing with PBS(-) for 5 minutes five times, an appropriate amount of Peroxidase Labelled Polymer Conjugated (Dako) was added dropwise thereto, and the glass slide was left standing at room temperature for 30 minutes in a moist chamber. After washing with PBS-T for 5 minutes six times, a DAB staining solution (Dako) was applied thereto, and the glass slide was left standing at room temperature for approximately 10 minutes. Then, the staining solution was discarded, and the glass slide was washed with PBS(-) for 5 minutes three times. Then, the section on the glass slide was embedded in Glycergel Mounting Medium (Dako), followed by observation. As a result of scoring as described in Example 4, the results of immunohistochemical staining using the antibody #8 exhibited score 0 for the melanoma cells B16 and score 1 for the large bowel cancer cells CT26. Thus, CAPRIN-1 expression was not detected. On the other hand, the results of immunohistochemical staining using the antibody #14 exhibited score 0 for the cancer cells B16, but exhibited score 2 and thus to be positive for the cancer cells CT26.
(2) Antitumor Effect of Antibody Against CAPRIN-1
The human-chicken chimeric anti-human CAPRIN-1 monoclonal antibody #13 was studied for its antitumor effect using the cancer-bearing mice prepared in the preceding paragraph (1). Of the cancer-bearing mice in which each cancer cell line (B16 or CT26) was transplanted, 5 cancer-bearing mice in each group underwent the intraperitoneal administration of the antibody #13 at a dose of 200 μg (200 μL) per mouse. Then, the antibody was intraperitoneally administered at the same dose as above to each cancer-bearing mouse a total of 3 times for 2 days. The size of tumor was measured every day, and the antitumor effect of the antibody #13 was observed (study group). On the other hand, PBS(-) was administered instead of the antibody to the remaining 5 cancer-bearing mice, which were in turn used as a control group.
As a result of observing the antitumor effect, the tumor volumes of the cancer cell B16-transplanted mice in the study group receiving the antibody #13 were increased to approximately 170%, 220%, 390%, and 680% at days 4, 6, 8, and 11, respectively, with the tumor volume at the start of antibody administration defined as 100%. On the other hand, the tumor volumes of the cancer cell CT26-transplanted mice in the study group were reduced to 65% at day 4, approximately 41% at day 6, and 17% at day 8 with the tumor volume at the start of antibody administration defined as 100%, and their tumors were almost completely regressed by days 10 to 14. The tumor volumes of both tumor-transplanted mice in the control group receiving PBS(-) were increased to approximately 230%, 290%, 470%, and 800% at days 4, 6, 8, and 11, respectively.
From the results mentioned above, the results of measuring the expression of CAPRIN-1 using the antibody #8 were not shown to correlate with cancer therapeutic effects based on the antitumor activity of the antibody, whereas the results of measuring the expression of CAPRIN-1 using the antibody #14 were shown to correlate with cancer therapeutic effects based on the antitumor activity of the antibody. Specifically, the results of measuring the expression level of CAPRIN-1 using the antibody #14 exhibited score 2 for the CT26 transplant-derived cancer tissues, which indicates CAPRIN-1 overexpression, and pharmacological effects based on the antitumor activity of the administered antibody were shown. On the other hand, the results of measuring the expression level of CAPRIN-1 using the antibody #14 exhibited score 0 for the transplanted B16-derived cancer tissues, which indicates that the expression of CAPRIN-1 was not observed. In addition, the antibody #13 having antitumor activity did not produce pharmacological effects when administered to the cancer-bearing mice in which the cancer cells B16 were transplanted.
These results indicated that a cancer or an individual determined to have a high expression level of CAPRIN-1 in a cancer tissue by detection of CAPRIN-1 in the cancer tissue using the antibody #14 of the present invention specifically binding to CAPRIN-1, can get high therapeutic effects by administering the anti-CAPRIN-1 antibody according to the present invention, based on the antitumor effect of the antibody.
INDUSTRIAL APPLICABILITY
The present invention can be utilized for the diagnosis of a cancer and for the determination of administration of a CAPRIN-1-targeting drug such as a CAPRIN-1-specific therapeutic drug.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
Free Text of Sequence Listing


    SEQ ID NOs: 31 to 36, and 38 to 42: Primer

## Claims

1. A method for detecting a cancer in an individual, comprising:
detecting an expression level of CAPRIN-1 in a biological sample from the individual above that of a healthy subject through an antigen-antibody reaction using
a monoclonal antibody having a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 70 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 71, or an antigen-binding fragment thereof.

2. The method according to claim 1, wherein the CAPRIN-1 to be detected is:
(a) a polypeptide consisting of the amino acid sequence shown in any even-numbered SEQ ID NO of SEQ ID Nos: 2 to 30 in the Sequence Listing, or
(b) a polypeptide consisting of 85% or higher sequence identity to the polypeptide consisting of the amino acid sequence shown in any even-numbered SEQ ID NO of SEQ ID Nos: 2 to 30 in the Sequence Listing.

3. The method according to claim 1, wherein the biological sample is derived from a human, a dog, or a cat.

4. The method according to claim 1, wherein the biological sample is derived from a dog, and the CAPRIN-1 to be detected consists of the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, or 14.

5. The method according to claim 1, wherein the biological sample is derived from a human, and the CAPRIN-1 to be detected consists of the amino acid sequence shown in SEQ ID NO: 2 or 4.

6. The method according to claim 1, wherein the detection of the expression level of CAPRIN-1 is carried out using an immunological assay method.

7. The method according to claim 6, wherein the immunological assay method is ELISA and/or an immunohistochemical staining method.

8. The method according to claim 1, wherein the biological sample is a body fluid, a tissue, or a cell.

9. The method according to claim 1, wherein the cancer is at least one cancer selected from the group consisting of breast cancer, brain tumor, esophagus cancer, stomach cancer, lung cancer, liver cancer, kidney cancer, thyroid gland cancer, spleen cancer, pancreas cancer, large bowel cancer, skin cancer, ovary cancer, uterus cancer, prostate cancer, bladder cancer, testis cancer, osteosarcoma, and fibrosarcoma.

10. The method according to claim 2, wherein the biological sample is derived from a human, a dog, or a cat.

11. The method according to claim 2, wherein the biological sample is derived from a dog, and the CAPRIN-1 to be detected consists of the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, or 14.

12. The method according to claim 3, wherein the biological sample is derived from a dog, and the CAPRIN-1 to be detected consists of the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, or 14.

13. The method according to claim 2, wherein the biological sample is derived from a human, and the CAPRIN-1 to be detected consists of the amino acid sequence shown in SEQ ID NO: 2 or 4.

14. The method according to claim 3, wherein the biological sample is derived from a human, and the CAPRIN-1 to be detected consists of the amino acid sequence shown in SEQ ID NO: 2 or 4.

15. A method for selecting an individual-specific therapeutic drug for a cancer, comprising:
detecting an expression level of CAPRIN-1 in a biological sample from the individual using a monoclonal antibody having a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 70 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 71, or an antigen-binding fragment thereof; and,
if the expression level is statistically significantly higher than that of a healthy individual, selecting a CAPRIN-1-targeting drug as a therapeutic drug for the cancer suitable for administration to the individual from which the biological sample was derived.

16. The method for selecting an individual-specific therapeutic drug for a cancer according to claim 15, wherein the CAPRIN-1-targeting drug is an antibody having immunological reactivity with CAPRIN-1, or an antigen-binding fragment thereof.

17. A method for detecting CAPRIN-1 in an individual comprising:
detecting CAPRIN-1 in a biological sample from the individual through an antigen-antibody reaction using a monoclonal antibody having a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 70 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 71, or an antigen-binding fragment thereof.