WO2013018885A1 - 膵臓癌の検出方法 - Google Patents
膵臓癌の検出方法 Download PDFInfo
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- WO2013018885A1 WO2013018885A1 PCT/JP2012/069824 JP2012069824W WO2013018885A1 WO 2013018885 A1 WO2013018885 A1 WO 2013018885A1 JP 2012069824 W JP2012069824 W JP 2012069824W WO 2013018885 A1 WO2013018885 A1 WO 2013018885A1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/303—Liver or Pancreas
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
Definitions
- the present invention relates to a method for detecting pancreatic cancer using CAPRIN-1 as a tumor marker.
- pancreatic cancer The number of patients with pancreatic cancer, one of the intractable cancers, has been reported to be over 10,000 in Japan, and its incidence has been increasing year by year and is expected to increase in the future. In the case of pancreatic cancer, even if surgery is possible, small cancers often infiltrate and metastasize to other organs and often recur, resulting in a 5-year survival rate of 9% and a very poor prognosis. .
- gemcitabine which is one of anticancer agents, has been applied for the purpose of preventing postoperative recurrence. However, the effect of gemcitabine administration is mainly painful, and tumor reduction effect and life extension effect can hardly be expected. There are other hospitals that use anti-cancer drug TS-1 that is currently used for gastric cancer, but it is still impossible to expect a therapeutic effect.
- Pancreatic cancer is also an intractable cancer in dogs, and the symptoms of pancreatic cancer are due to hypoglycemia such as sudden loss of energy, wandering, or abnormal gait, although the abdomen can be confirmed to be lumped. It is a symptom, and in most cases, such symptoms are finally noticed. In addition, when such symptoms are seen, pancreatic cancer is already in an advanced state, and the treatment is not only removing pancreatic cancer by surgery, but also supportive therapy and anticancer agents are administered. Early detection is important for dogs with pancreatic cancer in order to enable effective treatment for pancreatic cancer in the same way as humans. There is no diagnostic agent, and examination methods such as X-ray, CT, and MRI imaging are not widely used in animal medicine.
- Diagnosis is highly dependent on veterinary experience through palpation, simple blood tests, and X-ray examinations. Sensitive and simple cancer detection applicable to canine pancreatic cancer diagnosis Providing the means makes it possible to treat appropriately and has great benefits for both owners and veterinarians.
- Cytoplasmic- and promotion-associated protein 1 (CAPRIN-1) is expressed when quiescent normal cells are activated or undergo cell division, and forms RNA and intracellular stress granules within the cell to transport mRNA. It is an intracellular protein known to be involved in the control of translation.
- the gene encoding the protein of CAPRIN-1 is specifically expressed in canine and human testis and malignant cancer cells. As a result of FCM analysis using breast cancer cells using antibodies against CAPRIN-1, CAPRIN-1 is expressed on the surface, and the results of immunohistochemical staining using breast cancer tissue indicate that CAPRIN-1 is strongly expressed in breast cancer.
- Patent Document 1 it has been reported that the above-mentioned antibody damages breast cancer cells through the function of lymphocytes, and that the antibody against CAPRIN-1 exerts a strong antitumor effect in a tumor-bearing mouse model transplanted with breast cancer cells.
- Patent Document 2 cancers such as breast cancer can be diagnosed by measuring an antibody induced in a subject against CAPRIN-1 in a cancer patient serum or a polypeptide that reacts with an antigen antibody.
- Patent Document 2 by measuring that CAPRIN-1 is expressed in pancreatic cancer and the antibody or antigen-antibody reactive polypeptide induced in the subject against CAPRIN-1 in pancreatic cancer patient serum, There has never been any report that diagnosis will be possible.
- An object of the present invention is to provide a means for detecting pancreatic cancer useful for diagnosis of pancreatic cancer.
- CAPRIN-1 is expressed in pancreatic cancer, and that an antibody against CAPRIN-1 induced in the serum of pancreatic cancer patients using CAPRIN-1 protein is measured. Furthermore, the inventors have found that antibodies produced using these proteins can be diagnosed, examined or detected by pancreatic cancer by binding to CAPRIN-1 in pancreatic cancer tissue, and the present invention has been completed.
- the present invention provides a method for detecting pancreatic cancer, which is a method performed on a sample isolated from a subject, which comprises measuring the expression of CAPRIN-1.
- the term “detection” can be replaced with inspection or evaluation.
- the present invention also provides a reagent or kit for detecting pancreatic cancer comprising a polypeptide that reacts with an antibody induced by CAPRIN-1 in a subject with an antigen.
- the present invention provides a pancreatic cancer detection reagent or kit comprising an antibody or antigen-binding fragment thereof that reacts with CAPRIN-1 in an antigen-antibody reaction.
- the present invention provides 15 to 19 bases or more, or 20 to 30 bases or more in the base sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,.
- a reagent or kit for detecting pancreatic cancer comprising a polynucleotide that specifically hybridizes with a partial sequence of
- the “reagent or kit for detecting pancreatic cancer” used in the specification can also be referred to as “reagent or kit for detecting pancreatic cancer”.
- the present invention has the following features.
- the polypeptide to be measured is CAPRIN-1 protein consisting of any amino acid sequence represented by an even sequence number among SEQ ID NOs: 2 to 30, or 85 to 90% of the CAPRIN-1 protein and The method according to (1) above, which is a polypeptide comprising an amino acid sequence having the above sequence identity.
- polypeptide to be measured comprises the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12 or 14.
- the presence or amount of the polypeptide is measured by immunologically measuring an antibody induced in the subject against the polypeptide to be measured, which can be contained in the sample. ) To (6).
- the presence or amount of the nucleic acid encoding the polypeptide is measured by measuring the nucleic acid encoding the polypeptide contained in the sample. That way.
- the subject is a dog, and the polynucleotide is 15 to 19 bases or more in the base sequence shown in SEQ ID NO: 5, 7, 9, 11 or 13 or its complementary sequence, preferably 20 to The method according to (9) above, which is a polynucleotide that specifically hybridizes with a partial sequence of 25 bases or more, more preferably 30 bases or more.
- the subject is a human, and the polynucleotide is 15 to 19 bases or more, preferably 20 to 25 bases or more, in the base sequence shown in SEQ ID NO: 1 or 3 or its complementary sequence, More preferably, the method according to (9), wherein the polynucleotide specifically hybridizes with a partial sequence of 30 bases or more.
- a reagent or kit for detecting pancreatic cancer comprising one or a plurality of polypeptides having a reactivity of binding to an antibody induced in a subject with CAPRIN-1 protein by an antigen-antibody reaction.
- a pancreatic cancer comprising one or more antibodies or antigen-binding fragments thereof that react with an antibody against the CAPRIN-1 protein by an antigen-antibody reaction and react with the polypeptide produced in a subject. Reagent or kit for detection.
- An antibody or antigen-binding fragment thereof that reacts with the polypeptide by antigen-antibody reaction binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 43, or an antigen-binding fragment thereof, and the amino acid sequences of SEQ ID NOs: 44 and 45 Monoclonal antibody or antigen-binding fragment thereof, monoclonal antibody comprising the amino acid sequence of SEQ ID NOs: 44 and 46, or antigen-binding fragment thereof, monoclonal antibody comprising the amino acid sequence of SEQ ID NOs: 44 and 47, or antigen-binding fragment thereof, SEQ ID NO: Monoclonal antibody comprising 44 and 48 amino acid sequences or antigen-binding fragment thereof; monoclonal antibody comprising amino acid sequences of SEQ ID NOs: 49 and 50 or antigen-binding fragment thereof; monoclonal antibody comprising amino acid sequences of SEQ ID NOs: 51 and 52 or Antigen-binding fragment, amino acids of SEQ ID NOs: 53 and 54 A monoclon
- a reagent or kit for detecting pancreatic cancer comprising one or more polynucleotides that specifically hybridize with a partial sequence of 20 to 25 bases or more, more preferably 30 bases or more.
- CAPRIN-1 protein an antibody against CAPRIN-1 protein, or CAPRIN-1 in a sample of a subject using at least one reagent or kit according to any one of (15) to (21) above
- a method for detecting pancreatic cancer comprising measuring the presence or amount of a nucleic acid encoding a protein.
- a method for detecting pancreatic cancer comprising measuring the presence or amount of CAPRIN-1 protein, an antibody against CAPRIN-1 protein, or a nucleic acid encoding CAPRIN-1 protein.
- the present invention provides a novel method for detecting pancreatic cancer.
- a recombinant polypeptide prepared based on the amino acid sequence of CAPRIN-1 (or “Caprin-1”) is an antibody present in the serum of pancreatic cancer patients. Can react specifically. Therefore, pancreatic cancer in a subject can be detected by measuring the antibody in a sample by the method of the present invention. In addition, pancreatic cancer in a subject can be detected by measuring CAPRIN-1 itself.
- CAPRIN-1 gene is specifically highly expressed in the testis and pancreatic cancer cells of the subject (in the specification, hereinafter, such an expression product is expressed as (Sometimes referred to as nucleic acid encoding CAPRIN-1 (protein)). Therefore, pancreatic cancer can also be detected by measuring the nucleic acid. It is also possible to measure the presence or amount of (expression of) CAPRIN-1 in pancreatic cancer tissue using an antibody against CAPRIN-1. Therefore, by performing the above-described measurement on pancreatic cancer patients in advance, it becomes possible to select pancreatic cancer patients to which a therapeutic drug targeting CAPRIN-1, such as an antibody drug, should be applied.
- a therapeutic drug targeting CAPRIN-1 such as an antibody drug
- the presence or amount of CAPRIN-1 (expression) is measured using a sample isolated from a subject.
- Methods for measuring the presence or amount of (expression) of CAPRIN-1 include a method of immunologically measuring an antibody against CAPRIN-1 contained in a sample (first method), a CAPRIN- contained in a sample A method for measuring 1 itself immunologically (second method) and a method for measuring a nucleic acid encoding CAPRIN-1 contained in a sample (for example, mRNA or cDNA synthesized from mRNA) (third method) Method).
- the presence or amount of (expression of) CAPRIN-1 may be measured by any of these methods.
- the term “measurement” includes detection, qualitative, quantitative, and semi-quantitative.
- the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12 or 14 is the amino acid sequence of canine CAPRIN-1.
- Canine CAPRIN-1 having the amino acid sequence has been identified as a polypeptide that binds to an antibody that specifically exists in serum derived from a cancer-bearing dog (see Example 1 described below), Then, an antibody against CAPRIN-1 having the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, or 14 is specifically induced. That is, canine pancreatic cancer can be detected by measuring the above antibody against CAPRIN-1 having the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, or 14 by the first method.
- canine pancreatic cancer can also be detected by measuring CAPRIN-1 itself having the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 12, or 14 as an antigen by the second method. Furthermore, in connection with the third method, since the CAPRIN-1 gene is significantly highly expressed in pancreatic cancer cells, canine pancreatic cancer can also be detected by measuring the nucleic acid. it can.
- a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 means 709 amino acids having the amino acid sequence of Met Pro Ser Ala, extended, Gln Gln Val Asn represented by SEQ ID NO: 2.
- a residue size polypeptide is meant.
- polypeptide having the amino acid sequence represented by SEQ ID NO: 2 may be abbreviated as “polypeptide of SEQ ID NO: 2”. The same applies to the expression “having a base sequence”.
- the term “comprising” may be replaced by the expression “comprising” or “consisting of”.
- polypeptide refers to a molecule formed by peptide bonding of a plurality of amino acids, and not only a polypeptide molecule having a large number of amino acids but also a low number of amino acids.
- oligopeptides oligopeptides
- full-length proteins are also included.
- an even sequence number among SEQ ID NOs: 2 to 30 ie, SEQ ID NOs: 2, 4, 6,..., 26, 28, 30.
- a full-length protein of CAPRIN-1 having the amino acid sequence shown in any of the above is also included.
- a “subject” as used herein is a vertebrate including mammals and birds, preferably a mammal, more preferably a human, a dog, a cow, a horse, and the like.
- sample used in the present specification is a biological sample for examination for detecting pancreatic cancer, and includes body fluid, tissue or cells separated from a subject.
- the body fluid includes, but is not limited to, blood, serum, plasma, ascites, pleural effusion, and the like.
- the tissue or cell includes pancreatic tissue or cell suspected of suffering from cancer.
- CAPRIN-1 not only canine CAPRIN-1 of SEQ ID NO: 6, 8, 10, 12 or 14 but also other mammalian CAPRIN-1 (hereinafter referred to as “homologous factor” (or “homologue for canine CAPRIN-1). Or “ortholog”).
- CAPRIIN-1 includes not only dogs but also CAPRIN-1 derived from other mammals including humans). It becomes a target.
- the human CAPRIN-1 gene is significantly highly expressed in human pancreatic cancer cells, and antibodies against the human CAPRIN-1 are not detected in healthy human bodies. Therefore, pancreatic cancer can be detected in mammals other than dogs by measuring the expression of CAPRIN-1.
- CAPRIN-1 other than dogs to be measured by the method of the present invention examples include, but are not limited to, human CAPRIN-1.
- the nucleotide sequence encoding human CAPRIN-1 and its amino acid sequence are as shown in SEQ ID NOs: 1, 3, 2 and 4, respectively, and the sequence identity with canine CAPRIN-1 is the nucleotide sequence. 94%, amino acid sequence 98%.
- sequence identity of the amino acid sequences of each CAPRIN-1 is very high at 98%. Therefore, even between mammals other than humans, It is considered that canine CAPRIN-1 and its homologous factor have a high sequence identity of about 85% or more.
- CAPRIN-1 for measuring expression in the method of the present invention is not particularly limited, but preferably has an amino acid sequence of canine CAPRIN-1 shown in SEQ ID NO: 6, 8, 10, 12 or 14 and preferably 85% or more The sequence identity is preferably 90% or more, more preferably 95% or more.
- measurement of the antibody that may be present in the sample can be easily performed by immunological measurement using an antigen substance that reacts with the antibody by antigen-antibody reaction.
- the immunological measurement method itself is a well-known conventional method as described in detail below.
- the antigen substance for immunological measurement include, for example, the canine CAPRIN-1 protein of SEQ ID NO: 6, 8, 10, 12, or 14 that has induced the antibody in cancer-bearing dogs, or an epitope of the protein Can be used.
- antibodies are cross-reactive, and even molecules other than the antigenic substance that actually became the immunogen can be used as immunogens if a structure similar to the epitope of the immunogen exists on the molecule.
- CAPRIN-1 can be used as an antigen for immunological measurement.
- an antibody produced against an antigenic substance such as a protein in a subject is a polyclonal antibody that is a mixture of a plurality of types of antibodies.
- the antibody found by the present inventors that is specifically present in serum derived from a tumor-bearing subject and specifically binds to a recombinant CAPRIN-1 protein by an antigen-antibody reaction is also a polyclonal antibody.
- the term “polyclonal antibody” refers to an antibody that is present in serum derived from a subject containing an antigen substance in the body, and that is induced in the subject with respect to the antigen substance. .
- polypeptides of SEQ ID NO: 6 and SEQ ID NO: 8 (canine CAPRIN-1), and SEQ ID NO: 2 (human CAPRIN) -1) polypeptides have been prepared, and the reactivity of these polypeptides with the antibodies in the serum from cancer-bearing subjects has been confirmed.
- the antibody since the antibody is a polyclonal antibody, it naturally binds if it is a polypeptide consisting of the homologous factor of SEQ ID NO: 6, 8 or 2, and even a fragment of the polypeptide is contained in the polyclonal antibody.
- the polypeptide used as an antigen for immunological measurement in the first method of the present invention is not limited to a polypeptide consisting of the full-length region of the CAPRIN-1 protein (eg, SEQ ID NO: 6, 8 or 2), A polypeptide fragment consisting of 7 to 12 or more, preferably 8 or more, 9 or more, or 10 or more consecutive amino acids in the amino acid sequence of a CAPRIN-1 protein, wherein the CAPRIN-1 protein Polypeptides that react with a polyclonal antibody against an antigen antibody (hereinafter sometimes referred to as “specifically reactive partial polypeptides” for convenience) are also included. It is known in the art that a polypeptide having about 7 to 12 amino acid residues or more exhibits antigenicity.
- the number of amino acid residues of the polypeptide fragment is preferably 20 or more, 30 or more, or 50 or more, more preferably 100 or more, or 150 or more, and more preferably 300. As described above, more preferably 600 or more, and even more preferably 1000 or more and 1500 or more.
- polypeptide used as an antigen include each of even-numbered SEQ ID NOs: SEQ ID NOs: 2 to 30, or a fragment containing an epitope thereof (ie, a polypeptide having about 7 to 12 amino acid residues or more). Peptide fragment).
- nucleotide sequences of the polynucleotides encoding the proteins consisting of the amino acid sequences of even sequence numbers (ie, SEQ ID NOs: 2, 4, 6,. It is shown by the odd number sequence number (namely, sequence number 1,3,5..27,29) among 29.
- a protein antigen has almost the same antigenicity as the original protein even when a few amino acid residues in the amino acid sequence of the protein are replaced, deleted, added, or inserted. It is well known to those skilled in the art that Thus, a polypeptide having a sequence in which a few (preferably one or several) amino acid residues of the CAPRIN-1 protein are substituted, deleted and / or inserted, 80% or more, 85-90% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more of the original sequence.
- a polypeptide that specifically binds to an antibody against CAPRIN-1 through an antigen-antibody reaction is also a cancer-like polypeptide.
- the specifically reactive modified polypeptide has an amino acid sequence in which one or several amino acid residues are substituted, deleted, added and / or inserted in the amino acid sequence of CAPRIN-1.
- “several” represents an integer of 2 to 10, preferably an integer of 2 to 6, and more preferably an integer of 2 to 4.
- sequence identity of amino acid sequences refers to the alignment of both amino acid sequences so that the amino acid residues of the two amino acid sequences to be compared match as much as possible. Is divided by the total number of amino acid residues and expressed as a percentage (%). In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary.
- alignment of sequences can be performed using, for example, a known program such as BLAST, FASTA, CLUSTAL W (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1993; Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997).
- the 20 amino acids constituting the natural protein are neutral amino acids having low polarity side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids having hydrophilic side chains (Asn). , Gln, Thr, Ser, Tyr, Cys, acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, Trp, His)
- Asp acidic amino acids
- Arg Lys, His
- aromatic amino acids Phe, Tyr, Trp, His
- the above-described variant may have a non-conservative substitution as long as it imparts immunity-inducing activity equivalent to or almost equivalent to that of the unmodified product.
- An antibody and an antigen against CAPRIN-1 containing the above-mentioned polypeptide used in the present invention as a partial sequence for example, one having both ends of the polypeptide used in the present invention added with other (poly) peptides
- a polypeptide that specifically binds by an antibody reaction hereinafter sometimes referred to as “specific reactive addition polypeptide” for convenience
- the polypeptide used in the present invention can be synthesized according to chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method) (edited by the Japanese Biochemical Society). Biochemistry Experiment Course 1, Protein Chemistry IV, Chemical Modification and Peptide Synthesis, Tokyo Chemical Doujin (Japan), 1981). Moreover, it can also synthesize
- a cDNA of the gene is prepared by RT-PCR from RNA extracted from a tissue expressing a gene encoding human CAPRIN-1 of SEQ ID NO: 2 or a homologous factor thereof, and the full length of the cDNA or a desired one is obtained.
- the part can be incorporated into an expression vector and introduced into a host cell to obtain the desired polypeptide.
- 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, and their human homologous factors SEQ ID NOs: 2 and 4 Since the nucleotide sequences of cDNAs encoding human CAPRIN-1 are shown in SEQ ID NOs: 1 and 3, respectively, primers used for RT-PCR can be easily designed with reference to these nucleotide sequences.
- the gene encoding CAPRIN-1 of mammals other than humans can be amplified with primers designed with reference to the nucleotide sequence of the odd sequence number among SEQ ID NOs: 1 to 29.
- a cDNA encoding CAPRIN-1 can also be easily prepared by the same method as described above. Extraction of RNA, RT-PCR, integration of cDNA into a vector, and introduction of a vector into a host cell can be performed by well-known methods, for example, as described below. Further, vectors and host cells to be used are well known, and various types are commercially available.
- the host cell may be any cell that can express the polypeptide.
- prokaryotic cells include Escherichia coli
- examples of eukaryotic cells include monkey kidney cells COS1 and Chinese hamster ovary cells.
- Examples include mammalian cultured cells such as CHO, human fetal kidney cell line HEK293, mouse fetal skin cell line NIH3T3, budding yeast, fission yeast, silkworm cells, and Xenopus egg cells.
- the expression vector When a prokaryotic cell is used as a host cell, the expression vector includes an origin, promoter, ribosome binding site, multicloning site, terminator, drug resistance gene, auxotrophic complementary gene, etc. that can be replicated in the prokaryotic cell. Is used. Examples of the expression vector for E. coli include pUC system, pBluescript II, pET expression system, pGEX expression system and the like. When the DNA encoding the above polypeptide is incorporated into such an expression vector, a prokaryotic host cell is transformed with the vector, and the resulting transformant is cultured, the polypeptide encoded by the DNA is prokaryotic. It can be expressed in a host cell.
- the polypeptide can also be expressed as a fusion protein with another protein.
- the DNA encoding the polypeptide can be obtained, for example, by preparing cDNA by RT-PCR as described above, or can be synthesized by a conventional method using a commercially available nucleic acid synthesizer as described later. it can.
- the nucleotide sequences of cDNAs of genes encoding CAPRIN-1 of SEQ ID NOs: 2 and 4 are shown in SEQ ID NOS: 1 and 3, respectively.
- an expression vector for a eukaryotic cell having a promoter, a splicing region, a poly (A) addition site and the like is used as an expression vector.
- expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pYES2, and the like.
- the DNA encoding the polypeptide used in the present invention is incorporated into such an expression vector, and after transforming a eukaryotic host cell with the vector, the obtained transformant is cultured.
- Polypeptides encoded by the DNA can be expressed in eukaryotic host cells.
- pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, etc. are used as an expression vector, a His tag (eg (His) 6 to (His) 10 ), FLAG tag, myc tag
- His tag eg (His) 6 to (His) 10
- FLAG tag eg (His) 6 to (His) 10
- the polypeptide can be expressed as a fusion protein to which various tags such as HA tag and GFP are added.
- a well-known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, virus infection, lipofection, binding to a cell membrane-permeable peptide, etc. can be used. .
- the target polypeptide from the host cell can be performed by combining known separation operations. For example, treatment with denaturing agents and surfactants such as urea, sonication, enzyme digestion, salting out and solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.
- Polypeptides obtained by the above methods include those in the form of fusion proteins with other arbitrary proteins. Examples thereof include glutathione-S-transferase (GST) and a fusion protein with a His tag. Such a polypeptide in the form of a fusion protein is also included in the above-mentioned specific reactive addition polypeptide, and can be used in the first detection method of the present invention. Furthermore, the polypeptide expressed in the transformed cell may be subjected to various modifications in the cell after being translated. Such a post-translationally modified polypeptide can also be used in the first detection method of the present invention as long as it has a specific binding property by an antigen-antibody reaction with an antibody against the CAPRIN-1 protein.
- translational modifications include elimination of N-terminal methionine, N-terminal acetylation, glycosylation, limited degradation by intracellular protease, myristoylation, isoprenylation, phosphorylation, and the like.
- the antibody in the sample can be easily measured by immunological measurement using the above-mentioned polypeptide as an antigen.
- Immunological measurement itself is well known in this field, and classified by reaction mode includes sandwich method, competition method, aggregation method, Western blot method and the like.
- the sandwich ELISA and the agglutination method can be preferably applied as an immunoassay method for the antibody in the method of the present invention because it is easy to operate and does not require a large-scale apparatus.
- an enzyme When an enzyme is used as a label for an antibody, the enzyme has special restrictions as long as it satisfies conditions such as a large turnover number, stability even when bound to the antibody, and specific coloring of the substrate.
- Use enzymes that are used in conventional enzyme immunoassays such as peroxidase, ⁇ -galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, etc. You can also.
- an enzyme inhibitor, a coenzyme, etc. can also be used.
- enzymes and antibodies can be bound by a known method using a crosslinking agent such as a maleimide compound, a biotin- (strept) avidin system, or the like.
- a crosslinking agent such as a maleimide compound, a biotin- (strept) avidin system, or the like.
- a known substance can be used according to the type of enzyme used. For example, when peroxidase is used as the enzyme, 3,3 ′, 5,5′-tetramethylbenzidine can be used, and when alkaline phosphatase is used as the enzyme, paranitrophenol or the like can be used.
- radioisotope those usually used in radioimmunoassay such as 125 I and 3 H can be used.
- fluorescent dye those used in usual fluorescent antibody methods such as fluoroless isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), cyanine fluorescent dye (Cy3, Cy5, etc.) can be used.
- FITC fluoroless isothiocyanate
- TRITC tetramethylrhodamine isothiocyanate
- Cy3, Cy5, etc. cyanine fluorescent dye
- the polypeptide used as an antigen is immobilized on a solid phase, After reacting with a sample such as serum and washing, an appropriate secondary antibody is reacted, and after washing, the secondary antibody bound to the solid phase is measured. Since the unbound secondary antibody can be easily removed by immobilizing the antigen polypeptide on the solid phase, it is preferable as an embodiment of the cancer detection method of the present invention.
- the secondary antibody for example, if the sample is derived from a dog, an anti-canine IgG antibody can be used. By labeling the secondary antibody with the labeling substance exemplified above, the secondary antibody bound to the solid phase can be measured.
- the amount of secondary antibody measured in this way corresponds to the amount of antibody in the serum sample.
- an enzyme used as the labeling substance
- the amount of antibody can be measured by adding a substrate that develops color by degradation by enzymatic action and optically measuring the amount of degradation of the substrate.
- a radioisotope used as the labeling substance
- the radiation dose emitted by the radioisotope can be measured with a scintillation counter or the like.
- CAPRIN-1 that can be contained in a sample obtained from a subject is measured.
- the amount of antibody that undergoes an antigen-antibody reaction with CAPRIN-1 is significantly higher. It shows that the accumulated amount of CAPRIN-1 which is an antigen is significantly large.
- the expression of CAPRIN-1 in a healthy subject is below the detection limit or weakly expressed in the tissue and remains in the cell.
- pancreatic cancer can be detected by measuring CAPRIN-1 itself. Therefore, pancreatic cancer in a subject can be detected by measuring CAPRIN-1 itself as in the first method.
- Measurement of a polypeptide in a sample can be easily performed by a well-known immunological measurement method.
- CAPRIN-1 that can exist in a sample is measured by preparing an antibody or antigen-binding fragment thereof that reacts with CAPRIN-1 and antigen-antibody, and performing immunological measurement using the antibody. can do.
- the antibody since the antibody has cross-reactivity, for example, using only the canine CAPRIN-1 of SEQ ID NO: 6 using an antibody or an antigen-binding fragment thereof that reacts with the dog CAPRIN-1 of SEQ ID NO: 6
- its homologous factors in other mammals such as human CAPRIN-1 of SEQ ID NO: 2 or 4 can also be measured.
- the immunological measurement method itself is a well-known conventional method as described above.
- CAPRIN-1 was found to be a cell membrane protein expressed on the surface of pancreatic cancer cells. Since cancer tissue of a subject having cancer contains many proteolytic enzymes, in cancer tissue, a portion of the CAPRIN-1 sequence that is expressed outside the cancer cell undergoes degradation and is removed from the cancer cell. There are more than parts that are isolated and expressed in cells of cancer cells in the CAPRIN-1 sequence. Therefore, if the antibody against CAPRIN-1 or its antigen-binding fragment used in this measurement is one that binds to the cell surface of pancreatic cancer cells, more CAPRIN-1 can be detected and pancreatic cancer can be detected with higher sensitivity. Can be diagnosed.
- an antibody that binds to a portion of the CAPRIN-1 protein molecule that is expressed on the cell surface of pancreatic cancer cells is preferably used.
- a partial peptide in CAPRIN-1 protein expressed on the cell surface of pancreatic cancer cells amino acid residues in the amino acid sequence represented by even numbers excluding SEQ ID NO: 6 and SEQ ID NO: 18 among SEQ ID NOS: 2 to 30 in the sequence listing
- Non-limiting examples of the amino acid sequence of the peptide include, for example, SEQ ID NO: 43 or SEQ ID NO: 61 (the amino acid sequence represented by SEQ ID NO: 62 or SEQ ID NO: 63 among the amino acid sequences represented by SEQ ID NO: 61). Or 80% or more, preferably 85% or more, more preferably 90% or more, and the amino acid sequence represented by Preferably include an amino acid sequence with a sequence identity of 95% or more.
- the antibody used in the present invention includes all antibodies that bind to these polypeptides.
- an antibody or antigen-binding fragment thereof that binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 43, a monoclonal antibody comprising the amino acid sequences of SEQ ID NOs: 44 and 45, or an antigen-binding fragment thereof, SEQ ID NOs: 44 and 46
- a monoclonal antibody comprising the amino acid sequence or antigen-binding fragment thereof; a monoclonal antibody comprising the amino acid sequence of SEQ ID NOs: 44 and 47 or an antigen-binding fragment thereof; a monoclonal antibody comprising the amino acid sequence of SEQ ID NOs: 44 and 48; Fragment, monoclonal antibody comprising the amino acid sequences of SEQ ID NOs: 49 and 50 or antigen-binding fragments thereof, monoclonal antibody comprising the amino acid sequences of SEQ ID NOs: 51 and 52, or antigen-binding fragments thereof, comprising the amino acid sequences of SEQ ID NOs: 53 and 54
- monoclonal antibody comprising the amino acid sequences of SEQ ID NOs: 57 and 58 or antigen-binding fragments thereof, or monoclonal antibody comprising the amino acid sequences of SEQ ID NOs: 59 and 60 or antigens thereof Examples include binding fragments.
- antigen-binding fragment means an antibody fragment having the ability to bind to an antigen, such as a Fab fragment, F (ab ′) 2 fragment, or Fv fragment contained in an antibody molecule.
- the antibody may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody with high reproducibility is preferable for immunological measurement. Methods for preparing polyclonal and monoclonal antibodies using a polypeptide as an immunogen are well known and can be easily performed by conventional methods.
- CAPRIN-1 or a fragment thereof alone or combined with a carrier protein such as keyhole limpet hemocyan (KLH), casein, serum albumin or the like as an immunogen and immunized to an animal together with an adjuvant Antibodies against -1 can be induced.
- a hybridoma is prepared by fusing an antibody-producing cell such as a spleen cell or lymphocyte collected from an immunized animal with a myeloma cell, and a hybridoma that produces an antibody that binds to CAPRIN-1 is selected and proliferated.
- a monoclonal antibody having CAPRIN-1 as a corresponding antigen can be obtained from the culture supernatant.
- the above method is a well-known ordinary method.
- a nucleic acid encoding CAPRIN-1 (for example, mRNA or cDNA synthesized from mRNA) that can be contained in a sample obtained from a living body is measured.
- CAPRIN-1 for example, mRNA or cDNA synthesized from mRNA
- the 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 significant in pancreatic cancer cells. Is highly expressed. Therefore, in vivo cancer can also be detected by measuring the nucleic acid in a sample.
- the mRNA in the sample can be quantified by a conventional method such as real-time detection RT-PCR using the mRNA as a template, and can be generally quantified also by a staining intensity in a Northern blot which is a conventional method.
- the sequences of cDNAs encoding CAPRIN-1 having an even sequence number among SEQ ID NOs: 2 to 30 are as shown in the odd sequence numbers in SEQ ID NOs: 1 to 29, respectively.
- polynucleotide for cancer detection Preparing a polynucleotide that specifically hybridizes with a partial region in the base sequence represented by an odd sequence number among SEQ ID NOs: 1 to 29 (hereinafter referred to as “polynucleotide for cancer detection”), It can be used as a probe or a primer in a nucleic acid amplification method to measure the abundance of the mRNA in a sample.
- any polynucleotide that specifically hybridizes with a partial region in the base sequence represented by an odd number of SEQ ID NOs: 1 to 29 encodes CAPRIN-1 in mammals other than dogs and humans. MRNA can also be measured.
- the polynucleotide may be RNA or DNA.
- “Conditions for stringent hybridization” refers to conditions used for normal PCR annealing and detection with a probe.
- PCR using Taq polymerase
- 50 mM KCl 10 mM Tris.
- the reaction is carried out using a general buffer solution such as HCl (pH 8.3 to 9.0) and 1.5 mM MgCl 2 at an appropriate annealing temperature of about 54 ° C to 60 ° C.
- hybridization 5 x SSPE, 50% formamide, 5 x Denhardt's solution, 0.1 to 0.5% SDS, or 0.1 to 5 x SSC, 0.1 to 0.5% SDS
- a general hybridization solution such as It refers to a reaction condition at hybridization temperature.
- washing is performed with, for example, 0.1 to 0.2 ⁇ SSC, 0.1% SDS.
- the appropriate annealing temperature or hybridization temperature is not limited to the above examples, and is determined based on the Tm value of the polynucleotide for cancer detection used as a primer or probe and the rule of thumb of the experimenter. Can be determined.
- “Substantially does not hybridize” means that it does not hybridize at all, or even if it is hybridized, it is much less than the amount hybridized to the target partial region, and hybridizes only in a relatively negligible amount. It means that.
- Examples of the polynucleotide that specifically hybridizes under such conditions include a polynucleotide having a certain sequence identity with the base sequence of the target partial region, for example, 70% or more, preferably 80% or more, A polynucleotide having a sequence identity of 85% or more, more preferably 90% or more, still more preferably 93% or more, still more preferably 95% or more, and still more preferably 98% or more.
- the polynucleotide has the same base sequence as that of the target partial region.
- sequence identity is the same as that of the amino acid sequence described above.
- partial sequence refers to a partial sequence in the base sequence represented by an odd sequence number among SEQ ID NOs: 1 to 29, It is a sequence of 19 bases or more, preferably 18 or more consecutive bases, more preferably 20 or more bases or 25 bases or more, more preferably 30, 40 or 50 bases or more.
- base sequence shown in SEQ ID NO: 5 includes the base sequence actually shown in SEQ ID NO: 5 as well as a complementary sequence thereto.
- polynucleotide having the base sequence shown in SEQ ID NO: 5 a single-stranded polynucleotide having the base sequence actually shown in SEQ ID NO: 5, its complementary base sequence And double-stranded polynucleotides comprising these.
- any base sequence will be selected as appropriate. You can easily make that choice.
- the number of bases of the polynucleotide for cancer detection is preferably 18 bases or more from the viewpoint of ensuring specificity.
- the size is preferably 18 bases or more, more preferably 20 bases or more and preferably not more than the entire length of the coding region.
- the size is preferably 18 bases or more, and preferably 50 bases or less.
- Preferable examples of the polynucleotide for cancer detection include polynucleotides comprising 18 or more consecutive bases in the base sequence represented by the odd sequence number among SEQ ID NOs: 1 to 29.
- the amount of nucleic acid (eg, mRNA or cDNA synthesized from mRNA) encoding the canine CAPRIN-1 protein of SEQ ID NO: 6, 8, 10, 12, or 14 as will be apparent to those skilled in the art with reference to this specification Is measured using a polynucleotide that specifically hybridizes to a partial region in SEQ ID NO: 5, 7, 9, 11 or 13, and a nucleic acid encoding human CAPRIN-1 of SEQ ID NO: 2 or 4 (
- a polynucleotide that specifically hybridizes with a partial region in SEQ ID NO: 1 or 3 is used for measuring the amount of mRNA or cDNA synthesized from mRNA.
- a protein derived from one mammal and its homologous factor derived from another mammal usually have high sequence identity even at the base sequence level, and the sequence identity with the base sequences of SEQ ID NOS: 1 to 13 is also 94-100. % And very high. Therefore, for example, a polynucleotide that specifically hybridizes with a partial region in SEQ ID NO: 5 can specifically hybridize with a partial region in an odd sequence number among SEQ ID NOS: 1 to 29 corresponding to the partial region. .
- a polynucleotide that specifically hybridizes with a partial region in SEQ ID NO: 5 not only mRNA encoding canine CAPRIN-1 in SEQ ID NO: 6, but also human CAPRIN-1 in SEQ ID NO: 2 or 4
- mRNA encoding CAPRIN-1 of other mammals such as cats can also be measured.
- a partial region having a particularly high sequence identity is selected between each SEQ ID NO (odd SEQ ID NO: 1 to 29). It is more desirable.
- a method for measuring a test nucleic acid using a polynucleotide that specifically hybridizes with a partial region of the test nucleic acid as a primer or probe for a nucleic acid amplification method such as PCR is well known.
- a nucleic acid amplification method such as PCR
- Northern blot, in situ hybridization and the like can be mentioned.
- any of these well-known measurement methods can be employed.
- Nucleic acid amplification methods such as PCR are well known in this field, and reagent kits and devices for that purpose are also commercially available and can be easily performed. That is, for example, a test nucleic acid as a template (for example, cDNA of a gene encoding a protein having an amino acid sequence represented by an even sequence number among SEQ ID NOs: 2 to 30) and a polynucleotide (primer) for cancer detection
- a test nucleic acid as a template for example, cDNA of a gene encoding a protein having an amino acid sequence represented by an even sequence number among SEQ ID NOs: 2 to 30
- a polynucleotide primer
- the denaturation step is 90 to 95 ° C.
- the annealing step is Tm of the template and the primer or in the vicinity thereof (preferably within ⁇ 4 ° C.)
- the extension step is the optimum temperature of a thermostable DNA polymerase such as Taq polymerase or Pfu polymerase.
- a thermostable DNA polymerase such as Taq polymerase or Pfu polymerase.
- Each step is appropriately selected in about 30 seconds to 2 minutes.
- the nucleic acid amplification method is not limited to PCR, and other nucleic acid amplification methods well known in this field can also be used.
- the test nucleic acid when nucleic acid amplification is performed using a pair of polynucleotides for cancer detection as primers and a test nucleic acid as a template, the test nucleic acid is amplified, whereas the sample contains the test nucleic acid. If not, amplification does not occur, and it can be determined whether or not the test nucleic acid is present in the sample by detecting the amplification product. Amplification products can be detected by electrophoresis of the reaction solution after amplification and staining the band with ethidium bromide, etc., or by immobilizing the amplified product on a solid phase such as nylon membrane, and specific to the test nucleic acid.
- test nucleic acid can be semi-quantified based on the intensity of the electrophoresis band.
- the test nucleic acid may be mRNA or cDNA reverse transcribed from mRNA.
- the NASBA method 3SR method, TMA method
- the NASBA method itself is well known, and kits therefor are also commercially available. Therefore, the NASBA method can be easily carried out using the above pair of primers.
- a labeled probe obtained by attaching a label such as a fluorescent label, a radioactive label, or a biotin label to a polynucleotide for cancer detection can be used.
- the polynucleotide labeling method itself is well known.
- To examine whether the test nucleic acid is present in the sample by immobilizing the test nucleic acid or its amplification product, hybridizing with the labeled probe, washing, and measuring the label bound to the solid phase Can do.
- the polynucleotide for cancer detection bound to the solid phase is also called a probe.
- a method for measuring a test nucleic acid using a polynucleotide probe is also well known in this field, and the polynucleotide probe is brought into contact with the test nucleic acid at or near Tm (preferably within ⁇ 4 ° C.) in a buffer solution. And washing, and then measuring the template nucleic acid bound to the hybridized labeled probe or solid phase probe.
- Tm preferably within ⁇ 4 ° C.
- Such methods include well-known methods such as Northern blot, in situ hybridization, Southern blot. In the present invention, any known method can be applied.
- pancreatic cancer In the detection method of the present invention, whether or not the subject animal (also referred to as “subject”) suffers from pancreatic cancer is determined based on the presence or amount of (expression) of CAPRIN-1 measured as described above. evaluate. Pancreatic cancer can be detected only by measuring the presence or amount of (expression) of CAPRIN-1 in the target animal, but from the viewpoint of improving the detection accuracy, CAPRIN-1 in one or more healthy human samples can be detected. It is preferable to examine the expression level (antibody level, polypeptide level or mRNA level) to obtain a healthy person reference value and compare the measured value of the target animal with the healthy person reference value.
- the CAPRIN-1 expression level is examined for samples obtained from a large number of patients known to suffer from pancreatic cancer to obtain a pancreatic cancer patient reference value, and You may compare a measured value with both a healthy subject reference value and a pancreatic cancer patient reference value.
- the reference value can be determined, for example, by quantifying the CAPRIN-1 expression level in each sample and calculating the average value.
- the healthy subject reference value and the pancreatic cancer patient reference value can be determined in advance by examining the CAPRIN-1 expression level for a large number of healthy subjects and pancreatic cancer patients. Therefore, when comparing with a reference value by the method of the present invention, a predetermined reference value may be used.
- diagnosis using other cancer antigens or cancer markers may be combined.
- the detection accuracy of pancreatic cancer can be further increased.
- another polypeptide that is highly expressed in cancer tissue may be used in combination as an antigen in the same manner as the above-described polypeptide. it can.
- pancreatic cancers expressing CAPRIN-1, pancreatic ductal cancer (Inductive pancreatic ductal carcinoma), pancreatic ductal carcinoma, pancreatic cancer Adenocarcinoma of cancer, acinar cell carcinoma (Acinar Cell Carcinoma), adenosquamous carcinoma (Adenosquamous Carcinoma), giant cell tumor (Giant cell Tumor) )), Mucinous cystadenocarcinoma (Mucinous cystic neoplasm (MCN)), pancreatic bud (Pancreatoblastoma), Serous cystadenocarcinoma, Solid papillary cancer (Solid-pseudopillary tumor (SPT)), Gastrinoma (Zollinger-Elsino-Golmonoma syndrome) Endocrine neoplasia 1 (Multiple Endocrine Neoplasia Type-1 (MEN
- Specimens used in the method of the present invention include blood, serum, plasma, ascites, pleural effusion and other body fluids, tissues, and cells.
- serum, plasma, ascites, pleural effusion, tissue sample and cell sample can be preferably used, and in the third method for measuring nucleic acid such as mRNA. Tissue samples and cell samples are preferred.
- polypeptides canine CAPRIN-1 of SEQ ID NO: 2 and its homologous factor, specific reactive partial polypeptide, specific reactive modified poly used as an antigen for immunological measurement in the first method
- the peptide and the specific reactive addition polypeptide can be provided as a pancreatic cancer detection reagent or a pancreatic cancer detection kit.
- the reagent may consist only of the above-mentioned polypeptide, and separately contains various additives useful for stabilizing the polypeptide, buffers necessary for measurement, secondary antibodies, enzyme substrates, and the like. You may go out.
- the reagent can also be provided in a state immobilized on a solid phase such as a plate or a membrane. Preferred examples of the polypeptide are as described above.
- An antibody or antigen-binding fragment thereof that reacts with CAPRIN-1 for antigen-antibody reaction which is used when immunologically measuring CAPRIN-1 itself in the second method, can also be provided as a pancreatic cancer detection reagent.
- the reagent for detecting pancreatic cancer in this case may also consist only of the antibody or antigen-binding fragment, and contains various additives useful for stabilizing the antibody or antigen-binding fragment. Also good.
- the antibody or antigen-binding fragment may be one obtained by binding a metal such as manganese or iron. When such a metal-binding antibody or antigen-binding fragment is administered into the body, a larger amount of the antibody or antigen-binding fragment accumulates at a site where a larger amount of antigen protein exists. The presence of pancreatic cancer cells that produce the protein can be detected.
- one or more of the above-mentioned polynucleotides for detecting pancreatic cancer used for measuring nucleic acids such as mRNA in the third method can also be provided as pancreatic cancer detection reagents or pancreatic cancer detection kits.
- the reagent for detecting pancreatic cancer may also consist of only the polynucleotide, and various additives useful for stabilizing the polynucleotide, buffers necessary for measurement, labels (for example, (Fluorescent label) etc. may be included separately.
- the polynucleotide for detecting pancreatic cancer contained in the reagent is preferably a primer or a probe.
- the conditions and preferred examples of the polynucleotide for detecting pancreatic cancer are as described above.
- RNA was extracted from testicular tissue of healthy dog by acid guanidinium-phenol-chloroform method (Acid guanidinium-Phenol-Chloroform method) PolyA RNA was purified using Oligotex-dT30 mRNA purification kit (Takara Shuzo) according to the protocol attached to the kit.
- a dog testis cDNA phage library was synthesized using the obtained mRNA (5 ⁇ g).
- the cDNA phage library was prepared using cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, ZAP-cDNA GigapackIII Gold Cloning Kit (manufactured by STRATAGENE) according to the protocol attached to the kit.
- the size of the prepared cDNA phage library was 7.73 ⁇ 10 5 pfu / ml.
- the membrane was recovered, immersed in TBS (10 mM Tris-HCl, 150 mM NaCl pH 7.5) containing 0.5% nonfat dry milk, and shaken at 4 ° C. overnight to suppress nonspecific reaction.
- TBS 10 mM Tris-HCl, 150 mM NaCl pH 7.5
- This filter was reacted with serum of a patient dog diluted 500 times at room temperature for 2 to 3 hours.
- the serum pretreatment method is as follows. That is, ⁇ ZAP Express phage into which no foreign gene was inserted was infected with host E. coli (XL1-BLue MRF ′), and then cultured overnight at 37 ° C. on NZY plate medium. Next, a buffer of 0.2 M NaHCO 3 pH 8.3 containing 0.5 M NaCl was added to the plate and allowed to stand at 4 ° C. for 15 hours, and then the supernatant was recovered as an E. coli / phage extract. Next, the recovered E.
- coli / phage extract was passed through an NHS-column (GE Healthcare Bio-Science) to immobilize the protein derived from E. coli / phage.
- Serum dog serum was passed through and reacted with this protein-immobilized column, and antibodies adsorbed to E. coli and phage were removed from the serum.
- the serum fraction passed through the column was diluted 500 times with TBS containing 0.5% nonfat dry milk, and this was used as an immunoscreening material.
- phagemid host Escherichia coli prepared to have an absorbance OD 600 of 1.0 and 10 ⁇ l of the purified phage solution were mixed and reacted at 37 ° C. for 15 minutes, and 50 ⁇ l was treated with ampicillin (final concentration 50 ⁇ g / ml).
- SOLR phagemid host Escherichia coli
- the purified plasmid was analyzed for the full-length insert sequence by the primer walking method using the T3 primer shown in SEQ ID NO: 31 and the T7 primer shown in SEQ ID NO: 32.
- the gene sequences described in SEQ ID NOs: 5, 7, 9, 11, and 13 were obtained by this sequence analysis.
- the homology search program BLAST search http://www.ncbi.nlm.nih.gov/BLAST/
- BLAST search http://www.ncbi.nlm.nih.gov/BLAST/
- sequence identity between this canine gene (any one of SEQ ID NOs: 5, 7, 9, 11, 13) and the gene encoding the human homologous factor is as follows: 94% nucleotide sequence and 98 amino acid sequence in the region translated into protein %Met.
- the base sequences of human homologous factors are shown in SEQ ID NOs: 1 and 3, and amino acid sequences are shown in SEQ ID NOs: 2 and 4.
- sequence identity between the obtained canine gene and the gene encoding the bovine homologous factor was 94% of the base sequence and 97% of the amino acid sequence in the region translated into protein.
- the base sequence of the bovine homologous factor is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16.
- sequence identity between the gene encoding the human homologous factor and the gene encoding the bovine homologous factor was 94% in the base sequence and 93-97% in the amino acid sequence in the region translated into the protein. Further, the sequence identity between the obtained canine gene and the gene encoding the equine homologous factor was 93% nucleotide sequence and 97% amino acid sequence in the region translated into protein.
- the base sequence of the equine homologous factor is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18.
- sequence identity between the gene encoding the human homologous factor and the gene encoding the equine homologous factor was 93% nucleotide sequence and 96% amino acid sequence in the region translated into protein.
- sequence identity between the obtained canine gene and the gene encoding the mouse homologous factor was 87 to 89% of the base sequence and 95 to 97% of the amino acid sequence in the region translated into the protein.
- the nucleotide sequence of the mouse homologous factor is shown in SEQ ID NO: 19, 21, 23, 25, 27, and the amino acid sequence is shown in SEQ ID NO: 20, 22, 24, 26, 28.
- the sequence identity between the gene encoding the human homologous factor and the gene encoding the mouse homologous factor was 89 to 91% of the base sequence and 95 to 96% of the amino acid sequence in the region translated into the protein.
- sequence identity between the obtained canine gene and the gene encoding the chicken homologous factor was 82% of the base sequence and 87% of the amino acid sequence in the region translated into protein.
- the base sequence of the chicken homologous factor is shown in SEQ ID NO: 29, and the amino acid sequence is shown in SEQ ID NO: 30.
- sequence identity between the gene encoding the human homologous factor and the gene encoding the chicken homologous factor was 81 to 82% of the base sequence and 86% of the amino acid sequence in the region translated into the protein.
- pancreatic cancer cell line Human normal tissue (mammary gland, brain, bone marrow, lung, esophagus, pancreas, testis), pancreatic cancer cell line 4 types (Capan) for the gene obtained by the above method -2, MIAPaCa-2, PANC-1, and BxPC-3) were examined by RT-PCR (Reverse Transcription-PCR) method.
- the reverse transcription reaction was performed as follows. That is, total RNA was extracted from 50 to 100 mg of each tissue and 5 to 10 ⁇ 10 6 cells of each cell line using TRIZOL reagent (manufactured by Invitrogen) according to the attached protocol.
- cDNA was synthesized according to the attached protocol by Superscript First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen).
- the PCR reaction was performed as follows using the obtained gene-specific primers (described in SEQ ID NOs: 33 and 34). That is, 0.25 ⁇ l of the sample prepared by the reverse transcription reaction, 2 ⁇ M each of the above primers, 0.2 mM of each dNTP, 0.65 U of ExTaq polymerase (Takara Shuzo Co., Ltd.), and each reagent and attached buffer were added to make a total volume of 25 ⁇ l.
- the cycle of 94 ° C./30 seconds, 60 ° C./30 seconds, and 72 ° C./30 seconds was repeated 30 times using a Thermal Cycler (manufactured by BIO RAD).
- the gene-specific primer was used to amplify a region of nucleotides 698 to 1124 in the nucleotide sequence of SEQ ID NO: 1 (human CAPRIN-1 gene).
- GAPDH specific primers (described in SEQ ID NOs: 35 and 36) were also used.
- Each organ was cut in PBS and fixed at reflux overnight with 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde (PFA). The reflux solution is discarded, and the tissue surface of each organ is rinsed with PBS.
- a PBS solution containing 10% sucrose is placed in a 50 ml centrifuge tube, and each tissue is placed therein and shaken at 4 ° C. for 2 hours using a rotor. That ’s it.
- the solution was replaced with a PBS solution containing 20% sucrose, allowed to stand at 4 ° C. until the tissue subsided, then replaced with a PBS solution containing 30% sucrose, and allowed to stand at 4 ° C. until the tissue subsided.
- the tissue was removed and the necessary part was cut out with a scalpel.
- an OCT compound manufactured by Tissue Tek
- Cryomold is placed on dry ice and rapidly frozen, then sliced into 10-20 ⁇ m using a cryostat (LEICA) and air-dried with a hair dryer for 30 minutes together with the slide glass, and the sliced tissue is placed.
- a slide glass was prepared.
- an operation of replacing PBS-T every 5 minutes in a staining bottle filled with PBS-T (physiological saline containing 0.05% Tween 20) was performed three times.
- the mouse tissue was MOM mouse Ig blocking reagent (VECTASTAIN), and the dog tissue was 10% fetal bovine serum.
- Each PBS-T solution was placed and allowed to stand for 1 hour at room temperature on a moist chamber.
- a monoclonal antibody prepared in Example 3 described later which reacts with the surface of cancer cells, the monoclonal antibody against CAPRIN-1 having a heavy chain variable region of SEQ ID NO: 55 and a light chain variable region of SEQ ID NO: 56
- a solution prepared by adding the antibody (monoclonal antibody # 8) to a concentration of 10 ⁇ g / ml with a blocking solution was placed thereon and allowed to stand overnight at 4 ° C. in a moist chamber. After washing with PBS-T three times for 10 minutes, MOM biotin-labeled anti-IgG antibody (VECTASTAIN) diluted 250 times with a blocking solution was placed thereon, and allowed to stand in a moist chamber at room temperature for 1 hour.
- VECTASTAIN MOM biotin-labeled anti-IgG antibody
- Example 2 Production of canine and human CAPRIN-1 protein (1) Production of recombinant protein Based on the gene of SEQ ID NO: 5 obtained in Example 1, a recombinant protein was produced by the following method. For PCR, 1 ⁇ l of a vector prepared from the phagemid solution obtained in Example 1 and subjected to sequence analysis, 2 types of primers containing NdeI and KpnI restriction enzyme cleavage sequences (described in SEQ ID NOs: 37 and 38) were each treated with 0.
- Reagents and attached buffers were added to give 4 ⁇ M, 0.2 mM dNTP, 1.25 U PrimeSTAR HS polymerase (Takara Shuzo) to make a total volume of 50 ⁇ l, and a thermal cycler (BIO RAD) was used at 98 ° C. / This was carried out by repeating the cycle of 10 seconds at 68 ° C./1.5 minutes 30 times.
- the above two kinds of primers amplify the region encoding the entire amino acid sequence of SEQ ID NO: 6 (canine CAPRIN-1). After PCR, the amplified DNA was electrophoresed on a 1% agarose gel, and a DNA fragment of about 1.4 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
- the purified DNA fragment was ligated to a cloning vector pCR-Blunt (manufactured by Invitrogen).
- the plasmid was recovered after transformation into E. coli, and it was confirmed by sequencing that the amplified gene fragment matched the target sequence.
- the plasmid corresponding to the target sequence was treated with NdeI and KpnI restriction enzymes, purified with QIAquick Gel Extraction Kit, and then inserted into the expression vector pET30b (manufactured by Novagen) for Escherichia coli treated with NdeI and KpnI restriction enzymes. .
- This vector a His-tagged recombinant protein can be produced.
- This plasmid was transformed into E. coli BL21 (DE3) for expression, and the target protein was expressed in E. coli by inducing expression with 1 mM IPTG.
- a canine homologous gene recombinant protein was prepared by the following method.
- 1 ⁇ l of cDNA whose expression by RT-PCR method was confirmed from various tissue and cell cDNAs prepared in Example 1 and two kinds of primers containing NdeI and KpnI restriction enzyme cleavage sequences (described in SEQ ID NOs: 39 and 40).
- the purified DNA fragment was ligated to a cloning vector pCR-Blunt (manufactured by Invitrogen). After transforming this into E. coli, the plasmid was recovered, and it was confirmed by sequencing that the amplified gene fragment matched the target sequence.
- the plasmid corresponding to the target sequence was treated with NdeI and KpnI restriction enzymes, purified with QIAquick Gel Extraction Kit, and then inserted into the expression vector pET30b (manufactured by Novagen) for Escherichia coli treated with NdeI and KpnI restriction enzymes. .
- This vector a His-tagged recombinant protein can be produced.
- This plasmid was transformed into E. coli BL21 (DE3) for expression, and the target protein was expressed in E. coli by inducing expression with 1 mM IPTG.
- a recombinant protein of human homologous gene was prepared by the following method. PCR was performed using 1 ⁇ l of cDNA that could be confirmed by RT-PCR method from various tissue and cell cDNAs prepared in Example 1, and two kinds of primers containing SacI and XhoI restriction enzyme cleavage sequences (described in SEQ ID NOs: 41 and 42). ) To 0.4 ⁇ M, 0.2 mM dNTP, 1.25 U PrimeSTAR HS polymerase (Takara Shuzo), add each reagent and attached buffer to make the total volume 50 ⁇ l, and use Thermal Cycler (BIO RAD).
- the purified DNA fragment was ligated to a cloning vector pCR-Blunt (manufactured by Invitrogen). After transforming this into E. coli, the plasmid was recovered, and it was confirmed by sequencing that the amplified gene fragment matched the target sequence.
- a plasmid corresponding to the target sequence was treated with SacI and XhoI restriction enzymes, purified with QIAquick Gel Extraction Kit, and then inserted into an expression vector pET30a (manufactured by Novagen) for Escherichia coli treated with SacI and XhoI restriction enzymes. .
- This vector a His-tagged recombinant protein can be produced.
- This plasmid was transformed into E. coli BL21 (DE3) for expression, and the target protein was expressed in E. coli by inducing expression with 1 mM IPTG.
- Each of the recombinant Escherichia coli expressing the genes of SEQ ID NOs: 1, 5, and 7 obtained above has an absorbance at 600 nm of about 0.7 in an LB medium containing 30 ⁇ g / ml kanamycin. After culturing at 37 ° C. until it reached a final concentration, isopropyl- ⁇ -D-1-thiogalactopyranoside was added to a final concentration of 1 mM, followed by culturing at 37 ° C. for 4 hours. Thereafter, the cells were collected by centrifugation at 4800 rpm for 10 minutes. The cell pellet was suspended in phosphate buffered saline, and further centrifuged at 4800 rpm for 10 minutes to wash the cell.
- the cells were suspended in phosphate buffered physiological saline and sonicated on ice.
- the E. coli sonication solution was centrifuged at 6000 rpm for 20 minutes, and the resulting supernatant was used as a soluble fraction and the precipitate was used as an insoluble fraction.
- the soluble fraction was applied to a nickel chelate column (carrier: Chelating Sepharose (trademark) Fast Flow (GE Health Care), column volume 5 mL, 50 mM hydrochloric acid buffer (pH 8.0) as an equilibration buffer) prepared according to a conventional method. Added. The unadsorbed fraction was washed with 10 mM column volume of 50 mM hydrochloric acid buffer (pH 8.0) and 20 mM imidazole-containing 20 mM phosphate buffer (pH 8.0), and immediately, 100 mM imidazole-containing 20 mM. Six beds were eluted with a phosphate buffer (pH 8.0).
- Example 3 Production of antibody against CAPRIN-1 (1) Production of polyclonal antibody against CAPRIN-1-derived peptide
- a CAPRIN-1-derived peptide (Arg-Asn-Leu- Glu-Lys-Lys-Lys-Gly-Lys-Leu-Asp-Asp-Tyr-Gln (SEQ ID NO: 43)) was synthesized. 1 mg of this peptide was mixed with an equal volume of incomplete Freund's adjuvant (IFA) solution as an antigen, and this was administered subcutaneously to rabbits 4 times every 2 weeks.
- IFA incomplete Freund's adjuvant
- the operation was repeated three times to obtain spleen cells.
- the obtained spleen cells and mouse myeloma cells SP2 / 0 (purchased from ATCC) were mixed at a ratio of 10: 1, and 200 ⁇ l of RPMI 1640 medium containing 10% FBS heated to 37 ° C. and PEG 1500 (Boehringer) PEG solution prepared by mixing 800 ⁇ l was added and allowed to stand for 5 minutes for cell fusion.
- the cells were suspended in 150 ml of RPMI 1640 medium (HAT selective medium) containing 15% FBS to which 2% equivalent of Gibco's HAT solution was added, and a 96-well plate (NUNC) 100 ⁇ l per well of (made) was seeded on 15 plates.
- RPMI 1640 medium HAT selective medium
- NUNC 96-well plate
- Hybridomas were selected using as an index the binding affinity of the antibody produced by the prepared hybridomas to the CAPRIN-1 protein.
- 100 ⁇ l of 1 ⁇ g / ml of the CAPRIN-1 protein solution prepared in Example 2 was added per well of a 96-well plate and allowed to stand at 4 ° C. for 18 hours. After washing each well 3 times with PBS-T, 400 ⁇ l of 0.5% Bovine Serum Albumin (BSA) solution (manufactured by Sigma) was added per well and allowed to stand at room temperature for 3 hours.
- BSA Bovine Serum Albumin
- hybridomas were added to the plate so that the number was 0.5 per well of the 96-well plate and cultured. One week later, hybridomas forming a single colony in the well were observed. The cells in these wells were further cultured, and hybridomas were selected using the binding affinity of the antibody produced by the cloned hybridomas to the CAPRIN-1 protein as an index. 100 ⁇ l of 1 ⁇ g / ml of the CAPRIN-1 protein solution prepared in Example 2 was added per well of a 96-well plate and allowed to stand at 4 ° C. for 18 hours. After washing each well 3 times with PBS-T, 400 ⁇ l of 0.5% BSA solution was added per well and allowed to stand at room temperature for 3 hours.
- the same operation as described above was performed using a medium added in place of the antibody as a control.
- 10 monoclonal antibodies (# 1 to # 10) having a higher fluorescence intensity than the control, that is, reacting with the cell surface of breast cancer cells were selected.
- the sequences of the heavy chain variable region and the light chain variable region of these monoclonal antibodies are shown in SEQ ID NOs: 44-60.
- the monoclonal antibody # 1 includes a heavy chain variable region of SEQ ID NO: 44 and a light chain variable region of SEQ ID NO: 45
- # 2 is a heavy chain variable region of SEQ ID NO: 44 and a light chain variable region of SEQ ID NO: 46.
- # 3 includes the heavy chain variable region of SEQ ID NO: 44 and the light chain variable region of SEQ ID NO: 47
- # 4 includes the heavy chain variable region of SEQ ID NO: 44 and the light chain variable region of SEQ ID NO: 48
- # 5 includes a heavy chain variable region of SEQ ID NO: 49 and a light chain variable region of SEQ ID NO: 50
- # 6 includes a heavy chain variable region of SEQ ID NO: 51 and a light chain variable region of SEQ ID NO: 52
- # 7 includes the heavy chain variable region of SEQ ID NO: 53 and the light chain variable region of SEQ ID NO: 54
- # 8 includes the heavy chain variable region of SEQ ID NO: 55 and the light chain variable region of SEQ ID NO: 56.
- # 9 contains the heavy chain variable region of SEQ ID NO: 57 and the light chain variable region of SEQ ID NO: 58
- # 10 comprises SEQ ID NO: 60 light chain variable region of: a heavy chain variable region of 59 and SEQ ID NO.
- DTT (manufactured by Fluka) was added to 100 ⁇ l of a recombinant CAPRIN-1 protein solution dissolved in PBS to a concentration of 1 ⁇ g / ⁇ l so as to have a final concentration of 10 mM, and reacted at 95 ° C. for 5 minutes to cause CAPRIN- Reduction of disulfide bonds in one protein was performed, and then iodoacetamide (manufactured by Wako Pure Chemical Industries, Ltd.) having a final concentration of 20 mM was added, and a thiol group alkylation reaction was performed at 37 ° C. under light-shielding conditions for 30 minutes.
- iodoacetamide manufactured by Wako Pure Chemical Industries, Ltd.
- each monoclonal antibody against # 1 to # 10 CAPRIN-1 was added, made up to 1 mL of 20 mM phosphate buffer (pH 7.0), and mixed by stirring. The reaction was allowed to proceed overnight at 4 ° C.
- trypsin manufactured by Promega
- 1% BSA manufactured by Sigma
- Protein A-glass beads blocked with PBS containing PBS and 1 mM calcium carbonate, NP-40 buffer (20 mM phosphate buffer (pH 7.4), 5 mM EDTA, 150 mM NaCl, 1% NP-40) and reacted for 30 minutes each.
- the antigen-antibody complex was eluted with 100 ⁇ l of 0.1% formic acid, and Q-TOF Premier (Waters-MicroMass) was used for the eluate. LC-MS analysis was performed using this. Analysis followed the protocol attached to the instrument.
- polypeptide of SEQ ID NO: 61 was identified as a partial sequence of CAPRIN-1 recognized by any of the monoclonal antibodies against CAPRIN-1 of # 1 to # 10. Furthermore, the peptide of SEQ ID NO: 62 was identified as a partial sequence in the polypeptide of SEQ ID NO: 61 recognized by the monoclonal antibodies # 1 to # 4, # 5 to # 7 and # 9, and the partial sequence peptide It was found that monoclonal antibody # 1 to # 4 recognize the peptide of SEQ ID NO: 63.
- Example 4 Diagnosis of pancreatic cancer using polypeptide of CAPRIN-1
- Diagnosis of pancreatic cancer in dog As a result of pathological diagnosis using excised tumor tissue, malignant pancreatic cancer was confirmed in the dog Blood was collected and serum was separated.
- the canine CAPRIN-1 protein (SEQ ID NO: 8) prepared in Example 2 and an anti-canine IgG antibody the IgG antibody titer in serum specifically reacting with the canine CAPRIN-1 protein was measured by ELISA.
- the prepared canine CAPRIN-1 protein was solid-phased by adding 100 ⁇ l / well of a recombinant protein solution diluted to 5 ⁇ g / mL with phosphate buffered saline to a 96-well immobilizer amino plate (manufactured by NUNK). It was allowed to stand overnight at 4 ° C. Blocking was performed by adding 100 ⁇ l / well of 50 mM sodium bicarbonate buffer solution (pH 8.4) (hereinafter blocking solution) containing 0.5% BSA (bovine serum albumin (bovine serum albumin), Sigma-Aldrich Japan). Shaking time.
- BSA bovine serum albumin
- the serum derived from these cancer-bearing dogs exhibited a higher antibody titer against the canine CAPRIN-1 protein than the control.
- Example 2 Diagnosis of human pancreatic cancer Using the human CAPRIN-1 protein (SEQ ID NO: 2) prepared in Example 2 and an anti-human IgG antibody, the IgG antibody titer in healthy human serum that specifically reacts with the polypeptide was measured.
- the solid phase of human CAPRIN-1 protein was prepared by adding 100 ⁇ l / well of a recombinant protein solution diluted to 100 ⁇ g / mL with phosphate buffered saline to a 96-well immobilizer amino plate (manufactured by Nunk). And left overnight.
- Blocking was performed by adding 100 ⁇ l / well of a solution prepared by dissolving 4 g of Block Ace powder (DS Pharma Biomedical Co., Ltd.) dissolved in 100 ml of purified water 4 times with purified water (hereinafter referred to as blocking solution), and shaking at room temperature for 1 hour. did. 100-fold dilution / well of a 1000-fold diluted serum using a blocking solution was added for dilution, and the reaction was performed by shaking at room temperature for 3 hours.
- Block Ace powder DS Pharma Biomedical Co., Ltd.
- HRP-modified anti-human IgG antibody HRP--washed 3 times with phosphate buffered saline (hereinafter PBS-T) containing 0.05% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) and diluted 10,000 times with blocking solution 100 ⁇ l / well of Goat Anti-Human IgG (H + L) Conjugate: manufactured by Zymed Laboratories) was added, and the reaction was performed by shaking at room temperature for 1 hour. After washing 3 times with PBS-T, 100 ⁇ l / well of HRP substrate TMB (1-Step Turbo TMB (tetramethylbenzidine), PIERCE) was added and allowed to react at room temperature for 30 minutes.
- HRP substrate TMB (1-Step Turbo TMB (tetramethylbenzidine), PIERCE
- pancreatic cancer can be detected by this technique even in humans.
- Example 5 Diagnosis of pancreatic cancer using antibody against CAPRIN-1 (1) Diagnosis of pancreatic cancer by measuring CAPRIN-1 protein CAPRIN-1-derived peptide (SEQ ID NO :) obtained in Example 3 (1) 43) and the CAPRIN-1 in Examples 4 (1) to (3) by sandwich ELISA using a combination of the polyclonal antibody against 43) and each monoclonal antibody obtained in Example 3 (2) against the CAPRIN-1 protein.
- the CAPRIN-1 protein itself contained in a specimen (serum derived from a cancer-bearing individual) that gave a positive reaction in cancer diagnosis using the protein was detected.
- a polyclonal antibody was used as the primary antibody, and each monoclonal antibody was used as the secondary antibody. The amount of the protein in serum that specifically reacts with each of the above antibodies was measured.
- cancer can be diagnosed or examined also in this technique of detecting CAPRIN-1 protein using an antibody against CAPRIN-1.
- a human pancreatic cancer tissue array was placed in a staining bottle filled with 10 mM citrate buffer (pH 6.0) containing 0.05% Tween 20, treated at 125 ° C. for 5 minutes, and allowed to stand at room temperature for 40 minutes or more. Excess water around the section was wiped off with Kimwipe, surrounded by DAKOPEN (made by DAKO), and a suitable amount of Peroxidase Block (made by DAKO) was dropped. After allowing to stand at room temperature for 5 minutes, it was placed in a staining bottle filled with PBS-T and replaced with PBS-T every 5 minutes three times.
- a blocking solution As a blocking solution, a PBS-T solution containing 10% FBS was placed and allowed to stand at room temperature for 1 hour in a moist chamber. Next, a solution prepared by adding 10 ⁇ g / ml of the monoclonal antibody # 1 to 10 prepared in Example 3 to a PBS-T solution containing 5% FBS was placed on the moist chamber at 4 ° C. overnight. After washing with PBS-T three times for 10 minutes, an appropriate amount of Peroxidase Labeled Polymer Conjugated (manufactured by DAKO) was dropped, and the mixture was allowed to stand at room temperature for 30 minutes in a moist chamber.
- DAKO Peroxidase Labeled Polymer Conjugated
- the results of immunohistochemical staining using antibody # 8 CAPRIN-1 was strongly expressed in 54 (54%) of 101 pancreatic cancer tissues.
- immunohistochemical staining was performed using a paraffin-embedded human normal tissue array (manufactured by BIOMAX) containing normal human pancreatic tissue. Excess water around the section was wiped off with Kimwipe, surrounded by DAKOPEN (made by DAKO), and a suitable amount of Peroxidase Block (made by DAKO) was dropped. After allowing to stand at room temperature for 5 minutes, it was placed in a staining bottle filled with PBS-T and replaced with PBS-T every 5 minutes three times.
- a blocking solution As a blocking solution, a PBS-T solution containing 10% FBS was placed and allowed to stand at room temperature for 1 hour in a moist chamber. Next, a solution prepared by adding 10 ⁇ g / ml of the monoclonal antibody # 1 to 10 prepared in Example 3 to a PBS-T solution containing 5% FBS was placed on the moist chamber at 4 ° C. overnight. After washing with PBS-T three times for 10 minutes, an appropriate amount of Peroxidase Labeled Polymer Conjugated (manufactured by DAKO) was added dropwise and allowed to stand at room temperature for 30 minutes in a moist chamber.
- DAKO Peroxidase Labeled Polymer Conjugated
- the present invention is industrially useful for diagnosis or detection of pancreatic cancer.
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Abstract
Description
(1)cDNAライブラリーの作製
健常な犬の精巣組織から酸グアニジウム-フェノール-クロロホルム法(Acid guanidium-Phenol-Chloroform法)により全RNAを抽出し、Oligotex-dT30 mRNA purification Kit(宝酒造社製)を用いてキット添付のプロトコールに従ってポリA RNAを精製した。
上記作製したイヌ精巣cDNAファージライブラリーを用いて、イムノスクリーニングを行った。具体的にはΦ90×15mmのNZYアガロースプレートに2210クローンとなるように宿主大腸菌(XL1-Blue MRF’)に感染させ、42℃、3~4時間培養し、溶菌斑(プラーク)を作らせ、IPTG(イソプロピル-β-D-チオガラクトシド)を浸透させたニトロセルロースメンブレン(Hybond C Extra: GE Healthecare Bio-Science社製)でプレートを37℃で4時間覆うことによりタンパク質を誘導・発現させ、メンブレンにタンパク質を転写した。その後メンブレンを回収し0.5% 脱脂粉乳を含むTBS(10mM Tris-HCl、150mM NaCl pH7.5)に浸し4℃で一晩振盪することによって非特異反応を抑制した。このフィルターを500倍希釈した患犬血清と室温で2~3時間反応させた。
上記方法により単離した5個の陽性クローンを塩基配列解析に供するため、ファージベクターからプラスミドベクターに転換する操作を行った。具体的には宿主大腸菌(XL1-Blue MRF’)を吸光度OD600が1.0となるよう調製した溶液200μlと、精製したファージ溶液250μlさらにExAssist helper phage(STRATAGENE社製)1μlを混合した後37℃で15分間反応後、LB培地を3ml添加し37℃で2.5~3時間培養を行い、直ちに70℃の水浴にて20分間保温した後、4℃、1000×g、15分間遠心を行い、上清をファージミド溶液として回収した。次いでファージミド宿主大腸菌(SOLR)を吸光度OD600が1.0となるよう調製した溶液200μlと、精製したファージ溶液10μlを混合した後37℃で15分間反応させ、50μlをアンピシリン(終濃度50μg/ml)含有LB寒天培地に播き37℃一晩培養した。トランスフォームしたSOLRのシングルコロニーを採取し、アンピシリン(終濃度50μg/ml)含有LB培地37℃にて培養後、QIAGEN plasmid Miniprep Kit(キアゲン社製)を使って目的のインサートを持つプラスミドDNAを精製した。
上記方法により得られた遺伝子に対しヒトの正常組織(乳腺、脳、骨髄、肺、食道、膵臓、精巣)、膵臓癌細胞株4種(Capan-2、MIAPaCa-2、PANC-1、BxPC-3)における発現をRT-PCR(Reverse Transcription-PCR)法により調べた。逆転写反応は以下の通り行なった。すなわち、各組織50~100mg及び各細胞株5~10×106個の細胞からTRIZOL試薬(invitrogen社製)を用いて添付のプロトコールに従い全RNAを抽出した。この全RNAを用いてSuperscript First-Strand Synthesis System for RT-PCR(invitrogen社製)により添付のプロトコールに従いcDNAを合成した。PCR反応は、取得した遺伝子特異的なプライマー(配列番号33及び34に記載)を用いて以下の通り行った。すなわち、逆転写反応により調製したサンプル0.25μl、上記プライマーを各2μM、0.2mM各dNTP、0.65UのExTaqポリメラーゼ(宝酒造社製)となるように各試薬と添付バッファーを加え全量を25μlとし、Thermal Cycler(BIO RAD社製)を用いて、94℃/30秒、60℃/30秒、72℃/30秒のサイクルを30回繰り返して行った。なお、上記遺伝子特異的プライマーは、配列番号1の塩基配列(ヒトCAPRIN-1遺伝子)中の698番~1124番塩基の領域を増幅するものであった。比較対照のため、GAPDH特異的なプライマー(配列番号35及び36に記載)も同時に用いた。ヒトCAPRIN-1遺伝子の発現を確認したところ、正常組織で発現が確認できたのは精巣のみだったが、膵臓癌細胞株では発現が検出された。この結果から、CAPRIN-1は精巣以外の正常組織では発現が見られず、一方、膵臓癌細胞株においても発現していることが確認された。
マウス(Balb/c、雌)およびイヌ(ビーグル犬、雌)をエーテル麻酔下およびケタミン/イソフルラン麻酔下で放血させ、開腹後、各臓器(胃、肝臓、眼球、胸腺、筋肉、骨髄、子宮、小腸、食道、心臓、腎臓、唾液腺、大腸、乳腺、脳、肺、皮膚、副腎、卵巣、膵臓、脾臓、膀胱)をそれぞれPBSの入った10cmディッシュに移した。PBS中で各臓器を切り開き、4% paraformaldehyde(PFA)を含む0.1Mリン酸緩衝液(pH7.4)で一晩還流固定した。還流液を捨て、PBSで各臓器の組織表面をすすぎ、10%ショ糖を含むPBS溶液を50ml容の遠心チューブに入れ、その中に各組織を入れて4℃で2時間ローターを用いて振とうした。20%ショ糖を含むPBS溶液に入れ替え、4℃で組織が沈むまで静置後、30%ショ糖を含むPBS溶液に入れ替え、4℃で組織が沈むまで静置した。組織を取り出し、必要な部分を手術用メスで切りだした。次に、OCTコンパウンド(Tissue Tek社製)をかけて組織表面になじませた後、クライオモルドに組織を配置した。ドライアイスの上にクライオモルドをおいて急速凍結させた後、クライオスタット(LEICA社製)を用いて10~20μmに薄切し、スライドガラスごとヘアードライアーで30分間風乾し、薄切組織がのったスライドガラスを作製した。次にPBS-T(0.05% Tween20を含む生理食塩水)を満たした染色瓶に入れて5分ごとにPBS-Tを入れ替える操作を3回行った。切片周囲の余分な水分をキムワイプでふき取り、DAKOPEN(DAKO社製)で囲んだ後、ブロッキング液として、マウス組織はMOMマウスIgブロッキング試薬(VECTASTAIN社製)を、イヌ組織は10%牛胎児血清を含むPBS-T溶液をそれぞれのせ、モイストチャンバー上で室温で1時間静置した。次に後述の実施例3で作製したモノクローナル抗体であって、癌細胞表面に反応する、配列番号:55の重鎖可変領域と配列番号:56の軽鎖可変領域を有するCAPRIN-1に対する該モノクローナル抗体(モノクローナル抗体#8)を、ブロッキング液で10μg/mlに調製した溶液をのせ、モイストチャンバー内で4℃下で一晩静置した。PBS-Tで10分間3回洗浄を行った後、ブロッキング液で250倍に希釈したMOMビオチン標識抗IgG抗体(VECTASTAIN社製)をのせ、モイストチャンバー内に室温で1時間静置した。PBS-Tで10分間3回洗浄を行った後、アビジンービオチンABC試薬(VECTASTAIN社製)をのせ、モイストチャンバー内で室温で5分間静置した。PBS-Tで10分間3回洗浄を行った後、DAB発色液(DAB 10mg+30%H2O2 10μl/0.05M Tris-HCl(pH7.6)50ml)をのせ、モイストチャンバー内に室温で30分間静置した。蒸留水でリンスし、ヘマトキシリン試薬(DAKO社製)を載せて室温で1分間静置後、蒸留水でリンスした。70%、80%、90%、95%、100%の各エタノール溶液に順番に1分間ずつ入れた後、キシレン中で一晩静置した。スライドガラスを取り出し、Glycergel Mounting Medium(DAKO社製)で封入後、観察を行った。その結果、CAPRIN-1は、唾液腺、腎臓、結腸、胃の各組織において細胞内で僅かに発現が認められたが、細胞表面での発現は認められず、また、その他の臓器由来の組織では全く発現が認められなかった。
(1)組換えタンパク質の作製
実施例1で取得した配列番号5の遺伝子を基に、以下の方法にて組換えタンパク質を作製した。PCRは、実施例1で得られたファージミド溶液より調製し配列解析に供したベクターを1μl、NdeI及びKpnI制限酵素切断配列を含む2種類のプライマー(配列番号37及び38に記載)を各0.4μM、0.2mM dNTP、1.25UのPrimeSTAR HSポリメラーゼ(宝酒造社製)となるように各試薬と添付バッファーを加え全量を50μlとし、Thermal Cycler(BIO RAD社製)を用いて、98℃/10秒、68℃/1.5分のサイクルを30回繰り返すことにより行った。なお、上記2種類のプライマーは、配列番号6(イヌCAPRIN-1)のアミノ酸配列全長をコードする領域を増幅するものであった。PCR後、増幅されたDNAを1%アガロースゲルにて電気泳動し、QIAquick Gel Extraction Kit(QIAGEN社製)を用いて約1.4kbpのDNA断片を精製した。
上記で得られた、配列番号1,5,7の遺伝子を発現するそれぞれの組換え大腸菌を30μg/mlカナマイシン含有LB培地にて600nmでの吸光度が0.7付近になるまで37℃で培養後、イソプロピル-β-D-1-チオガラクトピラノシド終濃度が1mMとなるよう添加し、37℃で4時間培養した。その後4800rpmで10分間遠心し集菌した。この菌体ペレットをリン酸緩衝化生理食塩水に懸濁し、さらに4800rpmで10分間遠心し菌体の洗浄を行った。
(1)CAPRIN-1由来ペプチドに対するポリクローナル抗体の作製
CAPRIN-1に結合する抗体を得るために、CAPRIN-1由来ペプチド(Arg-Asn-Leu-Glu-Lys-Lys-Lys-Gly-Lys-Leu-Asp-Asp-Tyr-Gln(配列番号43))を合成した。このペプチド1mgを抗原として、等容量の不完全フロイントアジュバント(IFA)溶液と混合し、これを2週間毎に4回、ウサギの皮下に投与を行った。その後血液を採取し、ポリクローナル抗体を含む抗血清を得た。さらにこの抗血清をプロテインG担体(GEヘルスケアバイオサイエンス社製)を用いて精製し、CAPRIN-1由来ペプチドに対するポリクローナル抗体を得た。次に得られたポリクローナル抗体の、癌細胞表面のCAPRIN-1に対する反応性を、乳癌細胞を用いて検討した。具体的には、106個のヒト乳癌細胞株MDA-MB-231Vを1.5ml容のミクロ遠心チューブにて遠心分離し、これに上記ポリクローナル抗体を含む0.1%牛胎児血清(FBS)入りPBS溶液を添加し、氷上で1時間静置した。PBSで洗浄した後、0.1%FBSを含むPBSで500倍希釈したFITC標識ヤギ抗マウスIgG抗体(インビトロジェン社製)を添加し、氷上で1時間静置した。PBSで洗浄後、ベクトンディッキンソン株式会社のFACSキャリバーにて蛍光強度を測定した。一方、上記と同様の操作を、ポリクローナル抗体の代わりに0.1%FBSを含むPBSを添加したものをコントロールとした。その結果、ポリクローナル抗体を処理した細胞は、コントロールの細胞に比べて蛍光強度が強く、従って、得られたポリクローナル抗体が乳癌細胞の細胞表面に結合するものであることが判った。
実施例2で調製した配列番号2に示される、抗原タンパク質(ヒトCAPRIN-1)100μgを等量のMPL+TDMアジュバント(シグマ社製)と混合し、これをマウス1匹当たりの抗原溶液とした。抗原溶液を6週齢のBalb/cマウス(日本SLC社製)の腹腔内に投与後、1週間毎にさらに3回投与を行った。最後の免疫から3日後に摘出した脾臓を滅菌した2枚のスライドガラスに挟んで擦り潰し、PBS(-)(日水社製)を用いて洗浄し1500rpmで10分間遠心して上清を除去する操作を3回繰り返して脾臓細胞を得た。得られた脾臓細胞とマウスミエローマ細胞SP2/0(ATCCから購入)とを10:1の比率にて混和し、そこに37℃に加温した10% FBSを含むRPMI1640培地200μlとPEG1500(ベーリンガー社製)800μlを混和して調製したPEG溶液を加えて5分間静置して細胞融合を行った。1700rpmで5分間遠心し、上清を除去後、ギブコ社製のHAT溶液を2%当量加えた15% FBSを含むRPMI1640培地(HAT選択培地)150mlで細胞を懸濁し、96穴プレート(ヌンク社製)の1ウェル当たり100μlずつ、プレート15枚に播種した。7日間、37℃、5%CO2条件で培養することで、脾臓細胞とミエローマ細胞が融合したハイブリドーマを得た。
上記で取得した、乳癌細胞の細胞表面に反応する#1~#10のCAPRIN-1に対するモノクローナル抗体を用いて、それらが認識するCAPRIN-1タンパク質中の部分配列の同定を行った。
(1)イヌの膵臓癌診断
摘出された腫瘍組織を用いた病理診断の結果、悪性の膵管癌と確定診断が患犬の血液を採取し、血清を分離した。実施例2で作製したイヌCAPRIN-1タンパク質(配列番号8)、抗イヌIgG抗体を用いてELISA法にてイヌCAPRIN-1タンパク質に特異的に反応する血清中のIgG抗体価を測定した。
実施例2で作製したヒトCAPRIN-1タンパク質(配列番号2)を用いて、上記と同様にして、ヒトCAPRIN-1タンパク質に反応する、イヌ血清中のIgG抗体価を測定した。健常犬血清を用いた検討結果では、上記と同様に450nmでの吸光度はほとんど検出されなかった。一方、(1)の膵臓癌の患犬の血清はコントロールよりも高いヒトCAPRIN-1タンパク質に対する抗体価を示した。
実施例2で作製したヒトCAPRIN-1タンパク質(配列番号2)と抗ヒトIgG抗体を用いて、該ポリペプチドに特異的に反応する健常人血清中のIgG抗体価を測定した。ヒトCAPRIN-1タンパク質の固相化は、リン酸緩衝化生理食塩水にて100μg/mLに希釈した組換えタンパク質溶液を96穴イモビライザーアミノプレート(ヌンク社製)に100μl/well添加し、4℃で一晩静置して行った。ブロッキングは、ブロックエース粉末(DSファーマバイオメディカル株式会社製)4gを精製水100mlに溶解した溶液を精製水で4倍希釈したもの(以下ブロッキング溶液)を100μl/well加え、室温で1時間振とうした。希釈にブロッキング溶液を用いた1000倍希釈血清を100μl/well添加し、室温で3時間振とうして反応させた。0.05%Tween20(和光純薬工業社製)含有リン酸緩衝化生理食塩水(以下PBS-T)で3回洗浄し、ブロッキング溶液にて10000倍希釈したHRP修飾抗ヒトIgG抗体(HRP-Goat Anti-Human IgG(H+L) Conjugate: Zymed Laboratories社製)を100μl/well加え、室温で1時間振とうして反応させた。PBS-Tで3回洗浄し、HRP基質 TMB(1-Step Turbo TMB(テトラメチルベンジジン)、PIERCE社)を100μl/well添加し、室温で30分間酵素基質反応させた。その後、0.5M硫酸溶液(シグマアルドリッチジャパン社製)を100μl/well加えて反応停止後、マイクロプレートリーダーにて450nmの吸光度測定を行った。ポジティブコントロールとしてリン酸緩衝化生理食塩水にて50μg/mlに調製した卵白アルブミン抗原を固相化したものを用いた。その結果、450nmでの吸光度は、卵白アルブミン抗原に対しては高い値を示した。一方、ヒトCAPRIN-1タンパク質に対してはゼロ(0)と全く検出されなかった。
(1)CAPRIN-1タンパク質を測定することによる膵臓癌診断
実施例3(1)で取得した、CAPRIN-1由来ペプチド(配列番号43)に対するポリクローナル抗体と実施例3(2)で取得した、CAPRIN-1タンパク質に対する各モノクローナル抗体を組み合わせて用いることによる、サンドイッチELISA法により、実施例4(1)~(3)においてCAPRIN-1タンパク質を用いた癌診断で陽性反応が得られた検体(担癌個体由来血清)中に含まれるCAPRIN-1タンパク質自体の検出を行った。1次抗体としてポリクローナル抗体を、2次抗体として各モノクローナル抗体を用いた。上記各抗体に特異的に反応する、血清中の該タンパク質量を測定した。
パラフィン包埋されたヒト膵臓癌組織アレイ(BIOMAX社製)の膵臓癌組織101検体を用いて、免疫組織化学染色を行った。ヒト膵臓癌組織アレイを60℃で3時間処理後、キシレンを満たした染色瓶に入れて5分ごとにキシレンを入れ替える操作を3回行った。次にキシレンの代わりにエタノールおよびPBS-Tで同様の操作を行った。0.05%Tween20を含む10mM クエン酸緩衝液(pH6.0)を満たした染色瓶にヒト膵臓癌組織アレイを入れ、125℃で5分間処理後、室温で40分以上静置した。切片周囲の余分な水分をキムワイプでふき取り、DAKOPEN(DAKO社製)で囲み、Peroxidase Block(DAKO社製)を適量滴下した。室温で5分間静置後、PBS-Tを満たした染色瓶に入れて5分ごとにPBS-Tを入れ替える操作を3回行った。ブロッキング液として、10%FBSを含むPBS-T溶液をのせ、モイストチャンバー内で室温で1時間静置した。次に実施例3で作製したモノクローナル抗体#1~10を5% FBSを含むPBS-T溶液で10μg/mlに調製した溶液をのせ、モイストチャンバー内に4℃で一晩静置した。PBS-Tで10分間3回洗浄を行った後、Peroxidase Labelled Polymer Conjugated(DAKO社製)適量滴下し、モイストチャンバー内で室温で30分間静置した。PBS-Tで10分間3回洗浄を行った後、DAB発色液(DAKO社製)をのせ、室温で10分程度静置した後、発色液を捨て、PBS-Tで10分間3回洗浄を行った後、蒸留水でリンスし、70%、80%、90%、95%、100%の各エタノール溶液に順番に1分間ずつ入れた後、キシレン中で一晩静置した。スライドガラスを取り出し、Glycergel Mounting Medium(DAKO社製)で封入後、観察を行った。その結果、いずれの抗体を用いても膵臓癌組織中の膵臓癌細胞膜および膵臓癌細胞内にCAPRIN-1の発現を確認することができ、例えば抗体#8を用いた免疫組織化学染色の結果では、CAPRIN-1は全膵臓癌組織101検体の内、54検体(54%)で強い発現が認められた。
同様の方法にてヒト膵臓正常組織を含むパラフィン包埋されたヒト正常組織アレイ(BIOMAX社製)を用いて、免疫組織化学染色を行った。切片周囲の余分な水分をキムワイプでふき取り、DAKOPEN(DAKO社製)で囲み、Peroxidase Block(DAKO社製)を適量滴下した。室温で5分間静置後、PBS-Tを満たした染色瓶に入れて5分ごとにPBS-Tを入れ替える操作を3回行った。ブロッキング液として、10%FBSを含むPBS-T溶液をのせ、モイストチャンバー内で室温で1時間静置した。次に実施例3で作製したモノクローナル抗体#1~10を5%FBSを含むPBS-T溶液で10μg/mlに調製した溶液をのせ、モイストチャンバー内に4℃で一晩静置した。PBS-Tで10分間3回洗浄を行った後、Peroxidase Labelled Polymer Conjugated(DAKO社製)適量滴下し、モイストチャンバー内で室温で30分間静置した。PBS-Tで10分間3回洗浄を行った後、DAB発色液(DAKO社製)をのせ、室温で10分程度静置した後、発色液を捨て、PBS-Tで10分間3回洗浄を行った後、蒸留水でリンスし、70%、80%、90%、95%、100%の各エタノール溶液に順番に1分間ずつ入れた後、キシレン中で一晩静置した。スライドガラスを取り出し、Glycergel Mounting Medium(DAKO社製)で封入後、観察を行った。その結果、いずれの抗体を用いても膵臓由来の正常組織は染色されず、CAPRIN-1の発現は認められなかった。
Claims (23)
- 被験体から分離された試料において、CAPRIN-1タンパク質に対する抗体と抗原抗体反応により特異的に結合する反応性を有するポリペプチド、又は該ポリペプチドをコードする核酸、の存在又は量を測定することを含む、膵臓癌の検出方法。
- 測定すべき前記ポリペプチドは、配列番号2~30のうち偶数の配列番号に示されるいずれかのアミノ酸配列からなるCAPRIN-1タンパク質、又は該CAPRIN-1タンパク質と85~90%又はそれ以上の配列同一性を有するアミノ酸配列からなるポリペプチドである、請求項1に記載の方法。
- 前記被験体が、ヒト又はイヌである、請求項1又は2に記載の方法。
- 前記被験体がイヌであり、測定すべき前記ポリペプチドは配列番号2~30のうち偶数の配列番号に示されるいずれかのアミノ酸配列を含む、請求項3に記載の方法。
- 前記被験体がイヌであり、測定すべき前記ポリペプチドは配列番号6、8、10、12又は14に示されるアミノ酸配列を含む、請求項4に記載の方法。
- 前記被験体がヒトであり、測定すべき前記ポリペプチドは配列番号2又は4に示されるアミノ酸配列を含む、請求項3に記載の方法。
- 前記ポリペプチドの存在又は量の測定は、前記試料に含まれ得る、測定すべき前記ポリペプチドに対し前記被験体内で誘導された抗体を免疫学的測定することにより行われる、請求項1~6のいずれか1項に記載の方法。
- 前記ポリペプチドをコードする核酸の存在又は量の測定は、前記試料中に含まれる、該ポリペプチドをコードする核酸を測定することにより行われる、請求項1~6のいずれか1項に記載の方法。
- 前記核酸の塩基配列又はその相補的配列中の15~19塩基又はそれ以上、或いは20~30塩基又はそれ以上、の部分配列と特異的にハイブリダイズするポリヌクレオチドを用いて、前記試料中の前記核酸の存在又は量を測定する、請求項8に記載の方法。
- 前記被験体がイヌであり、前記ポリヌクレオチドは配列番号5、7、9、11又は13に示される塩基配列又はその相補的配列中の15~19塩基又はそれ以上、或いは20~30塩基又はそれ以上、の部分配列と特異的にハイブリダイズするポリヌクレオチドである、請求項9に記載の方法。
- 前記被験体がヒトであり、前記ポリヌクレオチドは配列番号1又は3に示される塩基配列又はその相補的配列中の15~19塩基又はそれ以上、或いは20~30塩基又はそれ以上、の部分配列と特異的にハイブリダイズするポリヌクレオチドである、請求項9に記載の方法。
- 前記ポリペプチドの存在又は量の測定は、前記試料中に含まれる、該ポリペプチドを測定することにより行われる、請求項1~6のいずれか1項に記載の方法。
- 前記測定が免疫学的測定である、請求項12に記載の方法。
- 前記試料が血液、血清、血漿、腹水、胸水、組織又は細胞である、請求項1~14のいずれか1項に記載の方法。
- CAPRIN-1タンパク質に対して被験体内で誘導される抗体と抗原抗体反応により結合する反応性を有する1もしくは複数のポリペプチドを含む、膵臓癌を検出するための試薬又はキット。
- CAPRIN-1タンパク質に対する抗体と抗原抗体反応により結合する反応性を有し被験体内で産生されるポリペプチドと抗原抗体反応する1もしくは複数の抗体又はその抗原結合性断片を含む、膵臓癌を検出するための試薬又はキット。
- CAPRIN-1タンパク質が、配列番号2~30のうち偶数の配列番号に示されるいずれかのアミノ酸配列からなる、請求項15又は16記載の試薬又はキット。
- 前記ポリペプチドと抗原抗体反応する抗体又はその抗原結合性断片が、膵臓癌細胞の表面に結合する抗体又はその抗原結合性断片である、請求項16又は17記載の試薬又はキット。
- 前記ポリペプチドと抗原抗体反応する抗体又はその抗原結合性断片が、配列番号2~30のうち配列番号6および配列番号18を除く偶数の配列番号で表されるいずれかのアミノ酸配列中のアミノ酸残基番号50~98又はアミノ酸残基番号233~344の領域内の連続する7~12個又はそれ以上のアミノ酸配列からなるポリペプチドと免疫学的反応性を有する抗体又はその断片を含むことを特徴とする、請求項16~18のいずれか1項記載の試薬又はキット。
- 前記ポリペプチドと抗原抗体反応する抗体又はその抗原結合性断片が、配列番号43のアミノ酸配列からなるポリペプチドに結合する抗体又はその抗原結合性断片、配列番号44と45のアミノ酸配列を含むモノクローナル抗体又はその抗原結合性断片、配列番号44と46のアミノ酸配列を含むモノクローナル抗体又はその抗原結合性断片、配列番号44と47のアミノ酸配列を含むモノクローナル抗体又はその抗原結合性断片、配列番号44と48のアミノ酸配列を含むモノクローナル抗体又はその抗原結合性断片、配列番号49と50のアミノ酸配列を含むモノクローナル抗体又はその抗原結合性断片、配列番号51と52のアミノ酸配列を含むモノクローナル抗体又はその抗原結合性断片、配列番号53と54のアミノ酸配列を含むモノクローナル抗体又はその抗原結合性断片、配列番号55と56のアミノ酸配列を含むモノクローナル抗体又はその抗原結合性断片、配列番号57と58のアミノ酸配列を含むモノクローナル抗体又はその抗原結合性断片、及び配列番号59と60のアミノ酸配列を含むモノクローナル抗体又はその抗原結合性断片からなる群から選択される1もしくは複数の抗体又はその抗原結合性断片である、請求項16~19のいずれか1項に記載の試薬又はキット。
- 配列番号1~29のうち奇数の配列番号に示されるかつCAPRIN-1タンパク質をコードするいずれかの塩基配列又はその相補的配列中の15~19塩基又はそれ以上、或いは20~30塩基又はそれ以上、の部分配列と特異的にハイブリダイズする1もしくは複数のポリヌクレオチドを含む、膵臓癌を検出するための試薬又はキット。
- 請求項15~21のいずれか1項に記載の少なくとも1つの試薬又はキットを用いて、被験体の試料中の、CAPRIN-1タンパク質、CAPRIN-1タンパク質に対する抗体、もしくはCAPRIN-1タンパク質をコードする核酸、の存在又は量を測定することを含む、膵臓癌の検出方法。
- 請求項1~14のいずれか1項に記載の方法において、請求項15~21のいずれか1項に記載の少なくとも1つの試薬又はキットを用いて、被験体の試料中の、CAPRIN-1タンパク質、CAPRIN-1タンパク質に対する抗体、もしくはCAPRIN-1タンパク質をコードする核酸、の存在又は量を測定することを含む、膵臓癌の検出方法。
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DK12819473.5T DK2741085T3 (en) | 2011-08-04 | 2012-08-03 | Method for detecting pancreatic cancer |
RU2014108048A RU2624040C2 (ru) | 2011-08-04 | 2012-08-03 | Способ обнаружения рака поджелудочной железы |
KR1020147005905A KR101984554B1 (ko) | 2011-08-04 | 2012-08-03 | 췌장암의 검출 방법 |
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US14/236,807 US9796775B2 (en) | 2011-08-04 | 2012-08-03 | Method for detecting pancreatic cancer |
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Cited By (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013125630A1 (ja) * | 2012-02-21 | 2013-08-29 | 東レ株式会社 | 癌の治療及び/又は予防用医薬組成物 |
WO2013147176A1 (ja) * | 2012-03-30 | 2013-10-03 | 東レ株式会社 | 胆嚢癌の治療及び/又は予防用医薬組成物 |
WO2013147169A1 (ja) * | 2012-03-30 | 2013-10-03 | 東レ株式会社 | 肝臓癌の治療及び/又は予防用医薬組成物 |
US8709418B2 (en) | 2010-02-04 | 2014-04-29 | Toray Industries, Inc. | Pharmaceutical composition for treating CAPRIN-1 expressing cancer |
US8828398B2 (en) | 2010-02-04 | 2014-09-09 | Toray Industries, Inc. | Pharmaceutical composition for treating and/or preventing cancer |
US8911740B2 (en) | 2010-02-04 | 2014-12-16 | Toray Industries, Inc. | Pharmaceutical composition for treating and/or preventing cancer |
US8937160B2 (en) | 2010-02-04 | 2015-01-20 | Toray Industries, Inc. | Pharmaceutical composition for treating and/or preventing cancer |
US9115200B2 (en) | 2010-02-04 | 2015-08-25 | Toray Industries, Inc. | Pharmaceutical composition for treating cancer using a monoclonal antibody having immunological reactivity with CAPRIN-1 |
US9175074B2 (en) | 2011-08-04 | 2015-11-03 | Toray Industries, Inc. | Pharmaceutical composition for treatment and/or prophylaxis of cancer |
US9180188B2 (en) | 2011-08-04 | 2015-11-10 | Toray Industries, Inc. | Pharmaceutical composition for treatment and/or prophylaxis of cancer |
US9181348B2 (en) | 2011-08-04 | 2015-11-10 | Toray Industries, Inc. | Pharmaceutical composition for treatment and/or prophylaxis of cancer |
US9181334B2 (en) | 2011-08-04 | 2015-11-10 | Toray Industries, Inc. | Pharmaceutical composition for treatment and/or prophylaxis of cancer |
US9180187B2 (en) | 2010-02-04 | 2015-11-10 | Toray Industries, Inc. | Medicament for treating and/or preventing cancer |
US9266958B2 (en) | 2012-02-21 | 2016-02-23 | Toray Industries, Inc. | Pharmaceutical composition for treatment and/or prevention of cancer |
US9273128B2 (en) | 2011-08-04 | 2016-03-01 | Toray Industries, Inc | Pharmaceutical composition for treatment and/or prophylaxis of cancer |
US9273130B2 (en) | 2012-02-21 | 2016-03-01 | Toray Industries, Inc. | Pharmaceutical composition for treatment and/or prevention of cancer |
US9409993B2 (en) | 2011-08-04 | 2016-08-09 | Toray Industries, Inc. | Pharmaceutical composition for treatment and/or prevention of pancreatic cancer |
US9416192B2 (en) | 2008-08-05 | 2016-08-16 | Toray Industries, Inc. | Pharmaceutical composition for treatment and prevention of cancers |
US9573993B2 (en) | 2012-02-21 | 2017-02-21 | Toray Industries, Inc. | Pharmaceutical composition for treatment of cancer comprising an anti-CAPRIN-1 peptide antibody |
US9753038B2 (en) | 2012-07-19 | 2017-09-05 | Toray Industries, Inc. | Method for detecting cancer via measurement of caprin-1 expression level |
US9772332B2 (en) | 2012-07-19 | 2017-09-26 | Toray Industries, Inc. | Method for detecting CAPRIN-1 in a biological sample |
US9796775B2 (en) | 2011-08-04 | 2017-10-24 | Toray Industries, Inc. | Method for detecting pancreatic cancer |
US9862774B2 (en) | 2013-08-09 | 2018-01-09 | Toray Industries, Inc. | Pharmaceutical composition for treatment and/or prevention of cancer |
RU2712921C1 (ru) * | 2019-08-12 | 2020-02-03 | Федеральное государственное бюджетное учреждение "Ростовский научно-исследовательский онкологический институт" Министерства здравоохранения Российской Федерации | Способ индивидуального прогнозирования сроков прогрессирования плоскоклеточного рака пищевода II-III стадии после хирургического лечения |
US11137401B2 (en) | 2008-08-05 | 2021-10-05 | Toray Industries, Inc. | Method for detecting cancer using CAPRIN-1 as a marker |
WO2022138460A1 (ja) * | 2020-12-24 | 2022-06-30 | ミナリスメディカル株式会社 | Dupan-2抗原の測定試薬及び測定キット |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2612082C1 (ru) * | 2015-10-14 | 2017-03-02 | Федеральное государственное бюджетное учреждение "Ростовский научно-исследовательский онкологический институт" Министерства здравоохранения Российской Федерации | Способ прогнозирования направленности патологического процесса при раке головки поджелудочной железы |
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KR20220004634A (ko) | 2019-03-15 | 2022-01-11 | 볼트 바이오테라퓨틱스 인코퍼레이티드 | Her2를 표적으로 하는 면역접합체 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080107668A1 (en) * | 2006-08-30 | 2008-05-08 | Immunotope, Inc. | Cytotoxic t-lymphocyte-inducing immunogens for prevention, treatment, and diagnosis of cancer |
WO2010016527A1 (ja) * | 2008-08-05 | 2010-02-11 | 東レ株式会社 | 癌の検出方法 |
Family Cites Families (79)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2161042C2 (ru) | 1994-09-19 | 2000-12-27 | Дж. Моро Рикардо | Обнаружение и лечение рака |
US5698396A (en) | 1995-06-07 | 1997-12-16 | Ludwig Institute For Cancer Research | Method for identifying auto-immunoreactive substances from a subject |
US20030118599A1 (en) | 1999-04-02 | 2003-06-26 | Corixa Corporation | Compositions and methods for the therapy and diagnosis of lung cancer |
RU2234942C2 (ru) | 1998-07-14 | 2004-08-27 | Корикса Корпорейшн | Выделенный опухолевый полипептид предстательной железы и кодирующий его полинуклеотид |
CA2334038A1 (en) | 1998-07-14 | 2000-01-27 | Corixa Corporation | Compositions and methods for therapy and diagnosis of prostate cancer |
GB9815909D0 (en) | 1998-07-21 | 1998-09-16 | Btg Int Ltd | Antibody preparation |
US6969518B2 (en) | 1998-12-28 | 2005-11-29 | Corixa Corporation | Compositions and methods for the therapy and diagnosis of breast cancer |
US6335170B1 (en) | 1999-02-22 | 2002-01-01 | Torben F. Orntoft | Gene expression in bladder tumors |
US6444425B1 (en) | 1999-04-02 | 2002-09-03 | Corixa Corporation | Compounds for therapy and diagnosis of lung cancer and methods for their use |
AU4185100A (en) | 1999-04-02 | 2000-10-23 | Corixa Corporation | Compounds for therapy and diagnosis of lung cancer and methods for their use |
US6949245B1 (en) | 1999-06-25 | 2005-09-27 | Genentech, Inc. | Humanized anti-ErbB2 antibodies and treatment with anti-ErbB2 antibodies |
JP2003528587A (ja) | 1999-10-29 | 2003-09-30 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | 27個のヒト分泌タンパク質 |
US20020006404A1 (en) | 1999-11-08 | 2002-01-17 | Idec Pharmaceuticals Corporation | Treatment of cell malignancies using combination of B cell depleting antibody and immune modulating antibody related applications |
AU2001261007A1 (en) | 2000-03-29 | 2001-10-08 | Corixa Corporation | Compositions and methods for the therapy and diagnosis of lung cancer |
US7919467B2 (en) | 2000-12-04 | 2011-04-05 | Immunotope, Inc. | Cytotoxic T-lymphocyte-inducing immunogens for prevention, treatment, and diagnosis of cancer |
US7408041B2 (en) | 2000-12-08 | 2008-08-05 | Alexion Pharmaceuticals, Inc. | Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof |
US20040029114A1 (en) | 2001-01-24 | 2004-02-12 | Eos Technology, Inc. | Methods of diagnosis of breast cancer, compositions and methods of screening for modulators of breast cancer |
RU2306952C2 (ru) | 2001-01-31 | 2007-09-27 | Байоджен Айдек Инк. | Лечение в-клеточных злокачественных опухолей с использованием комбинации применений, связанных с антителами, уменьшающими количество b-клеток, и с иммуномодулирующими антителами |
US20040236091A1 (en) | 2001-03-28 | 2004-11-25 | Chicz Roman M. | Translational profiling |
WO2002083070A2 (en) | 2001-04-10 | 2002-10-24 | Corixa Corporation | Compounds for immunotherapy and diagnosis of colon cancer and methods for their use |
EP1516049A4 (en) | 2001-05-11 | 2006-01-11 | Corixa Corp | COMPOSITIONS AND METHODS FOR THE TREATMENT AND DIAGNOSIS OF LUNG CANCER |
US20030190640A1 (en) | 2001-05-31 | 2003-10-09 | Mary Faris | Genes expressed in prostate cancer |
JP2004535202A (ja) | 2001-07-17 | 2004-11-25 | リサーチ ディベロップメント ファンデーション | アポトーシス促進性蛋白質を含む治療剤 |
RU2319709C2 (ru) | 2001-07-17 | 2008-03-20 | Рисерч Дивелопмент Фаундейшн | Терапевтические агенты, содержащие проапоптозные белки |
US20040142325A1 (en) | 2001-09-14 | 2004-07-22 | Liat Mintz | Methods and systems for annotating biomolecular sequences |
EP2388265A1 (en) | 2002-02-22 | 2011-11-23 | Genentech, Inc. | Compositions and methods for the treatment of immune related diseases |
US20050003390A1 (en) | 2002-05-17 | 2005-01-06 | Axenovich Sergey A. | Targets for controlling cellular growth and for diagnostic methods |
US20040126379A1 (en) | 2002-08-21 | 2004-07-01 | Boehringer Ingelheim International Gmbh | Compositions and methods for treating cancer using cytotoxic CD44 antibody immunoconjugates and chemotherapeutic agents |
WO2004047728A2 (en) | 2002-11-26 | 2004-06-10 | Genentech, Inc. | Compositions and methods for the treatment of immune related diseases |
WO2004076682A2 (en) | 2003-02-26 | 2004-09-10 | Surromed, Inc. | Targets for controlling cellular growth and for diagnostic methods |
EP1629119A2 (en) | 2003-04-29 | 2006-03-01 | Wyeth | Methods for diagnosing aml and mds by differential gene expression |
US20050009067A1 (en) | 2003-05-19 | 2005-01-13 | Craig Logsdon | Expression profile of pancreatic cancer |
CA2528669A1 (en) | 2003-06-09 | 2005-01-20 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
US20070048738A1 (en) | 2003-07-14 | 2007-03-01 | Mayo Foundation For Medical Education And Research | Methods and compositions for diagnosis, staging and prognosis of prostate cancer |
US20050032113A1 (en) | 2003-07-17 | 2005-02-10 | Mitsubishi Pharma Corporation | Membrane protein library for proteome analysis and method for preparing same |
EP1711525A2 (en) | 2004-01-26 | 2006-10-18 | Debiovision Inc. | Neoplasm-specific polypeptides and their uses |
PL1735348T3 (pl) | 2004-03-19 | 2012-11-30 | Imclone Llc | Ludzkie przeciwciało przeciwko receptorowi naskórkowego czynnika wzrostu |
WO2005100998A2 (en) | 2004-04-16 | 2005-10-27 | Europroteome Ag | Membrane markers for use in cancer diagnosis and therapy |
WO2006002378A2 (en) | 2004-06-23 | 2006-01-05 | Avalon Pharmaceuticals | Determining cancer-linked genes and therapeutic targets using molecular cytogenetic methods |
DK1839662T3 (da) | 2005-01-19 | 2010-05-10 | Zeria Pharm Co Ltd | Antitumormiddel |
CN102580084B (zh) | 2005-01-21 | 2016-11-23 | 健泰科生物技术公司 | Her抗体的固定剂量给药 |
JP5006802B2 (ja) | 2005-02-18 | 2012-08-22 | チルドレンズ メディカル センター コーポレイション | 上皮由来の癌診断および予後診断のためのバイオマーカーとしてのCyr61 |
CA2602088C (en) | 2005-03-11 | 2021-07-27 | Ciphergen Biosystems, Inc. | Biomarkers for ovarian cancer and endometrial cancer |
JP2006316040A (ja) | 2005-05-13 | 2006-11-24 | Genentech Inc | Herceptin(登録商標)補助療法 |
US8014957B2 (en) | 2005-12-15 | 2011-09-06 | Fred Hutchinson Cancer Research Center | Genes associated with progression and response in chronic myeloid leukemia and uses thereof |
EP2586454B1 (en) * | 2006-01-05 | 2014-10-08 | The Ohio State University Research Foundation | MicroRNA expressions abnormalities in pancreatic endocrine and acinar tumors |
CA2642342A1 (en) * | 2006-02-14 | 2007-08-23 | Dana-Farber Cancer Institute, Inc. | Compositions, kits, and methods for identification, assessment, prevention, and therapy of cancer |
WO2008073162A2 (en) | 2006-08-17 | 2008-06-19 | Cell Signaling Technology, Inc. | Lysine acetylation sites |
WO2008031041A2 (en) | 2006-09-07 | 2008-03-13 | H. Lee Moffitt Cancer Center And Research Institute, Inc. | Melanoma gene signature |
WO2008059252A2 (en) | 2006-11-15 | 2008-05-22 | Oxford Biomedica (Uk) Limited | Methods and composition fro t cell receptors which recognize 5t4 antigen |
PT3106874T (pt) | 2007-10-25 | 2020-05-08 | Toray Industries | Método para a deteção de cancro |
JP5663834B2 (ja) | 2008-03-14 | 2015-02-04 | 東ソー株式会社 | 遺伝子組換え抗体の製造方法 |
EP3692988A3 (en) | 2008-03-18 | 2020-10-14 | Genentech, Inc. | Combinations of an anti-her2 antibody-drug conjugate and 5-fu, anti-vegf antibody, carboplatin or abt-869 and methods of use |
CN106039307A (zh) * | 2008-08-05 | 2016-10-26 | 东丽株式会社 | 用于治疗和预防癌症的药物组合物 |
MX348464B (es) | 2008-08-05 | 2017-06-14 | Toray Industries | Agente inductor de inmunidad. |
BR112012003686B1 (pt) | 2009-08-19 | 2021-09-21 | Merck Patent Gmbh | Anticorpos monoclonais, seu uso para detecção de complexos de integrina em material ffpe, e polinucleotídeo |
MY160680A (en) | 2009-09-22 | 2017-03-15 | Probiogen Ag | Process for producing molecules containing specialized glycan structures |
BR112012018951C8 (pt) | 2010-02-04 | 2020-06-23 | Toray Industries | anticorpo monoclonal, composição farmacêutica, combinação farmacêutica, uso de um anticorpo, uso de uma composição farmacêutica e uso de uma combinação farmacêutica |
CN102822335B (zh) | 2010-02-04 | 2015-09-30 | 东丽株式会社 | 癌的治疗和/或预防用药物组合物 |
CN102821788B (zh) | 2010-02-04 | 2016-11-16 | 东丽株式会社 | 癌的治疗和/或预防用药物组合物 |
KR101758117B1 (ko) | 2010-02-04 | 2017-07-14 | 도레이 카부시키가이샤 | 암의 치료 및/또는 예방용 의약 조성물 |
HUE030102T2 (en) | 2010-02-04 | 2017-04-28 | Toray Industries | A pharmaceutical composition comprising anti-caprin-1 antibodies for the treatment and / or prevention of cancer |
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AU2011284908B2 (en) | 2010-07-26 | 2015-05-21 | Centre National De La Recherche Scientifique (Cnrs) | Methods and compositions for liver cancer therapy |
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BR112014021103A2 (ja) * | 2012-02-21 | 2019-10-15 | Toray Industries, Inc | The medical treatment and/or the medicine constituent for prevention of cancer |
EP2818482B1 (en) * | 2012-02-21 | 2019-05-22 | Toray Industries, Inc. | Pharmaceutical composition for treatment of cancer |
PL2818483T3 (pl) * | 2012-02-21 | 2018-01-31 | Toray Industries | Kompozycja lecznicza do leczenia i/lub zapobiegania nowotworowi |
KR102052400B1 (ko) | 2012-03-30 | 2019-12-06 | 도레이 카부시키가이샤 | 담낭암의 치료 및/또는 예방용 의약 조성물 |
HUE036424T2 (hu) | 2012-03-30 | 2018-07-30 | Toray Industries | Gyógyászati készítmény májrák kezelésére és/vagy megelõzésére |
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-
2012
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- 2012-08-03 EP EP12819473.5A patent/EP2741085B1/en active Active
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080107668A1 (en) * | 2006-08-30 | 2008-05-08 | Immunotope, Inc. | Cytotoxic t-lymphocyte-inducing immunogens for prevention, treatment, and diagnosis of cancer |
WO2010016527A1 (ja) * | 2008-08-05 | 2010-02-11 | 東レ株式会社 | 癌の検出方法 |
Non-Patent Citations (1)
Title |
---|
See also references of EP2741085A4 * |
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Also Published As
Publication number | Publication date |
---|---|
RU2624040C2 (ru) | 2017-06-30 |
PT2741085T (pt) | 2017-06-30 |
CA2844033C (en) | 2021-07-27 |
EP2741085A1 (en) | 2014-06-11 |
US20140179558A1 (en) | 2014-06-26 |
RU2014108048A (ru) | 2015-09-10 |
DK2741085T3 (en) | 2017-06-19 |
KR20140048318A (ko) | 2014-04-23 |
JP6094220B2 (ja) | 2017-03-15 |
CA2844033A1 (en) | 2013-02-07 |
ES2629061T3 (es) | 2017-08-07 |
EP2741085A4 (en) | 2015-03-04 |
HUE033628T2 (en) | 2017-12-28 |
KR101984554B1 (ko) | 2019-05-31 |
US9796775B2 (en) | 2017-10-24 |
BR112014002616A2 (pt) | 2019-03-19 |
BR112014002616B1 (pt) | 2022-01-18 |
CN103718045B (zh) | 2016-08-17 |
JPWO2013018885A1 (ja) | 2015-03-05 |
MX2014001377A (es) | 2014-03-21 |
CN103718045A (zh) | 2014-04-09 |
AU2012290948B2 (en) | 2017-05-25 |
MX349907B (es) | 2017-08-18 |
AU2012290948A1 (en) | 2014-03-20 |
EP2741085B1 (en) | 2017-04-05 |
PL2741085T3 (pl) | 2017-09-29 |
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