METHOD TO DETECT PANCREATIC CANCER

专利摘要:
methods to detect pancreatic cancer and reagents or kits to detect pancreatic cancer. this invention relates to a method for detecting pancreatic cancer using novel tumor markers. specifically, the invention provides a method for detecting pancreatic cancer, comprising measuring the presence or amount of a polypeptide that has a binding reactivity via an antigen-antibody reaction to an antibody against caprin-1 protein in a separate sample from a subject, and a reagent or kit for detecting pancreatic cancer, which comprises a caprin-1 protein or a fragment thereof, an antibody thereto, or a polynucleotide encoding the same.
公开号:BR112014002616B1
申请号:R112014002616-5
申请日:2012-08-03
公开日:2022-01-18
发明作者:Takayoshi Ido；Fumiyoshi Okano
申请人:Toray Industries, Inc；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The present invention relates to a method for detecting pancreatic cancer with the use of CAPRIN-1 as a tumor marker. BACKGROUND OF THE TECHNIQUE
[002] It is reported that there are over 10,000 patients with refractory pancreatic cancer in Japan whose occurrence of the same is increasing year by year, and it is assumed that the number of patients will continue to increase. Even if the pancreatic cancer was surgically removed, the small cancer cells often infiltrated and metastasized to other organs. Consequently, pancreatic cancer often recurs, and the 5-year survival rate is as low as 9%, that is, the prognosis of pancreatic cancer is very poor. For the purpose of preventing postoperative recurrence, gemcitabine, an anticancer agent, was used. However, the primary goal of gemcitabine administration is pain relief, and tumor size reduction or survival advantage can hardly be expected. In some hospitals, another anticancer agent, TS-1, is used, which is currently used for gastric cancer, although it is difficult to expect any therapeutic effects.
[003] In order to improve the prognosis for pancreatic cancer, early detection is important, as for other types of cancer; however, early detection is difficult because pancreatic cancer shows substantially no early symptoms. To date, pancreatic cancer detection methods using carcinoembryonic antigen (CEA) and glycoproteins (CA19-9 and Dupan-2) in biological samples as tumor markers of pancreatic cancer have been actively employed. However, levels of these tumor markers do not become elevated unless pancreatic cancer advances, and these markers occasionally show normal values in the progressive stage. Consequently, these tumor markers are not considered sufficient for the accurate detection of pancreatic cancer. Furthermore, most tumor markers that are currently known are present in very small amounts in body fluids (at the pg/ml level). In order to detect small amounts of these markers, therefore, detection techniques with high sensitivity or special techniques are required. Under such circumstances, it is expected that a new technique used for the detection of pancreatic cancer in a simple way, with high sensitivity, is expected to be applicable for the diagnosis of pancreatic cancer. It is necessary to undergo periodic thorough examinations in order to detect pancreatic cancer at an early stage. Consequently, a method for detecting cancer has been awaited that can be performed in a simple way, with the use of blood serum or urine samples, without imposing material or financial burdens, both in healthy individuals without pancreatic cancer or in patients with cancer.
[004] In addition, pancreatic cancer is refractory in dogs. Although a lump can be seen in the abdominal region of a dog sick with pancreatic cancer, the main symptoms are rapid loss of energy, unsteady gait, and gait abnormalities resulting from hypoglycemia. In most cases, the development of cancer would not be detected until these symptoms were observed. In addition, pancreatic cancer is usually likely to be advanced when these symptoms are observed. In addition to surgical removal of pancreatic cancer, therefore, therapeutic techniques are limited to supportive therapy and administration of anticancer agents. As with human patients, early detection is important for dogs suffering from pancreatic cancer in order to effectively treat this pancreatic cancer. As in the case of humans, there were no diagnostic agents for dogs in the past that allowed the detection of pancreatic cancer at an early stage in a simple way. In the field of veterinary medicine, detection techniques such as radiographic techniques using X-rays, CT or MRI have not yet become common. At present, detection is performed by palpation, simple blood testing, and X-ray photography, and diagnosis is heavily dependent on the expertise of veterinarians. If a simple means of detecting cancer with high sensitivity that can be applied to the diagnosis of pancreatic cancer in dogs is provided, appropriate treatment can be carried out, which has great advantages for dog owners and veterinarians.
[005] Cytoplasmic and proliferation-associated protein 1 (CAPRIN-1) is an intracellular protein that is expressed when normal cells, in the resting phase, are activated or undergo cell division. CAPRIN-1 is also known to be involved in controlling the transport and translation of mRNAs through the formation of cytoplasmic stress granules and RNA in a cell. In addition, genes encoding CAPRIN-1 proteins have been shown to be specifically expressed in canine and human testis and malignant tumor cells, FCM analysis of breast cancer cells using antibodies against CAPRIN-1 demonstrates high expression of CAPRIN-1 on breast cancer cell surfaces, and immunohistochemical staining using breast cancer tissues demonstrates high-level expression of CAPRIN-1 in breast cancer cells. Furthermore, it was reported that the aforementioned antibodies would damage breast cancer cells through lymphocyte functions, and that antibodies against CAPRIN-1 exert potent antitumor effects in cancer-bearing mouse models in which breast cancer cells were transplanted (Patent Literature 1). Furthermore, it was reported that cancers such as breast cancer could be diagnosed by measuring both antibodies induced in a subject's body against CAPRIN-1 present in blood serum, and polypeptides that undergo antigen-antibody reactions with CAPRIN. -1 (Patent Literature 2). To date, however, there have been no reports of the fact that pancreatic cancer can be diagnosed by measuring either antibodies to CAPRIN-1 induced in the blood serum of a pancreatic cancer patient or polypeptides that undergo antigen-antibody reactions. with CAPRIN-1. BACKGROUND ART LITERATURES Patent Literature 1: international publication document WO 2010/016526Patent Literature 2: international publication document WO 2010/016527 BRIEF DESCRIPTION OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION
[006] It is an object of the present invention to provide a means for detecting pancreatic cancer, which is useful for diagnosing pancreatic cancer. MEANS TO SOLVE THE PROBLEM
[007] The present inventors have conducted concentrated studies. As a result, the present inventors have now discovered that pancreatic cancer can be diagnosed, examined, or detected based on the expression of CAPRIN-1 in pancreatic cancer, by measuring (or assaying) antibodies against CAPRIN-1 induced in a patient's blood serum. with pancreatic cancer using a CAPRIN-1 protein, and in binding antibodies produced using these proteins to CAPRIN-1 in pancreatic cancer tissue. This led to the completion of the present invention.
[008] Specifically, the present invention provides a method for detecting pancreatic cancer, which comprises measuring CAPRIN-1 expression in a separate sample from a subject. The term "detection", as used in the present application, may be used interchangeably with the term "analysis" or "evaluation". Furthermore, the present invention provides a reagent or kit for detecting pancreatic cancer, which comprises a polypeptide that is subjected to an antigen-antibody reaction with an antibody to CAPRIN-1 induced or produced in a subject's body. Furthermore, the present invention provides a reagent or kit for detecting pancreatic cancer, which comprises an antibody that is subjected to an antigen-antibody reaction with CAPRIN-1 or an antigen-binding fragment of the antibody. Furthermore, the present invention provides a reagent or kit for detecting pancreatic cancer, which comprises a polynucleotide that specifically hybridizes to a partial sequence comprising at least 15 to 19 nucleotides or at least 20 to 30 nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, ..., 29. The “reagent or kit for detecting pancreatic cancer” used in the present application may also be referred to as a “reagent or kit for detecting pancreatic cancer”. for pancreatic cancer detection.
[009] Specifically, the present invention has the features described below. (1) A method, for detecting pancreatic cancer, which comprises measuring the presence or amount of a polypeptide that has a reactivity to specifically bind an antibody against a protein CAPRIN-1 via an antigen-antibody reaction, or the presence or amount of a nucleic acid encoding the polypeptide in a separate sample from a subject.(2) Method according to (1), wherein the polypeptide to be measured is a CAPRIN-1 protein, which consists of an amino acid sequence represented by any of even-numbered SEQ ID NOs: 2 to 30, or a polypeptide consisting of an amino acid sequence that has 85 to 90% or greater identity sequence with CAPRIN-1 protein.(3) Method according to (1) or (2), wherein the subject is a human or a dog.(4) Method according to (3), wherein the subject is a dog and the polypeptide to be measured comprises an amino acid sequence represented by any of even-numbered SEQ ID NOs: 2 to 30. (5) Method according to (4), wherein the subject is a dog and the polypeptide to be measured comprises the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12 or 14. (6) The method according to (3), wherein the subject is a human and the polypeptide to be measured comprises the amino acid sequence represented by SEQ ID NO: 2 or 4.(7) A method according to any one of (1) to (6), wherein the presence or amount of the polypeptide is determined by immunological measurement of an antibody induced against the polypeptide to be measured in the body of a subject, the which may be contained in the sample.(8) A method according to any one of (1) to (6), wherein the presence or amount of a nucleic acid encoding the polypeptide is determined by measuring a nucleic acid encoding the polypeptide that polypeptide contained in the sample.(9) Method according to (8), wherein the presence or amount of the nucleic acid in the sample is measured with the use of a polynucleotide that specifically hybridizes to a partial sequence comprising at least 15 to 19 nucleotides, preferably at least 20 to 25 nucleotides, and more preferably at least 30 nucleotides of the nucleotide sequence in the nucleic acid or a sequence complementary thereto. (10) The method according to (9), wherein the subject is a dog and the polynucleotide specifically hybridizes to a partial sequence comprising at least 15 to 19 nucleotides, preferably at least 20 to 25 nucleotides, and more preferably at least 30 nucleotides in the nucleotide sequence represented by SEQ ID NO: 5, 7, 9, 11 or 13 or a sequence complementary thereto.(11) Method according to (9), wherein the subject is a human and the polynucleotide specifically hybridizes to a partial sequence comprising at least 15 to 19 nucleotides, preferably at least 20 to 25 nucleotides, and more preferably at least 30 nucleotides in the nucleotide sequence represented by SEQ ID NO: 1 or 3 or a sequence complementary thereto.(12) A method according to any one of (1) to (6), wherein the presence or amount of the polypeptide is determined by measurement of the polypeptide contained in the sample.(13) Method according to (12), wherein the assay is an immunological assay.(14) Method, according to any one of (1) to (13), wherein the sample is blood, serum, blood plasma, ascitic fluid, pleural effusion, tissue or cells.(15) Reagent or kit to detect pancreatic cancer, which comprises one or more polypeptides that have a binding reactivity through an antigen-antibody reaction to a antibody induced against a CAPRIN-1 protein in a subject's body.(16) A reagent or kit for the detection of pancreatic cancer, which comprises one or more antibodies that undergo an antigen-antibody reaction with a polypeptide that has a binding reactivity through an antigen-antibody reaction to an antibody with a CAPRIN-1 protein and produced in a subject's body or with an antigen-binding fragment of the antibody.(17) Reagent or kit, according to (15) or (16), wherein the CAPRIN-1 protein has an amino acid sequence represented by any one of even-numbered SEQ ID NOs: 2 to 30. (18) Reagent or kit, according to (16) or (17), wherein the antibody or an antigen-binding fragment of the that is subjected to an antigen-antibody reaction with the polypeptide is an antibody or an antigen-binding fragment thereof that binds to the surface of a pancreatic cancer cell.(19) Reagent or kit, according to any of (16) to (18), wherein the antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with the polypeptide comprises an antibody or a fragment thereof that has an immunological reactivity with a polypeptide that consists of an amino acid sequence comprising at least 7 to 12 contiguous residues. amino acid residues in the region of amino acid residues No. 50 to 98 or amino acid residues No. 233 to 344 of the amino acid sequence represented by any of the even-numbered SEQ ID NOs: 2 to 30, except SEQ ID NOs: 6 and 18 or with a polypeptide comprising the polypeptide as a partial sequence.(20) A reagent or kit according to any one of (16) to (19), wherein the antibody or an antigen-binding fragment thereof which is subjected to an antigen-antibody reaction with the polypeptide is one or more antibodies selected from the group consisting of: an antibody that binds to a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 43 or a binding fragment of antigen thereof; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOs: 44 and 45 or an antigen-binding fragment thereof; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOs: 44 and 46 or an antigen-binding fragment thereof; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOs: 44 and 47 or an antigen-binding fragment thereof; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOs: 44 and 48 or an antigen-binding fragment thereof; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOs: 49 and 50 or an antigen-binding fragment thereof; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOs: 51 and 52 or an antigen-binding fragment thereof; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOs: 53 and 54 or an antigen-binding fragment thereof; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOs: 55 and 56 or an antigen-binding fragment thereof; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOs: 57 and 58 or an antigen-binding fragment thereof; and a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOs: 59 and 60 or an antigen-binding fragment thereof.(21) Reagent or kit, for detecting pancreatic cancer, comprising one or more polynucleotides that specifically hybridize with a partial sequence comprising at least 15 to 19 nucleotides, preferably at least 20 to 25 nucleotides, and more preferably at least 30 nucleotides in the nucleotide sequence represented by any of the odd-numbered and encoding SEQ ID NOs: 1 to 29 a CAPRIN-1 protein or in a sequence complementary to the nucleotide sequence.(22) A method to detect pancreatic cancer, which comprises measuring the presence or amount of a CAPRIN-1 protein, an antibody against a CAPRIN-1 protein, or a a nucleic acid encoding the CAPRIN-1 protein in a sample from a subject using at least one reagent or kit according to any one of (15) to (21).(23) A method for detecting pancreatic cancer according to any one of (1) to (14), which comprises measuring the presence or amount of a CAPRIN-1 protein, an antibody against a CAPRIN-1 protein, or an acid nucleic acid encoding CAPRIN-1 protein in a sample from a subject using at least one reagent or kit according to any one of (15) to (21).
[010] According to the present invention, a new method for detecting pancreatic cancer is provided. As specifically described in the examples below, a recombinant polypeptide prepared based on the amino acid sequence of CAPRIN-1 (or Caprin-1) is capable of reacting specifically with an antibody that exists in the serum of a pancreatic cancer patient. Thus, pancreatic cancer existing in a subject can be detected by measuring the antibody in a sample by the method of the present invention. In addition, existing pancreatic cancer in a subject can be detected by measuring (or assaying) CAPRIN-1 itself. As described in the examples below, in addition, high levels of CAPRIN-1 gene expression are specifically observed in testis and pancreatic cancer cells of subjects (from this point forward, this expression product is occasionally referred to as “nucleic acid encoding a CAPRIN-1 (protein)”). Therefore, pancreatic cancer can also be detected by measuring a nucleic acid. In addition, the presence or amount of CAPRIN-1 (expression) in pancreatic cancer tissue can be measured using an antibody against CAPRIN-1. Pancreatic cancer patients can undergo this measurement in advance, so that patients to whom a CAPRIN-1-targeted therapeutic agent (eg, medicinal antibody) is applicable can be selected. METHODS FOR CARRYING OUT THE INVENTION
[011] According to the method of the present invention, the presence or amount of CAPRIN-1 (expression) is measured using a separate sample from a subject. Examples of methods for measuring the presence or amount of CAPRIN-1 (expression) include: a method for immunologically measuring an antibody to CAPRIN-1 contained in a sample (first method); a method for measuring the CAPRIN-1 itself contained in a sample (second method); and a method for measuring a nucleic acid encoding CAPRIN-1 contained in a sample, such as mRNA or cDNA synthesized from the mRNA (third method). In the present invention, the presence or amount of CAPRIN-1 (expression) can be measured by any of the above methods. In the present invention, the term "measurement" is intended to include any of the following meanings: detection, qualitative measurement, quantitative measurement, and semiquantitative measurement.
[012] The amino acid sequence represented by SEQ ID NOs: 6, 8, 10, 12 or 14 is a canine CAPRIN-1 amino acid sequence. Canine CAPRIN-1 with this amino acid sequence has been identified as a polypeptide that binds to an antibody specifically found in serum derived from a dog with cancer (see Example 1). An antibody against CAPRIN-1 having the amino acid sequence represented by SEQ ID NOs: 6, 8, 10, 12 or 14 is specifically induced or stimulated in the body of a dog bearing cancer. Specifically, canine pancreatic cancer can be detected by measuring the antibody against CAPRIN-1 having the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12 or 14 by the first method. Furthermore, canine pancreatic cancer can be detected by measuring CAPRIN-1 itself which has the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12 or 14 by the second method. Since the CAPRIN-1 gene is expressed at significantly high levels in pancreatic cancer cells, canine pancreatic cancer can be detected by nucleic acid measurement according to the third method.
[013] The term "with an amino acid sequence", used in the present application, refers to amino acid residues aligned in a certain order. Therefore, for example, the term "polypeptide having the amino acid sequence represented by SEQ ID NO: 2" refers to a polypeptide of 709 amino acid residues consisting of the amino acid sequence of Met Pro Ser Ala... (partially omitted ) ... Gln Gln Val Asn represented by SEQ ID NO: 2. In addition, the term "polypeptide having the amino acid sequence represented by SEQ ID NO: 2" may also be abbreviated as "the polypeptide of SEQ ID NO: 2". 2,” for example. The same applies to the expression “which has a sequence of nucleotides”. In this case, the term “which has” can be replaced by the expression “which comprises” or “which consists of”.
[014] Furthermore, the term "polypeptide", used in the present application, refers to a molecule that is formed through the peptide bond of a plurality of amino acids. Examples of such a molecule include not only polypeptide molecules with a large number of constituent amino acids, but also low molecular weight molecules (oligopeptides) with a small number of amino acids and entire proteins. The present invention further encompasses full-length CAPRIN-1 proteins having an amino acid sequence represented by any of the even-numbered SEQ ID NOs: 2 to 30 (i.e., SEQ ID NOs: 2, 4, 6, -26, 28, and 30 ).
[015] The term "subject" as used in the present application refers to vertebrates, including mammals and birds, preferably mammals, and more preferably, humans, dogs, cattle and horses.
[016] The term “sample”, used in the present application, refers to a biological sample submitted to an examination aimed at the detection of pancreatic cancer. Sample examples include body fluids, tissues and cells separated from a subject. Examples of body fluids include, but are not limited to, blood, serum, blood plasma, ascitic fluid, and pleural effusion. Pancreatic tissues or cells suspected of being affected by cancer are within the scope of the “sample”.
[017] In the method of the present invention, the targets to be measured are not only canine CAPRIN-1 of SEQ ID NO: 6, 8, 10, 12 or 14, but also CAPRIN-1 from other mammals (which from this point onwards, they may also be called the “homologous” (or “orthologous”) of canine CAPRIN-1). When simply called “CAPRIN-1”, CAPRIN-1 from another mammal, including a human, is also a target to be measured, in addition to CAPRIN-1 from a dog. As specifically described in the examples below, expression of the human CAPRIN-1 gene is significantly high in human pancreatic cancer cells, while no antibodies against the CAPRIN-1 gene are detected in a healthy human organism. In this way, pancreatic cancer of a mammal other than a dog can be detected by measuring the expression of CAPRIN-1 in the mammal. An example of a mammalian CAPRIN-1, other than a dog, to be measured by the method of the present invention is, but is not limited to, human CAPRIN-1. The nucleotide sequences encoding human CAPRIN-1 and the amino acid sequences thereof are represented by SEQ ID NOs: 1 and 3 and SEQ ID NOs: 2 and 4 and in the Sequence List. The sequence identity between human CAPRIN-1 and canine CAPRIN-1 is 94% for the nucleotide sequence and 98% for the amino acid sequence. The sequence identity of CAPRIN-1 amino acid sequences is as high as 98% between genetically distant mammals such as a dog and a human. Therefore, sequence identity is considered to be about 85% or greater between a dog and a mammal other than a human; that is, canine CAPRIN-1 and its counterpart. CAPRIN-1, the expression of which can be measured by the method of the present invention, preferably has 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity to the the canine CAPRIN-1 amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12 or 14, although sequence identities are not limited thereto.
[018] In the first method, the antibody that may be present in a sample can be easily measured by immunoassay using an antigenic substance that undergoes an antigen-antibody reaction with the antibody. The immunoassay itself is a well known conventional method, as specifically described below. As an antigenic substance for the immunoassay, for example, the canine CAPRIN-1 protein of SEQ ID NO: 6, 8, 10, 12 or 14 which induces the antibody within the body of a dog bearing cancer or a fragment that contains a epitope of that protein can be used. In addition, the antibody shows cross-reactivity. A molecule other than the antigenic substance that actually serves as an immunogen can also bind, through an antigen-antibody reaction, to an antibody induced against an immunogen, provided that the molecule has a structure analogous to an epitope of the immunogen. Between a protein from a certain type of mammal and a homologue of it from another mammal, in particular, their amino acid sequence identity is high and the epitope structures are often analogous to each other. As specifically described in the examples below, canine CAPRIN-1 of SEQ ID NO: 6, 8, 10, 12 or 14 is subjected to an antigen-antibody reaction with an antibody induced against canine CAPRIN-1 within the body of a dog. cancer carrier. In addition, human CAPRIN-1 undergoes an antigen-antibody reaction with the antibody induced within the body of a cancer-carrying dog. Consequently, CAPRIN-1 from any mammal can be used as an antigen for the immunoassay according to the first method of the present invention.
[019] When an antigenic substance is a protein or a similar one with a complicated structure and a high molecular weight, in general, a number of sites with different structures are present in the molecule. Therefore, a series of types of antibodies capable of recognizing and binding to different sites of these antigenic substances are produced in a subject's body. Specifically, an antibody that is produced in the subject against an antigenic substance, such as a protein, is a polyclonal antibody that is a mixture of a number of antibody types. An antibody recently discovered by the present inventors is also a polyclonal antibody, which is specifically present in serum obtained from a cancer-bearing subject and which specifically binds, via an antigen-antibody reaction, to a recombinant CAPRIN-1 protein. The term "polyclonal antibody" as used in the present invention refers to an antibody that exists in serum obtained from a subject that contains an antigenic substance and is induced against that antigenic substance.
[020] In the examples below, the polypeptides of SEQ ID NO: 6 and SEQ ID NO: 8 (both from canine CAPRIN-1) and the polypeptide of SEQ ID NO: 2 (human CAPRIN-1) were prepared as antigens for the immunoassay of specific antibodies in live cancer-bearing animals. Reactivity between these polypeptides and antibodies in serum obtained from a subject with cancer was then confirmed. However, the antibodies mentioned above are polyclonal antibodies and naturally bind to polypeptides consisting of homologs of SEQ ID NOs: 6, 8 and 2. Even in the case of a fragment of this polypeptide, it can bind to an antibody present in the serum obtained from a subject with cancer, since some polyclonal antibodies are able to recognize the structure of the fragment. In this way, both the polypeptide (i.e., the full-length CAPRIN-1 protein) of the homolog of SEQ ID NO: 6, 8, or 2 and a fragment thereof can similarly be used for the assay of a polyclonal antibody present specifically in the serum of a subject with cancer, and they are useful for cancer detection. Consequently, a polypeptide to be used as an antigen for the immunoassay in the first method of the present invention is not limited to just one polypeptide consisting of the entire region of a CAPRIN-1 protein (e.g., SEQ ID NO: 6, 8 or 2) . It can be a polypeptide fragment consisting of at least 7 to 12, and preferably at least 8, 9, or 10 continuous amino acids of the amino acid sequence of a CAPRIN-1 protein that undergoes an antigen-antibody reaction. with a polyclonal antibody against the CAPRIN-1 protein (which, hereafter, may be called a “partially reactive specifically polypeptide” for convenience). It is known in the art that a polypeptide comprising about 7 to 12 or more amino acid residues can exert antigenicity. If the number of amino acid residues is too low, however, that polypeptide most likely cross-reacts with an antibody against a protein other than the CAPRIN-1 protein that exists in the sample. In order to improve the accuracy of the immunoassay, therefore, the number of amino acid residues of a polypeptide fragment is preferably 20 or more, 30 or more, 50 or more, more preferably, 100 or more or 150 or more, even more. more preferably 300 or more and more preferably 600 or more. The number of amino acid residues can be 1000 or more or 1500 or more.
[021] Preferred examples of polypeptides to be used as antigens include polypeptides of even numbers of SEQ ID NOs: 2 to 30 or fragments thereof that comprise epitopes (e.g., a polypeptide fragment comprising about 7 to 12 or more amino acid residues).
[022] Nucleotide sequences of protein-coding polypeptides consisting of the even-numbered amino acid sequences of SEQ ID NOs: 2 to 30 (i.e., SEQ ID NOs: 2, 4, 6...28 and 30) are represented by the odd-numbered SEQ ID NOs: 1 to 29 (i.e., SEQ ID NOs: 1, 3, 5...27 and 29).
[023] In general, it is known in the art that protein antigens maintain antigenicity nearly equivalent to that of the original protein, even if a small number of amino acid residues are substituted, deleted, added, or inserted into the amino acid sequence of the protein. Therefore, a polypeptide with a sequence derived from the amino acid sequence of a CAPRIN-1 protein by substitution, deletion and/or insertion of a small number of (preferably one or more) amino acid residues with 80% or more, 85 to 90% or more, preferably 90% or more, more preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more sequence identity to the original sequence, and which specifically binds via an antigen-antibody reaction to an antibody against CAPRIN-1 (which hereafter may be called a “specifically reactive modified polypeptide” for convenience) can be used for cancer detection in a similar manner to the case of polypeptides. described above. Preferably, a specifically reactive modified polypeptide has an amino acid sequence derived from an amino acid sequence of a CAPRIN-1 protein by substitution, deletion and/or insertion of one or more amino acid residues. The term "various" as used in the present application refers to an integer from 2 to 10, preferably an integer from 2 to 6, and even more preferably an integer from 2 to 4.
[024] The term "sequence identity", used in the present application with respect to amino acid sequences, is determined by aligning two amino acid sequences to be compared so that as many amino acid residues as possible combine, by dividing the number of amino acid residue matches by the total number of amino acid residues and then expressing the results in terms of percentage (%). Through the above alignment, gaps are inserted where appropriate in one or both sequences to be compared, as needed. Such sequence alignment can be performed using a well-known program or algorithm such as BLAST, FASTA or CLUSTAL W (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1993; Altschul et al. al., Nucleic Acids Res., 25: 3389-3402, 1997).
[025] The twenty types of naturally occurring constituent amino acids can be divided into groups of amino acids with properties analogous to each other: neutral amino acids with side chains with low polarity (Gly, Ile, Val, Leu, Ala, Met and Pro); neutral amino acids with hydrophilic side chains (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). Substitution between these amino acids (ie, conservative substitution) is known to rarely change the properties of the resulting polypeptide. When CAPRIN-1 amino acid residues are substituted, consequently, the substitution is carried out between members of the same group, so that the possibility of maintaining binding with the corresponding antibody becomes greater. In the present invention, however, the above variant may involve non-conservative substitution, provided that immunity-inducing activity equivalent or nearly equivalent to that of an unmodified polypeptide is conferred.
[026] A polypeptide (which, from this point forward, may be called a “specifically reactive addition polypeptide” for convenience) that contains, as a partial sequence, the above polypeptide to be used in the present invention (e.g., prepared by adding another (poly)peptide to one or both ends of a polypeptide to be used in the present invention) and which specifically binds through an antigen-antibody reaction to an antibody against CAPRIN-1 can also be used for cancer detection pancreatic tissue in a manner similar to the above polypeptide cases.
[027] The polypeptides used in the present invention can be synthesized according to a chemical synthesis method, such as the Fmoc method (the fluorenylmethyloxycarbonyl method) or the tBoc method (the t-butyloxycarbonyl method) (the Japanese Biochemical Society (ed. .), Seikagaku Jikken Koza (Biochemical Experimental Lecture Series) 1, Tanpakushitsu no Kagaku (Protein Chemistry) IV, Kagaku Shushoku to Peptide Gousei (Chemical Modification and Peptide Synthesis), TOKYO KAGAKU DOZIN CO., LTD, Japan, 1981). Furthermore, polypeptides can be synthesized by a conventional method using various commercially available peptide synthesizers. Alternatively, polypeptides can be readily prepared by genetic engineering techniques (e.g., Sambrook et al., Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology, 1989, Cold Spring Harbor Laboratory Press; Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, A Compendium of Methods from Current Protocols in Molecular Biology, 1995, John Wiley & Sons). From RNA extracted from a tissue that expresses a gene encoding human CAPRIN-1 of SEQ ID NO: 2 or a homolog thereof, for example, cDNA of the gene is prepared by RT-PCR, the entire sequence or a The desired partial sequence of the cDNA is incorporated into an expression vector, and the vector is then introduced into a host cell. In this way, a polypeptide of interest can be obtained. The nucleotide sequences of cDNAs encoding canine CAPRIN-1 of SEQ ID NOs: 6, 8, 10, 12 and 14 are shown in SEQ ID NOs: 5, 7, 9, 11 and 13, respectively. Nucleotide sequences of human homologs thereof, i.e., the cDNAs encoding human CAPRIN-1 of SEQ ID NOs: 2 and 4, are shown in SEQ ID NOs: 1 and 3, respectively. Consequently, the primers used for RT-PCR can be easily designed with reference to these nucleotide sequences. As described below, a gene encoding CAPRIN-1 from a non-human mammal can be amplified using primers designed with reference to these odd-numbered nucleotide sequences of SEQ ID NOs: 1 to 29. Consequently, cDNA encoding, for example, feline CAPRIN-1 can be readily prepared by techniques similar to the above techniques. RNA extraction, RT-PCR, incorporation of cDNA into a vector and introduction of a vector into a host cell can be performed, for example, by well known techniques as described below. Furthermore, the vectors and host cells used in the present application are well known, and a number of vectors and host cells are commercially available.
[028] The above host cells can be any cells as long as they can express the above polypeptides. Examples of prokaryotic cells include Escherichia coli. Examples of eukaryotic cells include mammalian cells in culture, such as monkey kidney cells (COS1), Chinese hamster ovary (CHO) cells, the human embryonic kidney cell line (HEK293), and the mouse embryonic skin cell line. (NIH3T3), germinating yeast, fissioning yeast, silkworm cells and Xenopus oocytes.
[029] When prokaryotic cells are used as host cells, an expression vector with an origin of replication in prokaryotic cells, a promoter, a ribosome binding site, a multicloning site, a terminator, a drug resistance gene, an auxotrophic complementary gene and the like are used. Examples of expression vectors for Escherichia coli include pUC vectors, pBluescriptII, pET expression systems, and pGEX expression systems. The DNA encoding the above polypeptide is incorporated into this expression vector, the prokaryotic cells are transformed with the vector, and thus the obtained transformant is cultured. In this way, the polypeptide encoded by the DNA can be expressed in prokaryotic host cells. At this point, the polypeptide can also be expressed as a fusion protein with another protein. DNA encoding the above polypeptide can be obtained by preparing the cDNA by, for example, RT-PCR, as described above. Alternatively, such DNA can be synthesized by a conventional technique using a commercially available nucleic acid synthesizer, as described below. The nucleotide sequences of cDNAs of the genes encoding CAPRIN-1 of SEQ ID NOs: 2 and 4 are shown in SEQ ID NOs: 1 and 3 and the Sequence List, respectively.
[030] When eukaryotic cells are used as host cells, an expression vector for eukaryotic cells with a promoter, a splicing region, an additional poly(A) site and the like are used. Examples of such an expression vector include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, an EBV vector, pRS, pcDNA3, and pYES2. The DNA encoding the above polypeptide is incorporated into this expression vector, the prokaryotic cells are transformed with the vector, and thus the obtained transformant is cultured. In this way, the polypeptide encoded by the above DNA can be expressed in eukaryotic host cells. When pIND/V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 and the like are used as an expression vector, the above polypeptide can be expressed as a fusion protein with various tags such as His tags (e.g. (His)6 to (His)10), a FLAG tag, a myc tag, an HA tag, or a GFP.
[031] An expression vector can be introduced into a host cell according to a well-known technique such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, lipofection, or ligation with a cell membrane permeable peptide.
[032] A polypeptide of interest can be isolated and purified from host cells with the combined use of known isolation techniques. Examples of these techniques include treatment using a denaturing agent such as urea or a surfactant, ultrasonication, enzymatic digestion, salting-out, solvent and precipitation fractionation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing. , ion exchange chromatography, hydrophobic chromatography, affinity chromatography and reversed-phase chromatography.
[033] Polypeptides obtained by the above methods include polypeptides in the form of fusion proteins with any other proteins. Examples of such fusion proteins include a glutathione-S-transferase fusion protein and a His-tag fusion protein. Polypeptides in the form of these fusion proteins are also within the scope of specifically reactive addition polypeptides, and these polypeptides can be used for the first detection method of the present invention. Furthermore, polypeptides expressed in transformed cells may occasionally undergo various types of modification within cells after translation. Such a post-translationally modified polypeptide can be used in the first detection method of the present invention, provided that it is capable of specifically binding, through an antigen-antibody reaction, to an antibody against a CAPRIN-1 protein. Examples of such post-translational modification include N-terminal methionine removal, N-terminal acetylation, glycosylation, intracellular protease limited proteolysis, myristoylation, isoprenylation, and phosphorylation.
[034] An antibody in a sample can be easily measured by immunoassay using the above polypeptide as an antigen. The immunoassay itself is well known in the art. The immunoassay is classified into sandwich method, competition method, agglutination method, Western blot method and the like based on the types of reaction. In addition, the immunoassay is classified, based on the markers, into radioimmunoassay, fluorescence immunoassay, enzyme immunoassay, and biotinylated immunoassay, for example. The above antibody immunoassay can be performed using any of these methods. The sandwich ELISA or the agglutination method is preferably used as an immunoassay technique for the above antibody in the method of the present invention, since the procedures of these methods are convenient and do not require any extensive apparatus and the like, although the techniques not be limited to these. When an enzyme is used as a marker for an antibody, that enzyme is not specifically limited as long as it satisfies conditions such as: high clearance number; remain stable even when bound to an antibody; and specifically promoting substrate color development. Enzymes that can be used for the common enzyme immunoassay include peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholine esterase, glucose-6-phosphate dehydrogenase, and malic acid dehydrogenase. In addition, substances that inhibit the enzyme, coenzymes and the like can be used. Binding of these enzymes to antibodies can be accomplished by methods known in the art, which involve the use of a cross-reactive agent such as a maleimide compound, the biotin-(strept)avidin system, or the like. As a substrate, a known substance can be used depending on the type of enzyme used. When peroxidase is used as an enzyme, for example, 3,3',5,5'-tetramethylbenzidine can be used. When alkaline phosphatase is used, for example, paranitrophenol can be used. A radioisotope that is commonly used for radioimmunoassay, such as 125I or 3H, can be used. A fluorescent dye that is used for common fluorescent antibody techniques, such as fluorescent isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), or a fluorescent cyanine dye (eg, Cy3 or Cy5), can be used.
[035] There is no need to explain the above immunoassay techniques in the present application, as these techniques are well known; however, briefly, for example, the sandwich method comprises immobilizing the above polypeptide used as an antigen on a solid phase, reacting the polypeptide with a sample such as serum, a wash, reacting a suitable secondary antibody with an antibody from the sample, washing again, and then measuring the secondary antibody bound to the solid phase. By immobilizing an antigenic polypeptide on a solid phase, an unbound secondary antibody can be easily removed. Accordingly, this is the preferred embodiment of the cancer detection method of the present invention. As a secondary antibody, an anti-canine IgG antibody, for example, can be used if the sample is obtained from a dog. A secondary antibody is pre-labeled with a labeling substance exemplified above, so that the secondary antibody bound to a solid phase can be measured. The amount of secondary antibody so measured corresponds to the amount of the above antibody in the serum sample. When an enzyme is used as a marker substance, the amount of antibody can be measured by adding a substrate which is degraded to develop color by enzymatic action and then optically measuring the amount of substrate degraded. When a radioisotope is used as a tracer substance, the amount of radiation from the radioisotope can be measured using a scintillation counter or the like.
[036] In the second method of the present invention, the CAPRIN-1 that may be contained in a sample obtained from a subject is measured. As described above, the amount of antibody that undergoes an antigen-antibody reaction with canine CAPRIN-1, human or similar, is significantly higher in subjects with pancreatic cancer compared to healthy subjects. This indicates that the amount of CAPRIN-1 accumulated as an antigen is significantly higher in pancreatic cancer cells. In the case of healthy subjects, the expression level of CAPRIN-1 is below the detection limit or the expression of CAPRIN-1 in tissue is weak and may occur merely within cells. Pancreatic cancer can also be detected by direct measurement of CAPRIN-1, as specifically described in the examples below. Therefore, pancreatic cancer can be detected in a subject by measuring CAPRIN-1 itself, as per the first method.
[037] A polypeptide in a sample can be easily measured by well-known immunoassay techniques. Specifically, an antibody that is subjected to an antigen-antibody reaction with CAPRIN-1, or an antigen-binding fragment thereof, is prepared, and immunoassay is performed using the same. In this way, the presence of CAPRIN-1 in the sample can be measured. As described above, an antibody is cross-reactive. With the use of an antibody that is subjected to antigen-antibody reaction with canine CAPRIN-1 of SEQ ID NO: 6 or an antigen-binding fragment thereof, therefore, not only canine CAPRIN-1 of SEQ ID NO: :6, but also its counterpart in other mammals (e.g., the human CAPRIN-1 of SEQ ID NO: 2 or 4) can be measured. The technique of the immunoassay itself is a well known conventional technique, as described above.
[038] This study shows that CAPRIN-1 is a cell membrane protein that is expressed on the surface of pancreatic cancer cells. A subject with cancer contains many proteases in the cancerous tissues. Consequently, the portion of the CAPRIN-1 sequence expressed outside the cancer cells is degraded and separated from the cancer cells, and that portion is greater in quantity than the portion of the CAPRIN-1 sequence expressed in the cancer cells. If an antibody capable of binding to the surface of cancer cells is used as an antibody against CAPRIN-1 in the measurement, or if an antigen-binding fragment of the same is used, consequently, a greater amount of CAPRIN-1 can be detected and pancreatic cancer can be diagnosed with greater sensitivity.
[039] In the present invention, therefore, the use of an antibody that binds to a portion expressed on the surface of a pancreatic cancer cell of a CAPRIN-1 protein molecule is preferred. An example of a partial peptide of a CAPRIN-1 protein expressed on the surface of a pancreatic cancer cell is a polypeptide consisting of an amino acid sequence of 7 to 12 or more continuous amino acid residues within the region of amino acid residues (aa ) 50 to 98 or from amino acid residues (aa) 233 to 305 in any of the amino acid sequences represented by even numbers of SEQ ID NOs: 2 to 30 in the Sequence List, excluding SEQ ID NO: 6 and SEQ ID NO: 18. A specific example thereof includes, but is not limited to, the amino acid sequence represented by SEQ ID NO: 43 or SEQ ID NO: 61 (in an amino acid sequence represented by SEQ ID NO: 61, a region of the amino acid sequence represented by SEQ ID NO: 62 or SEQ ID NO: 63 is preferred) or an amino acid sequence of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably, 95% or more sequence identity with the relevant amino acid sequence. Furthermore, all antibodies that bind to these polypeptides are within the scope of the antibodies used in the present invention. Specific examples include an antibody that binds a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 43 or an antigen-binding fragment thereof; a monoclonal antibody having the amino acid sequences represented by SEQ ID NOs: 44 and 45 or an antigen-binding fragment thereof, a monoclonal antibody having the amino acid sequences represented by SEQ ID NOs: 44 and 46 or a fragment of antigen binding thereof, a monoclonal antibody having the amino acid sequences represented by SEQ ID NOs: 44 and 47 or an antigen-binding fragment thereof, a monoclonal antibody having the amino acid sequences represented by SEQ ID NOs: 44 and 48 or an antigen-binding fragment thereof, a monoclonal antibody having the amino acid sequences represented by SEQ ID NOs: 49 and 50 or an antigen-binding fragment thereof, a monoclonal antibody having the amino acid sequences represented by SEQ ID NOs: 51 and 52 or an antigen-binding fragment thereof, a monoclonal antibody having the amino acid sequences represented by SEQ ID NOs: 53 and 54, or a fragment antigen-binding fragment thereof, a monoclonal antibody having the amino acid sequences represented by SEQ ID NOs: 55 and 56 or an antigen-binding fragment thereof, a monoclonal antibody having the amino acid sequences represented by SEQ ID NOs :57 and 58 or an antigen-binding fragment thereof, and a monoclonal antibody having the amino acid sequences represented by SEQ ID NOs: 59 and 60 or an antigen-binding fragment thereof.
[040] The term "antigen-binding fragment", as used in the present application, refers to an antibody fragment capable of binding an antigen, such as a Fab fragment, an F(ab')2 fragment or a fragment Fv, contained in an antibody molecule. An antibody can be a polyclonal antibody or a monoclonal antibody. For immunoassay, a monoclonal antibody with high reproducibility is preferred. Methods of preparing a polyclonal antibody and a monoclonal antibody, using a polypeptide as an immunogen, are well known and can readily be carried out in a conventional manner. For example, an animal is immunized with CAPRIN-1 or a fragment thereof alone, or with CAPRIN-1 or a fragment thereof linked to a carrier protein such as keyhole limpet hemocyanin (KLH), casein or serum albumin, as an immunogen, along with an adjuvant, and an antibody against CAPRIN-1 can then be induced. Antibody-producing cells, such as splenocytes or lymphocytes, collected from the immunized animal are fused with myeloma cells to prepare hybridomas, and hybridomas producing an antibody that binds to CAPRIN-1 are selected and then cultured. In this way, a monoclonal antibody whose corresponding antigen is CAPRIN-1 can be obtained from the culture supernatant. The method described above is a well known conventional method.
[041] In the third method of the present invention, a nucleic acid encoding CAPRIN-1 (eg, mRNA or cDNA synthesized from mRNA) that may be contained in a sample obtained from a living organism is measured. As specifically described in the examples below, a nucleic acid encoding canine CAPRIN-1 of SEQ ID NO: 6, 8, 10, 12 or 14 or human CAPRIN-1 of SEQ ID NO: 2 or 4 is expressed at a level significantly high in pancreatic cancer cells. Therefore, a cancer existing in the living organism can be detected by measuring this nucleic acid in a sample.
[042] The mRNA in a sample can be quantitatively measured, for example by a conventional method such as real-time RT-PCR detection using the mRNA as a template. Such mRNA can generally be quantitatively measured based on staining intensity or similar in the conventional Northern blot method. The even-numbered CAPRIN-1 encoding cDNA sequences of SEQ ID NOs: 2 to 30 are represented by the odd numbers of SEQ ID NOs: 1 to 29, respectively. Consequently, a polynucleotide that specifically hybridizes to a partial region of the nucleotide sequence represented by any odd-numbered sequence of SEQ ID NOs: 1 to 29 (hereinafter referred to as a “cancer detection polynucleotide”) is prepared based on in these sequences, and this polynucleotide is used as a probe or primer for nucleic acid amplification to determine the amount of mRNA in a sample. If a polynucleotide is capable of specifically hybridizing to a partial region of the nucleotide sequence represented by any odd-numbered sequence of SEQ ID NOs: 1 to 29, the mRNA encoding CAPRIN-1 in a mammal other than dogs and humans can also be measured, as described later. In the present invention, a polynucleotide may be RNA or DNA.
[043] The term "specifically hybridizes to", used in the present application, refers to a situation in which said polynucleotide only hybridizes to a partial target region and does not substantially hybridize to any other region under stringent hybridization conditions.
[044] The term “under stringent hybridization conditions”, used in the present application, refers to conditions used for annealing in ordinary PCR or detection using a probe. In the case of PCR using Taq polymerase, for example, a reaction is carried out at a suitable annealing temperature in the range of about 54°C to about 60°C using a common buffer, such as a buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3 to 9.0) and 1.5 mM MgCl 2 . In the case of Northern blot hybridization, for example, a reaction is performed using a common hybridization solution such as 5x SSPE, 50% formamide, 5x Denhardt's solution, and 0.1 to 0.5% SDS, or SSC 0.1 to 5x and 0.1 to 0.5% SDS at a suitable annealing temperature in the range from 42 to 65°C. After hybridization, washing is carried out, for example, with 0.1 to 0.2x SSC and 0.1% SDS. Suitable annealing temperatures or annealing temperatures are not limited to the examples above, and such temperatures are determined based on the Tm value of a polynucleotide used as a primer or probe for cancer detection and the empirical rules of researchers. One skilled in the art can easily determine this temperature range.
[045] The expression "does not substantially hybridize to", used in the present application, refers to a situation in which said polynucleotide does not hybridize to a target partial region at all or an extremely low amount of the polynucleotide hybridizes to a partial region target, i.e. a relatively insignificant amount, even when it hybridizes to a partial target region. An example of a polynucleotide that specifically hybridizes under these conditions is a polynucleotide with a specific or greater level of sequence identity to the nucleotide sequence of a target partial region. For example, such a polynucleotide is 70% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, more preferably 95% or more, and specifically preferably, 98% or more sequence identity. Most preferably, the polynucleotide has a nucleotide sequence identical to the nucleotide sequence of a target partial region. Sequence identity is defined in the same way as the sequence identity of amino acid sequences described above. Even if a terminus of a polynucleotide for detecting cancer contains a region that does not hybridize to it, in the case of a probe, it can be used for detection, provided that a hybridization region occupies about half or more of the entire probe. . In the case of a primer, it can be used for detection as long as a hybridization region occupies about half or more of the entire primer and is located at the 3' end, as this allows for normal annealing and reaction to occur. of extension. When a terminus of a polynucleotide for cancer detection contains a non-hybridizing region, as described above, sequence identity to a target nucleotide sequence is calculated based on the hybridizing region alone, without regard to the non-hybridizing region.
[046] In the present invention, the term "partial sequence" (or "partial region") refers to a portion of a nucleotide sequence represented by any odd-numbered sequence of SEQ ID NOs: 1 to 29. Specifically, the partial sequence comprises at least 15 to 19 continuous nucleotides, preferably 18 or more continuous nucleotides, more preferably at least 20 or 25 continuous nucleotides, and more preferably at least 30, 40 or 50 continuous nucleotides. The expression "the nucleotide sequence represented by SEQ ID NO: 5", used in the present application, refers to a sequence complementary to the nucleotide sequence actually shown in SEQ ID NO: 5, a sequence complementary thereto. Accordingly, the term "polypeptide having the nucleotide sequence represented by SEQ ID NO: 5" refers, for example, to a single-stranded polynucleotide having the nucleotide sequence actually represented by SEQ ID NO: 5, a polynucleotide of single-stranded with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 5 or to a double-stranded polynucleotide consisting of two single-stranded polynucleotides When a polynucleotide for use in the present invention is prepared either a polynucleotide encoding a polypeptide to be used in the present invention is prepared, any one of the nucleotide sequences is suitably selected, and one skilled in the art can readily perform such selection.
[047] The number of nucleotides in a polynucleotide for cancer detection is preferably 18 or more to ensure specificity. When the polynucleotide is used as a probe, it preferably comprises 18 or more nucleotides, and it further preferably comprises 20 nucleotides up to the entire coding region. When the polynucleotide is used as a primer, it preferably comprises 18 to 50 nucleotides. A preferred example of a polynucleotide for detecting cancer is a polynucleotide comprising 18 or more contiguous nucleotides in a nucleotide sequence represented by any of the odd-numbered SEQ ID NOs: 1 to 29.
[048] It is apparent to one skilled in the art, upon consulting the description of the present invention, that: a polynucleotide that specifically hybridizes to a partial region in SEQ ID NO: 5, 7, 9, 11 or 13 is used to measure the amount a nucleic acid (e.g., mRNA or cDNA synthesized from the mRNA) encoding the canine CAPRIN-1 protein of SEQ ID NO: 6, 8, 10, 12 or 14, respectively; and a polynucleotide that specifically hybridizes to a partial region in SEQ ID NO: 1 or 3 is used to measure the amount of a nucleic acid (e.g., mRNA or cDNA synthesized from the mRNA) that encodes the human CAPRIN-1 protein of SEQ ID NO: 2 or 4, respectively. However, a protein from one mammal and a homologue of it from another mammal generally share a high sequence identity, even at the nucleotide sequence level. Thus, the sequence identity between the nucleotide sequences of SEQ ID NOs: 1 to 13 is also as high as 94 to 100%. Accordingly, a polynucleotide that specifically hybridizes to a partial region of the sequence of SEQ ID NO: 5 can also hybridize to a partial region corresponding to the partial region of any of the odd-numbered SEQ ID NOs: 1 to 29.
[049] In fact, a primer pair with the nucleotide sequences represented by SEQ ID NOs: 33 and 34 specifically hybridizes to both partial regions of any odd-numbered sequence of SEQ ID NOs: 1 to 29, and to a region partial sequence of SEQ ID NO: 5, as described in the examples below. In this way, both the mRNA encoding the canine CAPRIN-1 of SEQ ID NO: 6 and the mRNA encoding a homolog thereof can be measured. Consequently, with the use of a polynucleotide that specifically hybridizes to a partial region of the sequence of SEQ ID NO: 5, not only the mRNA encoding canine CAPRIN-1 of SEQ ID NO: 6, but also the mRNA encoding CAPRIN -1 human of SEQ ID NO: 2 or 4 can be measured. Similarly, mRNA encoding CAPRIN-1 from another mammal, such as a cat, can also be measured. When a polynucleotide for cancer detection is designed, it is more desirable to select partial regions with high sequence identity, specifically, between the odd-numbered sequences of SEQ ID NOs: 1 to 29 (and identical nucleotide sequences are preferred). If, specifically, there is high sequence identity with the partial region between the canine and human CAPRIN-1 proteins, a region that shows very high sequence identity with that region is expected to also be present in a homologous gene from another animal species. . By selecting this partial region, the accuracy for measuring mRNA encoding CAPRIN-1 from an animal species other than dogs or humans can be increased.
[050] A method for measuring a nucleic acid analyte using a polynucleotide that specifically hybridizes to a partial region of the nucleic acid analyte as a probe or primer for the nucleic acid amplification method, such as PCR, is well known. Examples of this method include, in addition to RT-PCR as specifically described in the examples below, Northern blotting and in situ hybridization. When the amount of mRNA is measured in the present invention, any well known measurement method can be used.
[051] A nucleic acid amplification method, such as PCR, is well known in the art, and the reagent kits and apparatus used therein are commercially available, so the method can be easily performed. Specifically, the denaturation, annealing, and extension steps are each performed using a nucleic acid analyte (e.g., the cDNA of a gene encoding a protein with an amino acid sequence represented by any of the sequences in SEQ ID NOs: 2 to 30 even-numbered) as a template and a pair of polynucleotides (primers) for detecting cancer, in a known buffer, in the presence of a thermostable DNA polymerase, such as Taq polymerase or Pfu polymerase, and dNTPs ( in the present application, N = A, T, C and G), by varying the temperature of the reaction solution at each step. In general, the denaturation step is carried out at 90°C to 95°C, the annealing step is carried out at or near the Tm of the mold and primers (preferably within ± 4°C), and the Extension is performed at 72°C, which is the optimal temperature for thermostable DNA polymerase such as Taq polymerase or Pfu polymerase, or a temperature close to the optimal temperature. The duration of each step is suitably set between about 30 seconds to 2 minutes. This heating cycle is repeated about 25 to 40 times, for example, so that the region of template nucleic acid present between a pair of primers is amplified. The nucleic acid amplification method is not limited to PCR, and any other nucleic acid amplification methods well known in the art can be employed. When a nucleic acid amplification method is performed using a pair of cancer detection polynucleotides as primers and a nucleic acid analyte as a template, as described above, the nucleic acid analyte is amplified. If a sample does not contain the test nucleic acid, however, amplification does not occur. Consequently, an amplification product can be detected in order to determine the presence or absence of the nucleic acid analyte in the sample. An amplification product can be detected by a method comprising subjecting a reaction solution after amplification to the electrophoresis process and then staining the band with ethidium bromide or the like, or a method comprising immobilizing an amplification product. of amplification after electrophoresis on a solid phase such as a nylon membrane, performing hybridization with a marker probe that specifically hybridizes to the nucleic acid analyte, washing, and then detecting the marker. Furthermore, so-called real-time PCR detection is performed using a fluorescent quencher dye and a fluorescent reporter dye, and the amount of the nucleic acid analyte in a specimen can thus be quantified. Since real-time detection PCR kits are commercially available, real-time detection PCR can be easily performed. In addition, semiquantitative measurement of an analyte nucleic acid analyte can be performed based on the intensity of the electrophoresis band. A nucleic acid analyte can be either mRNA or cDNA reverse transcribed from mRNA. When mRNA is amplified as a nucleic acid analyte, a NASBA method (the 3SR method or TMA method) using the above primer pair can also be employed. The NASBA method is well known, and kits for the same are also commercially available, so the method can be easily performed using the above pair of primers.
[052] As a probe, a labeled probe prepared by labeling a polynucleotide for the detection of cancer with a fluorescent marker, a biotin marker or the like can be used. Methods for labeling a polynucleotide are well known. The presence or absence of a nucleic acid analyte in a sample can be examined by immobilizing a nucleic acid analyte or an amplification product thereof, performing hybridization with a labeled probe, washing, and then measuring the bound marker. to the solid phase. Alternatively, a polynucleotide for detecting cancer is immobilized, a nucleic acid analyte is hybridized thereto, and solid phase bound test nucleic acid can then be detected using the labeled probe or the like. In this case, a polynucleotide for detecting cancer linked to the solid phase is also called a “probe”. Methods for measuring a nucleic acid analyte using a polynucleotide probe are also well known in the art. This method can be performed, in a buffer, by contacting the polynucleotide probe with the nucleic acid analyte at or near Tm (preferably ±4°C) for hybridization, washing, and then measuring the hybridized labeled probe or of the template nucleic acid bound to the solid phase probe. Examples of this method include well-known methods such as Northern blot, in situ hybridization, and Southern blot. In the present invention, any well-known method is applicable.
[053] According to the detection method of the present invention, whether or not an animal subject (or subject) is afflicted with pancreatic cancer is assessed based on the presence or amount of CAPRIN-1 expression as described above. While pancreatic cancer can be detected only by measuring the presence or amount of CAPRIN-1 expression in an animal subject, it is preferable that the expression levels (the antibody level, polynucleotide level or mRNA level) of CAPRIN-1 in one or more samples from healthy subjects are examined and the value determined from an animal subject is compared to the standard value obtained from healthy subjects in order to improve detection accuracy. To further improve detection accuracy, CAPRIN-1 expression levels are measured in samples obtained from many pancreatic cancer patients, so as to obtain a default value for pancreatic cancer patients, and the determined value of a animal subject can then be compared with both the standard value for healthy subjects and the standard value for pancreatic cancer patients. The above standard values can be determined, for example, by quantifying the level of CAPRIN-1 expression in each sample and calculating their mean value. The default value for healthy subjects and the same for pancreatic cancer patients can be previously determined by measuring CAPRIN-1 expression levels in many healthy subjects and pancreatic cancer patients. When comparison with the default value is performed by the method of the present invention, therefore, a predetermined default value can be used.
[054] The detection method of the present invention may comprise diagnosis based on other cancer antigens or cancer markers combined. This may further improve the accuracy of pancreatic cancer detection. When an antibody that is specifically present in patients with pancreatic cancer is measured by the method of the present invention, for example, another polypeptide that is often expressed in a cancerous tissue can be used in combination with an antigen in a manner similar to that used for the polypeptides described above. Furthermore, the method of the present invention can be performed in combination with diagnosis using a previously known cancer marker.
[055] The pancreatic cancer subjected to the method for detecting pancreatic cancer of the present invention is pancreatic cancer that expresses CAPRIN-1. Examples of such cancer include, but are not limited to, pancreatic duct carcinoma, invasive pancreatic duct carcinoma, adenocarcinoma, acinar cell carcinoma, adenosquamous carcinoma, giant cell tumor, intraductal papillary mucosal neoplasm (IPMN), mucosal cystic neoplasm (MCN). ), pancreatoblastoma, serous cystadenocarcinoma, solid pseudopapillary tumor (PTS), gastrinomas (Zollinger-Ellison syndrome), glucagonomas, insulinomas, multiple endocrine neoplasia type 1 (MEN1) (Wermer syndrome), non-functional islet cell tumor, somatostatinomas and VIPomas. A subject of the method of the present invention is a mammal, preferably a human or a dog.
[056] Examples of samples subjected to the method of the present invention include body fluids such as blood, serum, blood plasma, ascitic fluid and pleural effusion, tissues and cells. In the first and second methods, specifically, samples of serum, blood plasma, ascitic fluid, pleural effusion, tissue and cells can preferably be used. In the third method, which comprises measuring a nucleic acid such as mRNA, tissue and cell samples are preferred.
[057] One or more polynucleotides used as antigens for the immunoassay in the first method described above (i.e. the canine CAPRIN-1 of SEQ ID NO: 2 and a homologue thereof, a specifically reactive partial polypeptide, a specifically reactive modified polynucleotide and a specifically reactive addition polynucleotide) can be supplied as reagents or kits for the detection of pancreatic cancer. Such a reagent may consist of the above polypeptides or may contain various additives useful for stabilizing the polypeptide, a buffer necessary for the assay, secondary antibodies, enzyme substrates or the like separately. Alternatively, this reagent can be immobilized on a solid phase, such as a plate or membrane. Preferred examples of such polynucleotides are given above.
[058] An antibody, or an antigen-binding fragment thereof, which is subjected to an antigen-antibody reaction with CAPRIN-1, used in CAPRIN-1 immunoassays by the second method may also be provided in the form of a reagent for the detection of pancreatic cancer. The reagent for detecting pancreatic cancer may consist of the antibody or antigen-binding fragment thereof above. The reagent may contain various additives useful for stabilizing that antibody or antigen-binding fragment thereof. Alternatively, a metal, such as manganese or iron, can be attached to the antibody or antigen-binding fragment thereof. When this metal-bound antibody or antigen-binding fragment thereof is administered to a living organism, the metal-bound antibody or antigen-binding fragment thereof is accumulated to an increased level at a site where the antigen protein is present. present at a higher level. When this metal-bound antibody or antigen-binding fragment thereof is administered to a living organism, the metal-bound antibody or antigen-binding fragment thereof is accumulated to an increased level at a site where the antigen protein is present. present at a higher level. Therefore, the metal is measured by MRI or similar method, and the presence of cancer cells that produce the antigen protein can thus be detected.
[059] In addition, one or more of the above polynucleotides for the detection of pancreatic cancer used for the measurement of a nucleic acid, such as mRNA, in the third method may also be provided as a reagent or kit for the detection of pancreatic cancer. In that case, the reagent for detecting pancreatic cancer may consist of the polynucleotide or may contain various additives useful for stabilizing the polynucleotide, a buffer necessary for the assay (eg, a fluorescent label), and the like, separately. The polynucleotide for the detection of pancreatic cancer contained in the reagent is preferably a primer or a probe. Preferred conditions and examples of the polynucleotide for detecting pancreatic cancer are described above. EXAMPLES
[060] The present invention will be described in more detail by reference to the following examples, although the technical scope of the present invention is not limited to the examples. EXAMPLE 1OBTAINING THE ANTIGEN PROTEIN OF PANCREATIC CANCER BY THE SEREX(1) METHOD CONSTRUCTION OF THE cDNA LIBRARY
[061] Total RNA was extracted from testicular tissue of a healthy dog by the guanidium-phenol-chloroform method and poly-A RNA was purified using an Oligotex-dT30 mRNA purification kit (Takara Shuzo , Co., Ltd.) according to the protocols attached to the kit.
[062] A canine testis cDNA phage library was synthesized using the obtained mRNA (5 μg). The cDNA phage library was constructed using cDNA Synthesis®, ZAP-cDNA Synthesis® and ZAP-cDNA GigapackIII Gold Cloning® (STRATAGENE) kits according to the protocols attached to the kits. The size of the constructed cDNA phage library was 7.73 x 10 5 pfu/ml.(2) SCREENING THE cDNA LIBRARY USING SERUM
[063] Immunoscreening was performed using the canine testis cDNA phage library constructed above. Specifically, host Escherichia coli (XL1-Blue MRF') was infected with the phage on an NZY agarose plate (Φ90 x 15 mm) to obtain 2210 clones. E. coli cells were grown at 42°C for 3 to 4 h to form plaques. The plate was coated with a nitrocellulose membrane (Hybond C Extra: GE Healthcare Bio-Science) impregnated with IPTG (isopropyl-β-D-thiogalactosidase) at 37°C for 4 h, so that the protein was induced to express and , then transferred to the membrane. Then, the membrane was collected and then impregnated with TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.5% skimmed milk powder, followed by stirring at 4°C overnight. for the day to suppress non-specific reactions. The filter was subjected to a reaction with serum from an affected dog diluted 500 times at room temperature for 2 to 3 h.
[064] As above affected dog serum, a serum collected from a dog with pancreatic cancer was used. The serum was stored at -80°C and then subjected to pretreatment immediately before use. A method for pre-treatment of serum is described below. Specifically, host Escherichia coli (XL1-Blue MRF') was infected with a ZAP Express phage into which no foreign gene had been inserted, and culture was performed overnight in an NZY plate medium at 37°C. . Subsequently, a buffer (0.2 M NAHCO3, pH 8.3) containing 0.5 M NaCl was added to the plate, which was incubated at 4°C for 15 h, and the supernatant was then collected as an Escherichia coli extract. /phage. The Escherichia coli/phage extract was then applied to an NHS column (GE Healthcare Bio-Science) so that a protein derived from Escherichia coli/phage was immobilized. Serum from the affected dog was applied to the immobilized protein column for the reaction, and Escherichia coli and a phage-absorbed antibody were then removed from the serum. The whey fraction that had been passed through the column was diluted 500-fold with TBS containing 0.5% skim milk powder. The diluted serum fraction was used as material for immunoscreening.
[065] A membrane onto which the treated serum and the above fusion protein had been transferred was washed 4 times with TBS-T (Tween 20/TBS 0.05%), and the membrane was then reacted with anti-IgG antibody goat canina (Goat anti-Dog IgG-h+I HRP conjugated, BETHYL Laboratories) diluted 5000-fold with TBC containing 0.5% skim milk powder as secondary antibody at room temperature for 1 h. Detection was performed by means of an enzymatic reaction with color development using the NBT/BCIP reaction solution (Roche). Colonies that showed positive sites for the color development reaction were collected from the NZY agarose plate (Φ90 x 15 mm) and then dissolved in 500 μl of an SM buffer (100 mM NaCl, 10 mM MgClSO4, Tris-HCl 50 mM, 0.01% gelatin, pH 7.5). Until colonies positive for the color development reaction were unified, secondary and tertiary screening were repeated by a similar method as described above, so that 30,940 phage clones that react with serum IgG were screened. Thus, 5 positive clones were isolated.(3) SEARCH FOR HOMOLOGY FOR THE ISOLATED ANTIGEN GENE
[066] For the analysis of the nucleotide sequences of the 5 positive clones isolated by the above method, a procedure for converting the phage vectors to plasmid vectors was performed. Specifically, 200 μl of a solution containing host Escherichia coli (XL1-Blue MRF') at an absorbance (OD 600) of 1.0 was prepared. The solution was mixed with 250 μl of a purified phage solution and 1 μl of ExAssist helper phage (STRATAGENE), the mixture was subjected to a reaction at 37°C for 15 min, 3 ml of LB medium was added thereto, and the culture was then carried out at 37°C for 2.5 to 3 h. Immediately afterwards, the temperature of the solution was maintained in a water bath at 70°C for 20 min, centrifugation was performed at 4°C and 1000 x g for 15 min, and the supernatant was then collected as a phagemid solution. Subsequently, 200 μl of a solution containing host Escherichia coli phagemids (SOLR) at an absorbance (OD600) of 1.0 were prepared. The resulting solution was mixed with 10 μl of a purified phage solution, followed by a reaction at 37°C for 15 min. The reaction product (50 ml) was seeded on an LB agar medium containing ampicillin (final concentration: 50 μg/ml), and the culture was carried out at 37°C overnight. The single colony of transformed SOLR was collected and then cultured in LB medium containing ampicillin (final concentration: 50 μg/ml) at 37°C. Subsequently, plasmid DNA containing an insert of interest was purified using the QIAGEN Plasmid Miniprep® Kit (QIAGEN).
[067] The purified plasmid was subjected to analysis of the entire insertion sequence by the primer walking method using the T3 primer represented by the sequence SEQ ID NO: 31 and the T7 primer represented by the sequence SEQ ID NO: 32. As a result of the In sequence analysis, the gene sequences represented by SEQ ID NOs: 5, 7, 9, 11 and 13 were obtained. A search in the BLAST homology search program (http://www.ncbi.nlm.nih.gov/BLAST/) was performed using the nucleotide sequences and amino acid sequences (SEQ ID NOs: 6, 8, 10, 12 and 14) of the genes. As a result of this search for homology with known genes, it was found that all 5 genes obtained encode CAPRIN-1. The sequence identity between the 5 genes was 100% for the nucleotide sequence and 99% for the amino acid sequence in the translated regions in proteins. In addition, the sequence identity between the canine gene (any one of SEQ ID NOs: 5, 7, 9, 11, or 13) and a gene encoding a human homolog thereof was 94% for the nucleotide and sequence sequence. 98% for the amino acid sequence in the translated regions in proteins. The nucleotide sequences of the human homolog are represented by SEQ ID NOs: 1 and 3, and the amino acid sequences thereof are represented by SEQ ID NOs: 2 and 4. In addition, sequence identity between the canine gene and a gene encoding a bovine homolog was 94% for the nucleotide sequence and 97% for the amino acid sequence in the translated regions in proteins. The nucleotide sequence of the bovine homolog is represented by SEQ ID NO: 15 and the amino acid sequence thereof is represented by SEQ ID NO: 16. The sequence identity between the gene encoding the human homolog and the gene encoding the homolog bovine was 94% for the nucleotide sequence and between 93 to 97% for the amino acid sequence in the translated regions in proteins. Furthermore, the sequence identity between the canine gene and a gene encoding an equine homolog was 93% for the nucleotide sequence and 97% for the amino acid sequence in the translated regions in proteins. The nucleotide sequence of the equine homolog is represented by SEQ ID NO: 17 and the amino acid sequence thereof is represented by SEQ ID NO: 18. The sequence identity between the gene encoding the human homolog and the gene encoding the equine homolog was 93% for the nucleotide sequence and 96% for the amino acid sequence in the translated regions in proteins. Furthermore, the sequence identity between the obtained canine gene and a gene encoding the mouse homolog varied between 87 and 89% in terms of nucleotide sequence and between 95 and 97% for the amino acid sequence in the translated regions in proteins. The nucleotide sequences of the mouse homolog are represented by SEQ ID NOs: 19, 21, 23, 25 and 27, and the amino acid sequences thereof are represented by SEQ ID NOs: 20, 22, 24, 26 and 28. A Sequence identity between the gene encoding the human homolog and the gene encoding the mouse homolog varied between 98 and 91% for the nucleotide sequence and between 95 and 96% for the amino acid sequence in the translated regions in proteins. Furthermore, the sequence identity between the obtained canine gene and a gene encoding a chicken homolog was 82% for the nucleotide sequence and 87% for the amino acid sequence in the translated regions in proteins. The nucleotide sequence of the chicken homolog is represented by SEQ ID NO: 29 and the amino acid sequence thereof is represented by SEQ ID NO: 30. The sequence identity between the gene encoding the human homolog and the gene encoding the human homolog chicken homolog ranged between 81 and 82% for the amino acid sequence and was 86% for the amino acid sequence in the translated regions in proteins.(4) GENE EXPRESSION ANALYSIS IN HUMAN PANCREATIC CANCER CELL LINES
[068] The expression of genes obtained by the above method in normal human tissues (i.e. mammary gland, brain, bone marrow, lung, esophagus, pancreas and testes) and in 4 types of human pancreatic cancer cell lines (i.e. Capan-2, MIAPaCa-2, PANC-1 and BxPC-3) was examined by RT-PCR (reverse transcription PCR). A reverse transcription reaction was performed as follows. Specifically, total RNA was extracted from each tissue (50 to 100 mg) and from each cell line (5-10 x 10 6 cells) using TRIZOL reagent (Invitrogen) according to the attached protocols. The cDNA was synthesized using total RNA and the Superscript First-Strand Synthesis System® for RT-PCR (Invitrogen) according to the attached protocols. PCR was performed, as follows, using specific primers for the genes obtained (represented by SEQ ID NOs: 33 and 34). Specifically, PCR was performed by preparing a reaction solution totaling 25 μl with the addition of reagents and an included buffer (i.e. 0.25 μl of a sample prepared by reverse transcription reaction, the above primers (2 μM each), dNTPs (0.2 mM each), 0.65 U ExTaq polymerase (Takara Shuzo, Co., Ltd.)), the resulting solution was cycled at 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds using a Thermal Cycler (BIO RAD), and this cycle was repeated 30 times. The gene-specific primers mentioned above were used to amplify the region between nucleotide 698 and nucleotide 1124 in the nucleotide sequence represented by SEQ ID NO: 1 (the human CAPRIN-1 gene). For comparison, GAPDH-specific primers (represented by SEQ ID NOs: 35 and 36) were used simultaneously. As a result of investigation of human CAPRIN-1 gene expression, expression of the same was observed only in testes for healthy canine tissues, although expression was observed in pancreatic cancer cells. The results demonstrate that CAPRIN-1 expression was not observed in normal tissues, with the exception of the testes, whereas CAPRIN-1 expression was observed in pancreatic cancer cells.(5) CAPRIN-1 EXPRESSION IN NORMAL TISSUES OF MOUSE AND DOG
[069] Mice (Balb/c, females) and dogs (beagle, females) were exsanguinated under ether and ketamine/isoflurane anesthesia. After laparotomy, the organs (stomach, liver, eyes, thymus, muscles, bone marrow, uterus, small intestine, esophagus, heart, kidney, salivary glands, large intestine, mammary glands, brain, lungs, skin, adrenal glands, ovaries, pancreas, spleen and bladder) were transferred to a 10 cm plate containing PBS. Each organ was cut in PBS and then perfused overnight with 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde (PFA). The perfusate was discarded, the tissue surface of each organ was washed with PBS and a PBS solution containing 10% sucrose was then introduced into a 50 ml centrifuge tube. Each tissue was then introduced into each tube, followed by agitation using a rotor at 4°C for 2 h. Each solution was replaced with a PBS solution containing 20% sucrose and then incubated at 4°C until tissue precipitation. Each solution was replaced with a PBS solution containing 30% sucrose and then incubated at 4°C until tissue precipitation. Each tissue was removed and a necessary portion was excised with a surgical scalpel. Subsequently, the OCT compound (Tissue Tek) was applied and spread over the surface of each tissue, which was then placed in Cryomold®. Cryomold® was placed on dry ice for rapid freezing. Tissues were cut into 10 to 20 μm long sections using a cryostat (LEICA), the cut tissue sections were then air-dried on glass slides for 30 min using a hair dryer, and the glass slides onto which the cut tissue sections had been applied were then prepared. Subsequently, each glass slide was introduced into a staining bottle filled with PBS-T (saline containing 0.05% Tween 20), a procedure involving the exchange of PBS-T for fresh PBS-T was performed every 5 min and repeated 3 times. Excess water around each specimen was removed using Kimwipes® and each section was then circled using DAKOPEN (DAKO). As blocking solutions, MOM® Mouse Ig Blocking Reagent (VECTASTAIN) was applied to the mouse tissue and a PBS-T solution containing 10% fetal bovine serum was applied to the canine tissue. The results were incubated in a humid chamber at room temperature for 1 h. Subsequently, a solution prepared with the blocking solution and an anti-CAPRIN-1 monoclonal antibody at 10 μg/ml (monoclonal antibody #8) with the heavy chain variable region of SEQ ID NO: 55 and the light chain variable region of SEQ ID NO: 56, which reacts with the surface of the cancer cells prepared in Example 3, was applied to each glass slide which was then incubated in a humid chamber at 4°C overnight. After 3 washes of 10 min with PBS-T, the biotin-labeled anti-IgG antibody MOM (VECTASTAIN) diluted 250-fold with the blocking solution was applied to each glass slide, which was then incubated in a humid chamber at room temperature. environment for 1 h. After 3 washes of 10 min with PBS-T, an avidin-biotin ABC reagent (VECTASTAIN) was applied to each glass slide which was then incubated in a humid chamber at room temperature for 5 min. After 3 washes of 10 min with PBS-T, a DAB staining solution (10 mg of DAB + 10 μl of 30% H2O2 and 50 ml of 0.05M Tris-HCl, pH 7.6) was applied, and the slides were glass were then incubated inside a glass chamber at room temperature for 30 min. The glass slides were washed with distilled water and a hematoxylin reagent (DAKO) was then applied. After incubation at room temperature for 1 min, the glass slides were washed with distilled water and immersed in 70%, 80%, 90%, 95% and 100% ethanol solutions, in this sequence, for 1 min each, and then incubated in xylene overnight. The glass slides were removed, covered with Glycergel Mounting Medium® (DAKO) and then observed. As a result, expression of CAPRIN-1 was observed to a mild degree within cells of salivary gland, kidney, colon and stomach tissues, but no cell surface expression was observed. Furthermore, absolutely no expression of CAPRIN-1 was observed in tissues of other organs. EXAMPLE 2 PREPARATION OF CANINE AND HUMAN CAPRIN-1 PROTEINS (1) PREPARATION OF RECOMBINANT PROTEIN
[070] A recombinant protein was prepared by the following method based on the gene of SEQ ID NO: 5 obtained in Example 1. PCR was performed by preparing a reaction solution of total amount of 50 μl with the addition of reagents and from an included buffer (i.e. 1 μl of a vector prepared from the phagemid solution obtained in Example 1 and then subjected to sequence analysis, 2 types of primers containing the restriction enzyme cleavage sequences NdeI and Kpnl (0.4 μM of each; SEQ ID NOs: 37 and 38), 0.2 mM dNTPs, 1.25 U PrimeSTAR HS® polymerase (Takara Shuzo, Co., Ltd.)), the resulting solution was subjected to a cycle of 98°C for 10 seconds and 65°C for 1.5 min using a Thermal Cycler (BIO RAD), and this cycle was repeated 30 times. The 2 types of primers were used to amplify the coding region of the entire amino acid sequence of SEQ ID NO: 6 (canine CAPRIN-1). After PCR, the amplified DNA was subjected to electrophoresis on a 1% agarose gel and a DNA fragment of about 1.4 kbp was then purified from the gel using the QIAquick Gel Extraction® Kit (QIAGEN) .
[071] The purified DNA fragment was coupled to a cloning vector pCR-Blunt (Invitrogen). The vector was transformed into Escherichia coli, the plasmid was collected and it was confirmed that the amplified gene fragment corresponded to the target sequence by sequencing. The plasmid compatible with the target sequence was treated with restriction enzymes NdeI and Kpnl, the resultant was purified using the QIAquick Gel Extraction® kit and the target gene sequence was inserted into a pET30b expression vector (Novagen) for Escherichia coli treated with restriction enzymes NdeI and Kpnl. Using the resulting vector, a His-tag fused recombinant protein can be produced. The plasmid was transformed into Escherichia coli BL21 (DE3) for expression, and the target protein was induced to express itself in Escherichia coli with the aid of 1 mM IPTG.
[072] Separately, the recombinant protein of a canine homolog gene was prepared by the following method based on the gene of SEQ ID NO: 7. PCR was performed by preparing a reaction solution of total amount of 50 μl with the addition of reagents and an included buffer (i.e. 1 μl of cDNA, its expression was confirmed by RT-PCR, which was selected among various tissues and the cellular cDNAs prepared in Example 1, 2 types of primers containing restriction enzyme cleavage sequences NdeI and Kpnl (0.4 µM each; SEQ ID NOs: 39 and 40), 0.2 mM dNTPs and 1.25 U PrimeSTAR HS® polymerase (Takara Shuzo, Co. , Ltd.)), the resulting solution was cycled at 98°C for 10 seconds and 68°C for 2.5 min using a Thermal Cycler (BIO RAD), and this cycle was repeated 30 times . The 2 types of primers were used to amplify the coding region of the entire amino acid sequence of SEQ ID NO: 8. After PCR, the amplified DNA was subjected to 1% agarose gel electrophoresis and a DNA fragment of about 2 .2 kbp was then purified from the gel using the QIAquick Gel Extraction® Kit (QIAGEN).
[073] The purified DNA fragment was coupled to a cloning vector pCR-Blunt (Invitrogen). The vector was transformed into Escherichia coli, the plasmid was collected and it was confirmed that the amplified gene fragment corresponded to the target sequence by sequencing. The plasmid compatible with the target sequence was treated with restriction enzymes NdeI and Kpnl, the resultant was purified using the QIAquick Gel Extraction® kit and the target gene sequence was inserted into a pET30b expression vector (Novagen) for Escherichia coli treated with restriction enzymes NdeI and Kpnl. Using the resulting vector, a His-tag fused recombinant protein can be produced. The plasmid was transformed into Escherichia coli BL21 (DE3) for expression, and the target protein was induced to express itself in Escherichia coli with the aid of 1 mM IPTG.
[074] Separately, recombinant protein from a human homolog gene was prepared by the following method based on the gene of SEQ ID NO: 1. PCR was performed by preparing a 50 μl total amount reaction solution with the addition of reagents and an included buffer (i.e. 1 μl of cDNA, its expression was confirmed by RT-PCR, which was selected among various tissues and the cellular cDNAs prepared in Example 1, 2 types of primers containing the restriction enzyme cleavage sequences Sacl and XhoI (0.4 µM each; SEQ ID NOs: 41 and 42), 0.2 mM dNTPs and 1.25 U PrimeSTAR HS polymerase (Takara Shuzo, Co., Ltd.)), the resulting solution was cycled at 98°C for 10 seconds and 68°C for 2.5 min using a Thermal Cycler (BIO RAD), and this cycle was repeated 30 times. The above 2 types of primers were used to amplify the coding region of the entire amino acid sequence of SEQ ID NO: 2. After PCR, the amplified DNA was subjected to 1% agarose gel electrophoresis and a DNA fragment of approx. 2.1 kbp was then purified from the gel using the QIAquick Gel Extraction® Kit (QIAGEN).
[075] The purified DNA fragment was coupled to a cloning vector pCR-Blunt (Invitrogen). The vector was transformed into Escherichia coli, the plasmid was collected and it was confirmed that the amplified gene fragment corresponded to the target sequence by sequencing. The plasmid compatible with the target sequence was treated with restriction enzymes Sacl and Xhol, the resultant was purified using the QIAquick Gel Extraction® kit and the target gene sequence was inserted into a pET30a expression vector (Novagen) for Escherichia coli treated with restriction enzymes Sacl and Xhol. Using the resulting vector, a His-tag fused recombinant protein can be produced. The plasmid was transformed into Escherichia coli BL21 (DE3) for expression, and the target protein was induced to express itself in Escherichia coli with the aid of 1 mM IPTG.(2) PURIFICATION OF RECOMBINANT PROTEIN
[076] The recombinant Escherichia coli strain obtained above expressing SEQ ID NOs: 1, 5 or 7 was cultured at 37°C in LB medium containing 30 μg/ml kanamycin until the absorbance at 600 nm reached about 0.7 . Then, isopropyl-β-D-1-thiogalactopyranosidase was added to a final concentration of 1 mM and the culture was performed at 37°C for 4 h. Subsequently, the culture was centrifuged at 4800 rpm for 10 min to collect the cells. The cell pellet was resuspended in phosphate-buffered saline and then centrifuged at 4800 rpm for 10 min to wash the cells.
[077] Cells were resuspended in phosphate-buffered saline and then subjected to ultrasonication on ice. The ultrasonic Escherichia coli solution was centrifuged at 6000 rpm for 20 min, the resulting supernatant was designated as the soluble fraction and the final precipitate was designated as the insoluble fraction.
[078] The soluble fraction was added to a nickel chelate column (carrier: Chelating SepharoseTM Fast Flow (GE Healthcare), column capacity: 5 ml, 50 mM hydrochloric acid buffer (pH 8.0) as calibration buffer ) prepared according to a conventional method. The unadsorbed fraction was washed with 10 column volumes of 50 mM hydrochloric acid buffer (pH 8.0) and 20 mM phosphate buffer (pH 8.0) containing 20 mM imidazole. Immediately afterwards, 6 beds were eluted with 20 mM phosphate buffer (pH 8.0) containing 100 mM imidazole. After the elution of the protein of interest was confirmed by Coomassie staining, an elution fraction of 20 mM phosphate buffer (pH 8.0) containing 100 mM imidazole was added to a strong ion exchange column (carrier: Q SepharoseTM Fast Flow (GE Healthcare), column volume: 5 ml and 20 mM phosphate buffer (pH 8.0) as calibration buffer). The unadsorbed fraction was washed with 10 column volumes of 20 mM phosphate buffer (pH 7.0) and 20 mM phosphate buffer (pH 7.0) containing 200 mM sodium chloride. Immediately afterwards, 5 beds were eluted using 20 mM phosphate buffer (pH 7.0) containing 400 mM sodium chloride. In this way, purified fractions of proteins with the amino acid sequences represented by SEQ ID NOs: 2, 6 and 8 were obtained, and these purified fractions were then used as material for the administration tests.
[079] Each of the purified preparations obtained by the above method (200 μl each) was dispensed in 1 ml of reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2, pH 7.4) and 2 μl of enterokinase (Novagen) were then added. The preparation was incubated at room temperature overnight for the reaction, a His tag was cleaved, and purification was then performed according to the protocols attached to the Enterokinase Cleavage Capture® kit (Novagen). Subsequently, 1.2 ml of the purified preparation obtained by the above method was replaced by physiological phosphate buffer (Nissui Pharmaceutical Co., Ltd.) using NANOSEP 10K OMEGA (PALL) ® ultrafiltration. Sterile filtration was performed using 0.22 μm HT Tuffryn Acrodisc® (PALL), and the supernatants were used for the following experiments. EXAMPLE 3PREPARATION OF ANTIBODY AGAINST CAPRIN-1(1) PREPARATION OF POLYCLONAL ANTIBODY AGAINST CAPRIN-1 DERIVED PEPTIDE
[080] To obtain an antibody that binds to CAPRIN-1, a peptide derived from CAPRIN-1(Arg-Asn-Leu-Glu-Lys-Lys-Lys-Gly-Lys-Leu-Asp-Asp-Tyr-Gln ; SEQ ID NO: 43) was synthesized. The peptide (1 mg) as antigen was mixed with an equivalent volume of Freund's adjuvant solution (IFA), and the mixture was administered subcutaneously to a rabbit 4 times every 2 weeks. Subsequently, blood was collected and an antiserum containing a polyclonal antibody was obtained. In addition, the antiserum was purified using a G protein carrier (GE Healthcare Bio-Sciences), and a polyclonal antibody against the CAPRIN-1-derived peptide was then obtained. Subsequently, the reactivity of the resulting polyclonal antibody to CAPRIN-1 on the surface of cancer cells was examined using breast cancer cells. Specifically, 106 cells of the MDA-MB-231 V breast cancer cell line were subjected to centrifugation in a 1.5 ml microcentrifuge tube, a PBS solution supplemented with 0.1% fetal bovine serum (FBS) containing the polyclonal antibody was added thereto and the resultant was then incubated on ice for 1 h. After washing with PBS, a FITC-labeled goat anti-mouse IgG (Invitrogen) diluted 500-fold with PBS containing 0.1% FBS was added to the solution, and the solution was then incubated on ice for 1 h. After washing with PBS, the fluorescence intensity was measured using FACS Calibur (Becton, Dickinson and Company). Separately, a control prepared according to a similar procedure as above, except that PBS containing 0.1% FBS was added instead of the polyclonal antibody. As a result, it was found that the fluorescence intensity in cells treated with the polyclonal antibody was stronger than in the control cells, and thus the obtained polyclonal antibody was found to bind to the surface of breast cancer cells.(2) PREPARATION OF MONOCLONAL ANTIBODY AGAINST CAPRIN-1 PROTEIN
[081] The antigenic protein (human CAPRIN-1) (100 μg) represented by SEQ ID NO: 2 prepared in Example 2 was mixed with an equivalent amount of an MPL+TDM adjuvant (Sigma), and the mixture was used as a solution. antigen for mice. The antigen solution was administered intraperitoneally to a 6-week-old Balb/c mouse (Japan SLC Inc.) and additionally administered 3 times every week. The spleen was removed 3 days after the final immunization, placed between two sterile glass slides, washed with PBS (-) (Nissui) and then centrifuged at 1500 rpm for 10 min to remove supernatants. This procedure was repeated 3 times to obtain spleen cells. The cells thus obtained were mixed with SP2/0 mouse myeloma cells (purchased from ATCC) at a ratio of 10:1. The PEG solution prepared by mixing 200 µl of RPMI1640 medium containing 10% FBS warmed to 37°C and 800 µl of PEG1500 (Boehringer) was added to the cells. The solution was incubated for 5 min for cell fusion. Centrifugation was performed at 1700 rpm for 5 min to remove supernatants, cells were resuspended in 150 ml of RPMI1640 medium (HAT selective medium) containing 15% FBS supplemented with 2% equivalent HAT solution (Gibco), and the suspension was then seeded in fifteen 96-well plates (Nunc) at 100 μl/well. Cells were cultured for 7 days at 37°C in the presence of 5% CO2. In this way, hybridomas resulting from the fusion of spleen cells with myeloma cells were obtained.
[082] Hybridomas were selected using as an indicator the binding affinity of the antibody produced to the hybridomas prepared for the CAPRIN-1 protein. The CAPRIN-1 protein solution (1 µg/ml) prepared in Example 2 was added to a 96-well plate at 100 µl/well, and the resultant was incubated at 4°C for 18 h. Each well was washed 3 times with PBS-T, a 0.5% fetal bovine albumin (BSA) solution (Sigma) was added at 400 μl/well, and the plate was then incubated at room temperature for 3 h. The solution was removed and each well was washed 3 times with 400 µl of PBS-T. Subsequently, each culture supernatant of the hybridomas obtained above was added to 100 μl/well, and the resultant was then incubated at room temperature for 2 h. Each well was washed 3 times with PBS-T, an HRP (H+L)-labeled anti-mouse IgG antibody (Invitrogen) diluted 5000 times with PBS was added thereto at 100 μl/well, and the resultant was incubated in room temperature for 1 h. Each well was washed 3 times with PBS-T, a TMB substrate solution (Thermo) was added at 100 µl/well, and the resultant was incubated for 15 to 30 min to allow color development. Subsequently, 1N sulfuric acid was added at 100 μl/well to stop the reaction. Absorbance was measured at 450 nm and 595 nm using a spectrophotometer. As a result, a series of antibody-producing hybridomas exhibiting high absorbance values were selected.
[083] The hybridomas so selected were added to a 96-well plate at 0.5 hybridoma per well and then cultured. After 1 week, single colony forming hybridomas in wells were observed. Cells from these wells were further cultured and hybridomas were selected using as an indicator the binding affinity of the antibody produced by the cloned hybridomas to the CAPRIN-1 protein. The CAPRIN-1 protein solution (1 µg/ml) prepared in Example 2 was added to a 96-well plate at 100 µl/well, and the resultant was incubated at 4°C for 18 h. Each plate was washed 3 times with PBS-T, a 0.5% BSA solution was added at 400 μl/well, and the plate was then incubated at room temperature for 3 h. The solution was removed and each well was washed 3 times with 400 µl of PBS-T. Subsequently, each culture supernatant of the hybridomas obtained above was added to 100 μl/well, and the resultant was then incubated at room temperature for 2 h. Each well was washed 3 times with PBS-T, an HRP (H+L)-labeled anti-mouse IgG antibody (Invitrogen) diluted 5000 times with PBS was added thereto at 100 μl/well, and the resultant was incubated in room temperature for 1 h. After each well was washed 3 times with PBS-T, a TMB substrate solution (Thermo) was added at 100 μl/well and then incubated for 15 to 30 min to allow color development. Subsequently, 1N sulfuric acid was added at 100 μl/well to stop the reaction, and the absorbance was measured at 450 and 595 nm using a spectrophotometer. As a result, a series of antibody-producing hybridomas exhibiting reactivity to CAPRIN-1 protein were selected, the hybridoma culture supernatant was purified using a G protein carrier, and 150 monoclonal antibodies that bind to CAPRIN-1 protein were selected. obtained.
[084] Subsequently, monoclonal antibodies that exhibit reactivity with the surface of cancer cells expressing CAPRIN-1 were selected from among the monoclonal antibodies with the use of breast cancer cells. Specifically, 106 cells of the MDA-MB-231 V breast cancer cell line were subjected to centrifugation in a 1.5 ml microcentrifuge tube, 100 μl of the hybridoma culture supernatant was added and the resultant was then incubated. on ice for 1 h. After washing with PBS, a FITC-labeled goat anti-mouse IgG (Invitrogen) diluted 500-fold with PBS containing 0.1% FBS was added to the solution, and the solution was then incubated on ice for 1 h. After washing with PBS, the fluorescence intensity was measured using FACS Calibur (Becton, Dickinson and Company). Separately, a control was prepared according to a similar procedure as above, except that a medium solution was added instead of the antibody. As a result, 10 monoclonal antibodies exhibiting a higher fluorescence intensity than the control (ie, 10 monoclonal antibodies exhibiting reactivity with the surface of breast cancer cells (#1 to #10)) were selected. The heavy chain variable regions and light chain variable regions of these monoclonal antibodies are shown in SEQ ID NOs: 44 to 60. The above monoclonal antibody No. 1 comprises the heavy chain variable region of SEQ ID NO: 44 and the variable region of the light chain of SEQ ID NO: 45, the above monoclonal antibody at 2 comprises the heavy chain variable region of SEQ ID NO: 44 and the light chain variable region of SEQ ID NO: 46, the above monoclonal antibody at 3 comprises the heavy chain variable region of SEQ ID NO: 44 and the light chain variable region of SEQ ID NO: 47, the above monoclonal antibody at 4 comprises the heavy chain variable region of SEQ ID NO: 44 and the variable region of light chain of SEQ ID NO: 48, the above monoclonal antibody at 5 comprises the heavy chain variable region of SEQ ID NO: 49 and the light chain variable region of SEQ ID NO: 50, the above monoclonal antibody at 6 comprises the heavy chain variable region of SEQ ID NO: 51 and the The light chain variable region of SEQ ID NO: 52, the above monoclonal antibody at 7 comprises the heavy chain variable region of SEQ ID NO: 53 and the light chain variable region of SEQ ID NO: 54, the above monoclonal antibody at 8 comprises the heavy chain variable region of SEQ ID NO: 55 and the light chain variable region of SEQ ID NO: 56, the above monoclonal antibody at 9 comprises the heavy chain variable region of SEQ ID NO: 57 and the light chain variable of SEQ ID NO: 58 and the above monoclonal antibody no 10 comprises the heavy chain variable region of SEQ ID NO: 59 and the light chain variable region of SEQ ID NO: 60.(3) IDENTIFICATION OF PEPTIDES IN THE CAPRIN-1 PROTEIN TO WHICH THE ANTIBODY AGAINST CAPRIN-1 THAT REACTS WITH THE SURFACE OF CANCEL CELLS OF MAMA CALLS
[085] With the use of monoclonal antibodies #1 to #10 against CAPRIN-1 that react with the surface of breast cancer cells obtained above, the partial sequences in the CAPRIN-1 protein recognized by these antibodies were identified.
[086] To 100 μl of a solution of recombinant CAPRIN-1 protein adjusted to a concentration of 1 μg/μl with PBS, first, DTT (Fluka) was added to result in a final concentration of 10 mM, and allowed to a reaction proceeded at 95°C for 5 min in order to reduce the disulfide bonds within the CAPRIN-1 protein. Subsequently, iodoacetamide (final concentration: 20 mM; Wako Pure Chemical Industries, Ltd.) was added and the thiol groups were subjected to alkylation at 37°C for 30 min in the dark. Monoclonal antibodies #1 to #10 against CAPRIN-1 (50 μg each) were added to 40 μg of reduced alkylated CAPRIN-1 protein, the volume of the mixture was adjusted to 1 ml with 20 mM phosphate buffer (pH 7.0) and the reaction was allowed to proceed at 4°C overnight with stirring and mixing.
[087] Subsequently, trypsin (Promega) was added to a final concentration of 0.2 μg. After allowing the reaction to proceed at 37°C for 1 h, 2 h, 4 h and then 12 h, the resultants were mixed with protein A (GE) coated glass microspheres that had been blocked with PBS containing 1% BSA. (Sigma) and washing with PBS previously in 1 mM calcium carbonate buffer and NP-40 (20 mM phosphate buffer (pH 7.4), 5 mM EDTA, 150 mM NaCl and 1% NP-40), and if the reaction still proceeds for another 30 min.
[088] The reaction solutions were washed with 25 mM ammonium carbonate buffer (pH 8.0), the antigen-antibody complexes were then eluted using 0.1% formic acid, and the eluates were subjected to LC analysis. -MS using Q-TOF Premier(Waters-MicroMass) according to the protocols attached to the instrument.
[089] As a result, the polypeptide of SEQ ID NO: 61 was identified as a partial sequence of CAPRIN-1, which was recognized by all monoclonal antibodies 1 to 10 against CAPRIN-1. In addition, the peptide of SEQ ID NO: 62 was identified as a partial sequence of the polypeptide of SEQ ID NO: 61 above, which was recognized by monoclonal antibodies #1 to #4, #5 to #7 and #9. , monoclonal antibodies #1 to #4 were found to recognize the peptide of SEQ ID NO:63, which was a partial sequence of the peptide. EXAMPLE 4 DIAGNOSIS OF PANCREATIC CANCER USING THE POLYPEPTIDE CAPRIN-1(1) DIAGNOSIS OF CANINE PANCREATIC CANCER
[090] As a result of pathological diagnosis using samples of removed tumor tissue, blood samples were collected from affected dogs bearing malignant pancreatic ductal carcinoma, and the sera were separated. As the use of the canine CAPRIN-1 protein (SEQ ID NO: 8) prepared in Example 2 and the anti-canine IgG antibody, the titer of the serum IgG antibody that specifically reacts with the canine CAPRIN-1 protein was measured by the ELISA method. .
[091] The prepared canine CAPRIN-1 protein was immobilized by adding a solution of the recombinant protein diluted to 5 μg/ml with phosphate-buffered saline to a 96-well immobilizing amine plate (Nunc) at 100 μl/well and, then, the plate was incubated at 4°C overnight. Blocking was performed by adding a 50 mM sodium bicarbonate buffer solution (pH 8.4) (hereinafter called “blocking solution”) containing 0.5 BSA (fetal bovine albumin, Sigma Aldrich, Japan) % at 100 μ/well, followed by stirring at room temperature for 1 h. The serum diluted 1000 times with the blocking solution was added to 100 μl/well and the mixture was then subjected to a reaction by stirring at room temperature for 3 h. The reaction product was washed 3 times with phosphate-buffered saline containing 0.05% Tween 20 (Wako Pure Chemical Industries, Ltd.; called "PBS-T" in the present application), an HRP-modified anti-canine IgG antibody. (Goat anti-Dog IgG-h+I HRP conjugated: BETHYL Laboratories) diluted 3000 times with the blocking solution was added at 100 μl/well, and the mixture was subjected to a reaction by stirring at room temperature for 1 h . After the reaction product was washed 3 times with PBS-T, HRP TMB substrate (1-Step Turbo TMB (tetramethylbenzidine), PIERCE) was added at 100 μl/well and an enzyme-substrate reaction was then carried out in room temperature for 30 min. Subsequently, a 0.5 M sulfuric acid solution (Sigma Aldrich, Japan) was added at 100 μl/well to stop the reaction, and the absorbance was measured using a microplate reader. As a control, a specimen in which no recombinant protein prepared was immobilized and a specimen with which serum from a cancer-bearing dog was not allowed to react were subjected to treatment and comparison in the same manner as described above.
[092] As a result, the antibody titer against a canine CAPRIN-1 protein of sera derived from cancer-bearing dogs was found to be higher than that of controls.(2) DIAGNOSIS OF CANINE PANCREATIC CANCER USING THE HUMAN CAPRIN-1 PROTEIN
[093] Using human CAPRIN-1 protein (SEQ ID NO: 2) prepared in Example 2, the titer of canine serum IgG antibody that reacts with human CAPRIN-1 protein was measured in the same manner as described above. When serum samples obtained from healthy dogs were subjected to the same measurement, the absorbance at 450 nm was not substantially observed as described above. Serum samples obtained from dogs with pancreatic cancer exhibited a higher antibody titer against human CAPRIN-1 protein (1) than that of the control.(3) DIAGNOSIS OF HUMAN PANCREATIC CANCER
[094] Using the human CAPRIN-1 protein (SEQ ID NO: 2) prepared in Example 2 and the anti-human IgG antibody, titration of the IgG antibody of serum samples obtained from healthy individuals that reacts with the polypeptide was measure. Human CAPRIN-1 protein was immobilized by adding a solution of recombinant protein diluted to 100 μg/ml with phosphate-buffered saline to an immobilizing amine plate (Nunc) at 100 μl/well and then the plate was incubated at 4°C overnight. Blocking was performed as follows. That is, 4 g of Block Ace® powder (DS PHARMA BIOMEDICAL Co., Ltd.) was dissolved in 100 ml of purified water, the solution was diluted 4 times with purified water (hereinafter referred to as “blocking solution” ), the blocking solution was added at 100 μl/well and the mixture was stirred at room temperature for 1 h. The serum diluted 1000 times with the blocking solution was added to 100 μl/well and then subjected to a reaction by stirring at room temperature for 3 h. After washing the resultant 3 times with phosphate-buffered saline containing 0.05% Tween 20 (Wako Pure Chemical Industries, Ltd.; solution called "TBS-T" in the present application), an HRP-modified anti-human IgG antibody ( HRP-Goat Anti-Human IgG (H+L) Conjugate: Zymed Laboratories) diluted 10000 times with the blocking solution was added to 100 μl/well and then subjected to a reaction by stirring at room temperature for 1 h . After the reaction product was washed 3 times with PBS-T, HRP TMB substrate (1-Step Turbo TMB (tetramethylbenzidine), PIERCE) was added at 100 μl/well, and an enzyme-substrate reaction was then performed. at room temperature for 30 min. Subsequently, a 0.5 M sulfuric acid solution (Sigma Aldrich, Japan) was added at 100 μl/well to stop the reaction, and the absorbance at 450 nm was then measured using a microplate reader. An ovalbumin antigen adjusted to 50 μg/ml with phosphate-buffered saline was immobilized and then used as a positive control. As a result, the absorbance at 450 nm was found to be high in the case of the ovalbumin antigen, although no absorbance (0) was detected in the case of the human CAPRIN-1 protein.
[095] In addition, serum samples obtained from patients with pancreatic ductal carcinoma were subjected to measurement of serum IgG antibody titer that specifically reacts with human CAPRIN-1 protein (the amino acid sequence of SEQ ID NO: 2) in the same way as described above. As a result, the absorbance at 450 nm was found to be lower than the lower limit of detection in the case of healthy subjects, although it was found to be high in the case of pancreatic cancer patients. Using the canine CAPRIN-1 protein (SEQ ID NO: 8) prepared in Example 2 and the anti-human IgG antibody, the titer of the serum IgG antibody that specifically reacts with the canine CAPRIN-1 protein was measured in the same way. as described above. As a result, pancreatic cancer patients were found to exhibit higher titers than healthy subjects.
[096] In this way, it was demonstrated that human pancreatic cancer could be detected by the method of the present invention. EXAMPLE 5 DIAGNOSIS OF PANCREATIC CANCER USING THE ANTIBODY AGAINST CAPRIN-1(1) DIAGNOSIS OF PANCREATIC CANCER BY MEASUREMENT OF CAPRIN-1 PROTEIN
[097] With the use of the polyclonal antibody against the peptide derived from CAPRIN-1 (SEQ ID NO: 43) obtained in Example 3 (1) in combination with each monoclonal antibody against the CAPRIN-1 protein obtained in Example 3 (2) , a sandwich ELISA was performed to detect whether the CAPRIN-1 protein (serum derived from cancer-bearing individuals) reacted positively upon cancer diagnosis using the CAPRIN-1 protein in Example 4 (1) to (3). The polyclonal antibody was used as the primary antibody and each monoclonal antibody was used as the secondary antibody. The amount of protein that specifically reacts with each of the above antibodies in the sera was measured.
[098] The primary antibody was immobilized by adding a polyclonal antibody solution diluted to 5 μg/ml with phosphate-buffered saline to a 96-well immobilizing amine plate (Nunc) at 100 μl/well and shaking the plate at room temperature. environment for 2 h. Blocking was performed by adding 50 mM sodium bicarbonate buffer solution (pH 8.4) (hereinafter called “blocking solution”) containing 0.5% BSA (fetal bovine albumin, Sigma Aldrich, Japan) to 100 μl/well, followed by stirring at room temperature for 1 h. Subsequently, serum samples obtained from cancer patients diluted with the blocking solution were added to 100 μl/well and then subjected to the reaction by stirring at room temperature for 3 h. The dilution ratio this time was adjusted with a 10-fold dilution series (ie, 10-fold to 1000-fold dilutions). The reaction product was washed 3 times with phosphate buffered saline containing 0.5% Tween 20 (Wako Pure Chemical Industries, Ltd.; solution called "PBS-T" in the present application), each monoclonal antibody as a secondary antibody diluted to a concentration of 1 μg/ml with the blocking solution was added to 100 μl/well, and the resultant was then stirred at room temperature for 1 h for the reaction. The reaction product was washed 3 times with PBS-T, an HRP-labeled (H+L) anti-mouse IgG antibody (Invitrogen) as a tertiary antibody diluted 5000 times was added at 100 μl/well, and the resulting was then incubated at room temperature for 1 h. After each well was washed 3 times with PBS-T, a solution of TMA substrate (Thermo) was added at 100 μl/well, and the resultant was incubated for 15 to 30 min to allow color development. Subsequently, 1N sulfuric acid was added at 100 μl/well to stop the reaction. Absorbance was measured at 450 nm using a spectrophotometer.
[099] When monoclonal antibodies #1 to #10 that react with the surface of cancer cells were used as secondary antibodies, as a result, high absorbance values were detected in all dogs with pancreatic ductal carcinoma, although no absorbance was found. detected in healthy dogs. When monoclonal antibodies that react with CAPRIN-1 proteins but do not react with the surface of cancer cells were used as secondary antibodies, polypeptide values were detected in all specimens. However, all absorbance values were lower than the detection limit, which were lower than the results for antibody combinations that react with the surface of cancer cells.
[0100] Subsequently, cancer can also be diagnosed or examined by this technique, which comprises the detection of CAPRIN-1 proteins using antibodies against CAPRIN-1.(2) DIAGNOSIS OR EXAMINATION OF CANCER BY MEASURING THE ANTIGEN POLYPEPTIDE IN TISSUE OF PANCREATIC CANCER BY IMMUNOHISTOCHEMICAL STAINING
[0101] Immunohistochemical staining was performed using an array (BIOMAX) with 101 paraffin-embedded human pancreatic cancer tissue specimens. The pancreatic cancer tissue array was treated at 60°C for 3 h, the resultant was immersed in a staining bottle filled with xylene, the xylene was replaced with fresh xylene every 5 min, and this procedure was repeated 3 times. Subsequently, a similar procedure was performed using ethanol and PBS-T instead of xylene. The human pancreatic cancer tissue array was immersed in a staining bottle filled with 10 mM citrate buffer (pH 6.0) containing 0.05% Tween 20, treated at 125°C for 5 min, and then incubated at room temperature. environment for 40 min or more. Excess water around each specimen was removed using Kimwipes®, each section was surrounded with DAKOPEN® (DAKO), and an appropriate amount of Peroxidase Block® (DAKO) was then added dropwise to the array. . The array was incubated at room temperature for 5 min and immersed in a staining bottle filled with PBS-T, which was replaced with fresh PBS-T every 5 min. This procedure was performed 3 times. As a blocking solution, a PBS-T solution containing 10% FBS was applied to the array, which was then incubated in a humid chamber at room temperature for 1 h. Thereafter, monoclonal antibodies #1 through #10 prepared in Example 3 adjusted to 10 μg/ml with a PBS-T solution containing 5% FBS were applied to the array, which was incubated in a humid chamber at 4°C overnight to the day. After the array was washed with PBS-T for 10 min 3 times, an appropriate amount of Peroxidase Labeled Polymer Conjugated® (DAKO) was added dropwise to the array, which was incubated in a humid chamber at room temperature for 30 min. After the array was washed with PBS-T for 10 min 3 times, a DAB color development solution (DAKO) was applied to the array, which was incubated at room temperature for about 10 min. After the color development solution was discarded, the array was washed with PBS-T for 10 min 3 times, rinsed with distilled water and successively immersed in 70%, 80%, 90%, 95% and 100% ethanol solutions for 1 min each and then incubated in xylene overnight. The glass slides were removed, covered with Glycergel Mounting Medium® (DAKO) and then observed. As a result, expression of CAPRIN-1 was observed in pancreatic cancer cell membranes and in pancreatic cancer cells in pancreatic cancer tissue samples with the use of any antibodies. When immunohistochemical staining was performed using antibody #8, for example, strong expression of CAPRIN-1 was observed in 54 specimens out of the total pancreatic cancer specimens (101 species men) (ie, 54%).
[0102] Similarly, immunohistochemical staining was performed using an array (BIOMAX) of normal human tissues embedded in paraffin, including normal human pancreatic tissues. Excess water around each specimen was removed using Kimwipes®, each section was surrounded with DAKOPEN® (DAKO), and an appropriate amount of Peroxidase Block® (DAKO) was then added dropwise to the array. . The array was incubated at room temperature for 5 min and immersed in a staining bottle filled with PBS-T, which was replaced with fresh PBS-T every 5 min. This procedure was performed 3 times. As a blocking solution, a PBS-T solution containing 10% FBS was applied to the array, which was then incubated in a humid chamber at room temperature for 1 h. Thereafter, monoclonal antibodies #1 through #10 prepared in Example 3 adjusted to 10 μg/ml with a PBS-T solution containing 5% FBS were applied to the array, which was incubated in a humid chamber at 4°C overnight to the day. After the array was washed with PBS-T for 10 min 3 times, an appropriate amount of Peroxidase Labeled Polymer Conjugated® (DAKO) was added dropwise to the array, which was incubated in a humid chamber at room temperature for 30 min. After the array was washed with PBS-T for 10 min 3 times, a DAB color development solution (DAKO) was applied to the array, which was incubated at room temperature for about 10 min. After the color development solution was discarded, the array was washed with PBS-T for 10 min 3 times, rinsed with distilled water and successively immersed in 70%, 80%, 90%, 95% and 100% ethanol solutions for 1 min each and then incubated in xylene overnight. The glass slides were removed, covered with Glycergel Mounting Medium® (DAKO) and then observed. As a result, none of the normal tissue samples derived from the pancreas were stained and no expression of CAPRIN-1 was observed regardless of the antibody used. INDUSTRIAL APPLICABILITY
[0103] The present invention is industrially useful for diagnosing or detecting pancreatic cancer.
[0104] All publications, patents and patent applications cited in this application are incorporated by reference in their entirety. FREE SEQUENCE LIST TEXT
[0105] SEQ ID NOs: 31 to 42: primers

权利要求:
Claims (11)
[0001]
1. METHOD FOR DETECTING PANCREATIC CANCER, characterized by comprising the following steps: (1) contacting a separate sample from the subject: (i) with an antibody against a CAPRIN-1 protein to measure the amount of a polypeptide that has a reactivity of specifically binding an antibody against a CAPRIN-1 protein via an antigen-antibody reaction, or (ii) with a CARPIN-1 protein to measure the amount of an antibody against a CAPRIN-1 protein; or (iii) with a polynucleotide that specifically hybridizes to a polynucleotide that encodes a CAPRIN-1 protein to measure the amount of a nucleic acid that encodes the CAPRIN-1 protein,(2) compare the polypeptide of (i), the antibody of (ii) or the nucleic acid of (iii) in terms of its amount with a default value; and (3) determine that the subject has pancreatic cancer when the amount of the polypeptide of (i), the antibody of (ii), or the nucleic acid of (iii) is greater than the default value, where the CAPRIN-1 protein consists of an amino acid sequence represented by any of the even-numbered sequences SEQ ID NO: 2 to 30, wherein the nucleic acid encoding the CAPRIN-1 protein consists of a nucleotide sequence represented by any of the sequences of SEQ ID NO: 1 to 29, odd-numbered, wherein the sample is selected from the group consisting of pancreatic tissue or cells, blood, serum, blood plasma, ascitic fluid, and pleural effusion.
[0002]
2. METHOD, according to claim 1, characterized in that the subject is a human or a dog.
[0003]
3. METHOD according to claim 2, characterized in that the subject is a dog and the CAPRIN-1 protein comprises the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12 or 14.
[0004]
4. METHOD according to claim 2, characterized in that the subject is a human and the CAPRIN-1 protein comprises the amino acid sequence represented by SEQ ID NO: 2 or 4.
[0005]
5. METHOD according to any one of claims 1 to 4, characterized in that the amount of nucleic acid in the sample is measured using a polynucleotide that specifically hybridizes to a partial sequence comprising 15-19 nucleotides, or 20-30 nucleotides in the nucleotide sequence of the nucleic acid or a sequence complementary thereto.
[0006]
6. METHOD according to claim 5, characterized in that the subject is a dog and the polynucleotide specifically hybridizes to a partial sequence comprising 15-19 nucleotides, or 20-30 nucleotides in the nucleotide sequence represented by SEQ ID NO: 5, 7 , 9, 11 or 13 or a sequence complementary thereto.
[0007]
METHOD according to claim 5, characterized in that the subject is a human and the polynucleotide specifically hybridizes to a partial sequence comprising 15-19 nucleotides, or 20-30 nucleotides in the nucleotide sequence represented by SEQ ID NO: 1 or 3 or a sequence complementary to it.
[0008]
8. METHOD according to any one of claims 1 to 4, characterized in that the amount of the CAPRIN-1 protein is determined by measuring the CAPRIN-1 protein contained in the sample by an immunological test.
[0009]
9. METHOD, according to any one of claims 1 to 8, characterized in that the antibody against the CAPRIN-1 protein that undergoes an antigen-antibody reaction with the polypeptide, is an antibody that binds to the surface of the pancreatic cancer cell.
[0010]
10. METHOD, according to any one of claims 1 to 9, characterized in that the antibody against the CAPRIN-1 protein that undergoes the antigen-antibody reaction with the polypeptide, comprises an antibody that has an immunological reactivity with a polypeptide consisting of a sequence of amino acids comprising 7 to 12 contiguous amino acid residues within the region of amino acid residues numbers 50 to 98 or amino acid residues numbers 233 to 344 of the amino acid sequences represented by any of the even-numbered SEQ ID NOs: 2 to 30 , except for SEQ ID NOs: 6 and 18, wherein the antibody that undergoes the antigen-antibody reaction with the polypeptide is one or more antibodies selected from the group consisting of: a monoclonal antibody comprising the amino acid sequences represented by SEQ ID Nos: 44 and 45, a monoclonal antibody comprising the amino acid sequences represented by SEQ ID Nos: 44 and 46, a monoclonal antibody comprising nding the amino acid sequences represented by SEQ ID NOS: 44 and 47; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOS: 44 to 48; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOS: 49 to 50; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID Nos: 50 to 51; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOS: 53 and 54; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOS: 55 and 56; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOS: 57 and 58; a monoclonal antibody comprising the amino acid sequences represented by SEQ ID NOS: 59 and 60.
[0011]
11. METHOD according to any one of claims 1 to 8, characterized by using a kit or reagent comprising one or more polynucleotides that specifically hybridize to a partial sequence comprising 15 to 19 nucleotides, or 20 to 30 nucleotides in the nucleotide sequence. represented by any one of odd-numbered SEQ ID Nos: 1 to 29, and which encodes a CAPRIN-1 protein, or in a sequence complementary to the nucleotide sequence.

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-08-13| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-03-02| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-09-21| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-11-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-18| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 03/08/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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JP2011-171364|2011-08-04|

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JP2011171364|2011-08-04|

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PCT/JP2012/069824|WO2013018885A1|2011-08-04|2012-08-03|Method for detecting pancreatic cancer|

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