JP2005295921A - Selective splicing variant of new bst1 and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new tumor marker antigen having high specificity to pancreatic cancer and to provide a method for detecting the new tumor marker. <P>SOLUTION: The BST1_ASV (splice variant of BAT1) gene and protein that do not exist in normal pancreatic tissue and are specifically expressed in cancerated pancreatic tissue or in a pancreatic cancer-derived cell are obtained. The method for confirming presence of crisis of pancreatic cancer comprises using the gene and protein as a pancreatic cancer specific tumor marker and confirming its presence. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel tumor marker substance that is specific for pancreatic cancer. The present invention also relates to an inspection method using the marker and a kit for the inspection. Furthermore, the present invention relates to a pharmaceutical composition for treating the cancer.

Recent advances in medical technology have greatly improved cancer treatment technology, and some cancers show a decreasing mortality rate. However, since early detection of the onset of cancer affects its therapeutic effect, establishment of a method that enables early detection of cancer is desired.
For cancer diagnosis, in addition to a method of visually capturing cancer tissue by ultrasound or X-ray examination, etc., a method of discovering abnormalities such as cell morphology of tissues collected from patients by immunohistological techniques, and identification Biochemical and immunochemical methods for detecting the dynamics of so-called tumor markers such as proteins or genes whose expression changes with the onset of cancer are known.

In these methods, the method of observing the morphology of the tissue by an immunohistological technique requires time for confirmation of the onset of cancer because it requires sampling of the tissue, and the observed morphology Therefore, considerable skill is required to determine the onset of cancer.
Therefore, the application of a method using a tumor marker, which can be relatively simple and objectively determine the presence or absence of cancer, has often been applied. However, when applying a method for detecting a tumor marker, the specificity of the marker for a particular cancer becomes very important. Many tumor markers used in the diagnosis of human cancer are also expressed to some extent in normal tissue cells. Therefore, it is often necessary to use fluctuations in the expression of the tumor marker as an indicator of canceration. There is always the possibility of misdiagnosis. In addition, currently used tumor markers, such as CEA and CA19-9, are produced by cells of many organs such as stomach, large intestine, pancreas and lung. It was difficult to determine which of the above organs had a cancer lesion.
Therefore, in order to more accurately and objectively detect the presence or absence of the onset of cancer, it is desirable to use as a tumor marker a substance produced only by cancerous cells without the presence of non-cancerous cells. Especially for pancreatic cancer, there is no effective screening test, so it is often too late when cancer symptoms appear. For early detection of pancreatic cancer, the use of tumor markers specific to pancreatic cancer is necessary. is necessary.

BST1, also referred to as BP-3 or CD157, is highly expressed in bone marrow stromal cell lines derived from rheumatoid arthritis (RA) and cloned as a glycosylphosphatidylinositol (GPI) -binding membrane protein involved in pre-B cell growth supportability (Non-patent Document 1), and is considered to be closely related to the pathology of rheumatoid arthritis. BST1 has also been reported to have ADP ribosyl cyclase activity and cyclic ADP ribose hydrolysis activity (Hirata et al., 1994).

BST1 expression in mice begins in the differentiation stage of pro-B cells and decreases in mature B cells in B cells, and in T cells, among fetal thymocytes and T precursor cells of adult thymocytes Only the CD44-CD25 + and CD44-CD25- stages were observed. Therefore, it is considered that mouse BST1 plays an important role in the rearrangement of IgH chain or TCRb chain generated in the early developmental stage of lymphocytes (Ishihara et al., 1996).
BST1 expression has also been confirmed in human peripheral blood granulocytes and monocytes. In these cells, it has also been suggested that BST1 plays a role as a receptor, but details have not been clarified.
Kaisho et al., Proc. Natl. Acad. Sci. USA 91: 5325-5329, 1994. Adkins et al., Mol Cell Proteomics. 12: 947-955, 2002 Anderson and Anderson, Mol Cell Proteomics. 11: 845-867, 2002

In view of the above, an object of the present invention is to provide a novel tumor marker antigen having high specificity for pancreatic cancer in order to solve the problems of the prior art relating to the tumor marker.
Furthermore, another object of the present invention is to provide a method for detecting pancreatic cancer using the tumor marker.
A further object of the present invention is to provide a kit that can be used in the detection method.
Another object of the present invention is to provide a medicament for tumor treatment targeting the tumor marker.

  In view of the above circumstances, the present inventors have conducted extensive research to identify an antigen that can be a tumor marker specific to pancreatic cancer. As a result, unexpectedly, a novel splicing variant of BST1 (hereinafter, referred to as “BST1”) was discovered. It was found that the protein (described as BST1_ASV) and the gene are specifically expressed in pancreatic cancer-derived cells.

That is, the said subject is solved by the following (1)-(12).
(1) The invention according to the first embodiment of the present invention is “a BST1_ASV polypeptide or a salt thereof comprising the following polypeptide (a) or (b)”:
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a polypeptide consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the sequence represented by SEQ ID NO: 2 and specifically present in pancreatic cancer cells ”.
Here, the “BST1_ASV polypeptide” means that the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 2 has one, several amino acids deleted, substituted, inserted or added. A polypeptide consisting of an amino acid sequence and present in pancreatic cancer cells. Furthermore, partial peptides and fragments of these polypeptides are also included in the “BST1_ASV polypeptide” of the present invention.
(2) The invention according to the second embodiment of the present invention is “a polynucleotide comprising a polynucleotide comprising the base sequence represented by SEQ ID NO: 1.” That is, in order to link a tag polypeptide known to those skilled in the art, for example, Flag, myc, HA, GST, etc., to the N-terminal or C-terminal part of the polypeptide encoded by the base sequence consisting of SEQ ID NO: 1, Polypeptides that link polypeptides other than the polynucleotide of No. 1 are also included in the polypeptides of the present invention.
(3) The invention according to the third embodiment of the present invention is “a gene encoding the BST1_ASV polypeptide”.
(4) The invention according to the fourth embodiment of the present invention is “a method for detecting pancreatic cancer cells, comprising the step of detecting the BST1_ASV polypeptide”.
Here, “pancreatic cancer” is a concept including pancreatic cancer in general (the same applies hereinafter). The method for detecting pancreatic cancer cells includes not only a method for detecting the presence or absence of pancreatic cancer cells but also a method for detecting the malignancy or tissue type of pancreatic cancer cells (hereinafter the same).
(5) The invention according to a fifth embodiment of the present invention is described above, wherein the step of detecting the BST1_ASV polypeptide uses an immunoassay method using an antibody that specifically binds to the BST1_ASV polypeptide. The method described in (4). "
Here, the “antibody” specifically binds to “BST1_ASV polypeptide” in the present invention, and binds to a known BST1 major form (BST1 polypeptide that is not a splice variant) and other BST1 selective splice variants. Means no antibody.
The method for “detecting the BST1_ASV polypeptide” described in (4) above includes all methods that can be performed based on ordinary knowledge of those skilled in the art, in addition to immunoassays using antibodies.
(6) The invention according to the sixth embodiment of the present invention is “the method according to (5) above, wherein serum or a pathological tissue section derived from a patient with pancreatic cancer is used as a sample”.
Samples used in the method described in (4) or (5) above are based on normal technical common knowledge in the art, in addition to the serum or pathological tissue section (such as pancreatic cancer tissue section) described in (6) above. Any sample that can be selected can be used.
(7) The invention according to the seventh embodiment of the present invention provides a pancreatic cancer cell comprising the step of detecting the polynucleotide according to (2) above or the gene according to (3) above. How to detect. "
(8) The invention according to the eighth embodiment of the present invention is such that “detection of the expression of the polynucleotide or the gene uses a probe that specifically amplifies the polynucleotide or a transcript of the gene or a fragment thereof. The method according to (7) above, which is carried out by the RT-PCR method.
The method for detecting “expression of the polynucleotide or the gene” described in the above (7) includes not only a method using PCR but also all methods that can be performed based on ordinary knowledge of those skilled in the art.
(9) The invention according to the ninth embodiment of the present invention is “the method according to (8) above, wherein a buffy coat derived from a patient with pancreatic cancer is used as a sample”.
Samples used in the method described in (7) or (8) above are based on common technical common knowledge in the art, such as pathological tissue sections (such as pancreatic cancer tissue sections) in addition to the buffy coat described in (9) above. Any sample that can be selected on the basis of it can be used.
(10) The invention according to the tenth embodiment of the present invention is "a kit for carrying out the method according to (4) above, wherein the kit is a monoclonal antibody against the polypeptide according to (1) above" And / or a kit comprising a polyclonal antibody.
(11) The invention according to the eleventh embodiment of the present invention is “a kit for carrying out the method according to (8) above, wherein the kit has a nucleotide sequence of SEQ ID NOS: 5 to 9”. A kit comprising at least one selected nucleic acid. "
(12) The invention according to the twelfth embodiment of the present invention is “a pharmaceutical composition comprising the antibody obtained by conjugating an anticancer compound to an antibody that specifically binds to the BST1_ASV polypeptide”.
Here, the “anticancer compound” is a substance that shows the effect of reducing or eliminating cancer foci by killing cancerous or tumorous cells, and any substance known to those skilled in the art can be used. Can do. Typically, the anticancer compound is selected from the group consisting of taxol, taxotere, cisplatin, adriamycin, epirubicin, cyclofasfamide, camptothecin, 5-FU (Ulorolacill), gemcitabine hydrochloride (Gemzar (trade name)). In particular, gemcitabine hydrochloride is preferable.

  By using the method provided by the present invention, pancreatic cancer can be reliably and rapidly detected. Moreover, effective treatment of pancreatic cancer is realized by using the pharmaceutical composition of the present invention.

1. Search for tumor marker candidates (1) Search for tumor marker candidates by in silico analysis In the present invention, BST1_ASV that can be used as a tumor marker is a search and search based on techniques in the field of bioinformatics and conventional molecular biological techniques. Can be identified.
First, in order to search for tumor marker candidates present in the blood, a published paper describing information on blood proteins is examined. The articles required for carrying out the present invention can be searched and collected by a well-known method. For example, NCBI (National Center for
PubMed (Biological Information Database) by Biotechnology Information may be used.
A list of proteins in the blood may be created with reference to the desired one from the collected papers. In this case, among the listed blood proteins, there is a certain standard, for example, a protein with little information as a tumor marker, a protein with little information such as the presence or absence of ASV, and the protein is secreted. It is possible to narrow down some proteins as tumor marker candidates by using the possibility as an index. BST1 according to the present invention was included in the protein list narrowed down by this method.

  As another method of the above method, it is also possible to use information of an existing tumor marker. Based on the characteristics of the existing tumor marker, although not limited, a desired tumor marker can be narrowed down from factors that are characterized by being an enzyme protein and a glycoprotein. For example, after searching Glycoprotein by Keyword search in the SWISS-PROT database, only proteins having the notation “_HUMAN” are extracted and listed. Furthermore, all the human proteins are extracted from the proteins existing in the Enzyme Nomenclature database in the SWISS-PROT database and listed. Proteins that overlap in the two lists can be narrowed down as tumor marker candidates. The BST1 according to the present invention was also included in the protein list narrowed down by this method.

(2) Examination of specificity as a tumor marker In order to be usable as a tumor marker, when a specific tumor (cancer) has developed, it is expressed with a certain degree of specificity and its expression has been confirmed. It is necessary to Confirmation of the expression of the tumor marker can be easily performed based on ordinary knowledge of those skilled in the art. For example, RT-PCR, Northern Confirmation by blotting, etc., or if the tumor marker is a protein secreted into the blood, secretion into the blood by immunoassay using an antibody that reacts specifically with the tumor marker protein against the blood sample Can be confirmed. In addition, when a specific tumor (cancer) has developed, any method can be used as long as it confirms that a desired tumor marker is specifically expressed in the tumor. In particular, RT-PCR method, immunoassay method and the like are preferable.

2. Acquisition of Sequence Information of Novel Tumor Marker BST1_ASV The BST1_ASV gene in the present invention can be cloned from a cDNA library, a genomic DNA library or the like using PCR primers prepared based on the known sequence information of BST1.
PCR primer design can be performed using primer design software such as OLIGO (Molecular Biology Insights. Inc). PCR primer synthesis can be performed using standard synthesis techniques such as an automatic DNA synthesizer, but may be obtained commercially. The length of the amplification product expected to be amplified by the PCR reaction is preferably such that the amplification efficiency, the subsequent separation ability by agarose gel and the base sequence analysis are easy. Using the created PCR primer, a PCR reaction is performed using a cDNA library or the like as a template, and it is confirmed by sequencing or the like that the amplified product is the target product.

2. BST1_ASV polypeptide of the present invention The BST1_ASV polypeptide of the present invention is a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. Here, the “polypeptide containing substantially the same amino acid sequence” means that the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 2 has one or several amino acids deleted, A polypeptide consisting of a substituted, inserted or added amino acid sequence and present in pancreatic cancer cells.

The polypeptide containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 is 1 or 2 or more (preferably about 1 to 30 in the amino acid sequence represented by SEQ ID NO: 2, more preferably 1 to 2 or more (preferably about 1 to 30 amino acids, more preferably about 1 to 10 amino acids, more preferably 1 to 5 amino acids), the amino acid sequence represented by SEQ ID NO: 2 Is an amino acid sequence to which about 1 to 10 amino acids, more preferably 1 to 5 amino acids are added, and 1 or 2 or more (preferably about 1 to 30) in the amino acid sequence represented by SEQ ID NO: 2 A polypeptide containing an amino acid sequence in which about 1 to 10 amino acids (more preferably 1 to 5) are substituted with other amino acids is included. The amino acid deletion, addition and substitution can be performed by modifying a gene encoding BST1_ASV polypeptide by a technique known in the art. For example, the substitution of a specific amino acid residue is performed by using a commercially available kit (for example, Mutan ^™ -G (TAKARA), Mutan ^™ -K (TAKARA)) or the like, and a known method such as the Gappedduplex method or the Kunkel method. Alternatively, it can be achieved by performing base substitution by a method according to them.

In addition, the C-terminal of the BST1_ASV polypeptide of the present invention and its partial peptide is usually a carboxyl group (—COOH) or a carboxylate (—COO ⁻ ), and the carboxyl group is an amide (—CONH ₂ ) or ester ( -COOR) or the like. Here, as R in the ester, a C _1-6 alkyl group (for example, methyl, ethyl, n-propyl, isopropyl, n-butyl), a C _3-8 cycloalkyl group (for example, cyclopentyl, cyclohexyl), C _1-6 aryl group (for example, phenyl, α-naphthyl), phenyl-C _1-2 alkyl group (for example, benzyl, phenethyl), α-naphthyl-C _1-2 alkyl group (for example, α-naphthylmethyl) and the like Is mentioned. In addition, pivaloyloxymethyl ester, which is widely used as an oral ester, can be used. When the BST1_ASV polypeptide of the present invention has a carboxyl group in the polypeptide chain in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the polypeptide of the present invention. Examples of the ester in this case include the above-mentioned esters. Similarly, the N-terminus of the novel BST1_ASV polypeptide of the present invention is usually an amino group (—NH ₂ ), but this amino group is chemically modified with a C _1-6 acyl group such as a formyl group or an acetyl group. It may be. In addition, a glutamyl group generated by cleavage of the N-terminal side in vivo is pyroglutamine oxidized, or a substituent on an amino acid side chain in the molecule (for example, —OH, —SH, amino group, imidazole group, indole group, Polypeptides of the present invention include those in which a guanidino group or the like) is chemically modified with an appropriate functional group (for example, formyl group, acetyl, etc.) or has a sugar chain attached thereto.

  Peptides (also referred to as partial peptides) containing partial amino acid sequences in any of the above BST1_ASV polypeptides are also included in the scope of the present invention. That is, the partial peptide of the present invention may be any peptide as long as it contains a partial amino acid sequence of the amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2.

The BST1_ASV polypeptide of the present invention or a partial peptide thereof can be provided in the form of a salt, preferably in the form of a physiologically acceptable acid addition salt, if necessary. Such salts include, but are not limited to, inorganic acids, such as salts such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, organic acids, but not limited to, for example, acetic acid, formic acid, propionic acid, fumaric acid. Examples thereof include salts such as acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid. In addition, inorganic bases, but not limited to, for example, salts with alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, organic bases, but not limited to, for example, trimethylamine, triethylamine, pyridine, Examples include salts with picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine and the like.
The BST1_ASV polypeptide of the present invention or a salt thereof is a human or animal (for example, mouse, rat, guinea pig, chicken, rabbit, pig, cow, monkey, sheep, dog, cat) expressing the novel BST1_ASV polypeptide of the present invention. Or the like) by culturing a transformant containing a DNA encoding the novel BST1_ASV polypeptide of the present invention as described later, or by culturing a transformant containing the DNA encoding the novel BST1_ASV polypeptide of the present invention as described later. It can also be produced by extraction and separation from the culture. When producing from human or animal tissues or cells, homogenize human or animal tissues or cells, extract with acid, etc., and use the resulting extract for hydrophobic chromatography, reverse phase chromatography, ion exchange chromatography, etc. It can be isolated and purified by combining various types of chromatography.

The partial peptide of the present invention or a salt thereof can be produced by a known peptide synthesis method or by cleaving the BST1_ASV polypeptide with an appropriate peptidase (eg, trypsin, chymotrypsin, arginyl endopeptidase). As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can constitute the novel BST1_ASV polypeptide of the present invention and the remaining portion, and removing the protective group when the product has a protective group. (See, eg, Bodanszky et al., 1996; Schroeder et al., 1965). After the synthesis reaction, the partial peptide of the present invention can be isolated and purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, high performance liquid chromatography, recrystallization and the like.
When the novel BST1_ASV polypeptide or its partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. When obtained by a salt, the free form is obtained by a known method. Can be converted to

3. A polynucleotide encoding the BST1_ASV polypeptide of the present invention or a partial peptide thereof The polynucleotide encoding the BST1_ASV polypeptide of the present invention or a partial peptide thereof is the nucleotide sequence (DNA or DNA encoding the BST1_ASV polypeptide of the above 2 or a partial peptide thereof. (RNA) or any complementary strand thereof may be used. Examples of the polynucleotide include DNA encoding the BST1_ASV polypeptide of the present invention, RNA such as mRNA, and may be double-stranded or single-stranded. In the case of a double strand, it may be a double strand DNA, a double strand RNA, or a hybrid of DNA and RNA.
The DNA encoding the BST1_ASV polypeptide of the present invention or a partial peptide thereof may be any of genomic DNA, genomic DNA library, those prepared from the aforementioned cDNA library, or synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, the RT-PCR method (Reverse Transcriptase Polymerase) can be directly used using the total RNA or mRNA fraction prepared from a desired cell or tissue.
It can also be amplified by chain reaction. Specifically, the polynucleotide encoding the novel BST1_ASV polypeptide of the present invention includes a polynucleotide containing the base sequence represented by SEQ ID NO: 1, and any of these polynucleotides and stringent conditions Any polynucleotide may be used as long as it is a polynucleotide encoding a BST1_ASV polypeptide that hybridizes underneath and has substantially the same activity as the BST1_ASV polypeptide of the present invention.

The DNA capable of hybridizing under stringent conditions with the polynucleotide containing the base sequence represented by SEQ ID NO: 1 is preferably about 70% or more, more preferably about 80%, with the base sequence represented by SEQ ID NO: 1. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, and most preferably, DNA containing a nucleotide sequence having a polynucleotide sequence homology of about 99%. Here, the “stringent conditions” are determined by common technical knowledge well-known in the technical field related to nucleic acid hybridization and cloning (see, for example, Sambrook et al., 1989), and are generally probe lengths. Empirical conditions, depending on the washing temperature and salt concentration. For example, the sodium concentration is about 20 to 40 mM, preferably about 20 to 25 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. Typically, “stringent conditions” refer to (1) conditions for washing at low ionic strength and high temperature (eg, 15 mM NaCl, 1.5 mM sodium citrate, 0.1% at 50 ° C. (2) Conditions using a denaturant during hybridization (eg, at 42 ° C., 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1 % Polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5; containing 750 mM NaCl, 75 mM sodium citrate), etc. In addition, typically with regard to washing conditions, 5 × SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% Contains sodium lophosphate, 5x denhardt solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate, 0.2x SSC at 42 ° C (sodium chloride / sodium citrate) Stringent wash conditions consisting of 0.1x SSC with medium wash and formamide at 55 ° C followed by EDTA at 55 ° C can be selected.
The polynucleotide encoding the partial peptide of the present invention may be any as long as it contains the nucleic acid sequence encoding the partial peptide of the present invention described above. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the aforementioned cells or tissues, cDNA isolated from the aforementioned cells or tissues, or a synthetic DNA isolated by a method known in the art. Good.

4). Production of Recombinant Vector and Transformant (1) Production of Recombinant Vector The recombinant vector used in carrying out the present invention is linked to a DNA encoding the BST1_ASV polypeptide of the present invention or a portion thereof to an appropriate vector. Can be obtained. The vector for inserting the DNA sequence encoding the BST1_ASV polypeptide of the present invention or a portion thereof is not particularly limited as long as it can be replicated in a host when used for cloning. Further, the vector for expressing the BST1_ASV polypeptide of the present invention or a part thereof is a promoter capable of replicating in a host and capable of expressing a DNA fragment encoding the polypeptide or part thereof. What you have can be used.
Examples of vectors that can be used include plasmid DNA and phage DNA. Plasmid DNA includes plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pCBD-C, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194, etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, YIp30, etc.) and the like, and examples of the phage DNA include λ phage. Furthermore, animal viruses such as retroviruses and vaccinia viruses, and insect virus vectors such as baculoviruses and togaviruses can also be used.

The promoter used in the present invention is not particularly limited as long as it is an appropriate promoter corresponding to the host used for gene expression.
For example, when animal cells are used as the host, SRα promoter, CMV promoter, SV40 promoter, LTR promoter, HSV-TK promoter and the like can be mentioned.
When the host is an E. coli, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter and the like, when the host is Bacillus subtilis, SPO1 promoter, SPO2 promoter, penP promoter.
When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like can be mentioned. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

The recombinant vector used in the present invention includes a novel BST1_ASV polypeptide or a coding sequence thereof, a promoter, an enhancer, a selection marker, a terminator, a splicing signal, a poly A addition signal, a ribosome binding sequence (SD sequence), An SV40 replication origin (SV40ori) or the like can be linked.
The selection markers include, but are not limited to, hygromycin resistance marker ^(Hyg r), dihydrofolate reductase gene (dhfr), the ampicillin resistance gene ^(Amp r), the kanamycin resistance gene ^(Kan r), neomycin resistant gene (Neo ^r , G418) can be used.
For the purpose of facilitating isolation and purification of the recombinant protein, an appropriate signal sequence may be added to the N-terminal side of the BST1_ASV polypeptide of the present invention or a portion thereof.
When the host is Escherichia coli, alkaline phosphatase signal, OmpA signal, etc. can be used. When the host is Bacillus subtilis, α-amylase signal sequence, subtilis signal sequence, etc. can be used. In some cases, α-factor signal sequence, invertase signal sequence, etc. can be used. When the host is an animal cell, for example, insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used. It is.

In order to insert the novel BST1_ASV gene of the present invention or a fragment thereof into the vector described above, the DNA encoding the novel BST1_ASV polypeptide cloned in the above 1 or a fragment thereof is digested as it is or with a restriction enzyme if desired. , By adding a linker and inserting it into a restriction enzyme site or multicloning site of the vector DNA. The DNA to be ligated may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter. The DNA to be ligated needs to be incorporated into a vector so that the novel BST1_ASV polypeptide of the present invention encoded in the DNA or a part thereof is expressed in the host cell.
By the above method, a vector containing a DNA sequence encoding the BST1_ASV polypeptide of the present invention can be constructed.

(2) Production of transformant The transformant of the present invention can be obtained by introducing the recombinant expression vector of the present invention into a host so that the target gene or a fragment thereof can be expressed. Here, the host is not particularly limited as long as it can express the DNA of the present invention. For example, E. coli (Escherichia coli) belonging to the genus Escherichia, such as, Bacillus subtilis (Bacillus subtilis) the genus Bacillus such as, Pseudomonas, such as Pseudomonas putida (Pseudomonas putida), Rhizobium meliloti (Rhizobium meliloti) bacteria belonging to the genus Rhizobium such as, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Schizosaccharomyces pombe ( Schizosaccharomyces pombe ), Pichia pastoris (Pichia pastoris), monkey cells COS-7, V cells Mouse AtT-20, human GH3, human FL cells, etc. Examples include animal cells or insect cells such as Sf9 and Sf21.

As a method for introducing a recombinant vector into Escherichia coli, a method using calcium ions (Cohen et al., 1972), an electroporation method (Shigekawa and Dower, 1988) and the like can be used. Methods for introducing recombinant vectors into yeast include electroporation (Becker et al., 1990), spheroplast method (Hinnen et al., 1978), lithium acetate method (Itoh et al.,
1983) can be used. As a method for introducing a recombinant vector into animal cells or animal cells, electroporation method, calcium phosphate method (Chen and Okayama,
1988), a method using a cationic lipid (Elroy-Stein and Moss, 1990) and the like.
As described above, a transformant containing an expression vector in which a DNA encoding the BST1_ASV polypeptide of the present invention or a partial peptide thereof is inserted can be obtained.

5). Production of novel BST1_ASV polypeptide or partial peptide thereof of the present invention The novel BST1_ASV polypeptide or partial peptide of the present invention can be produced by culturing the transformant obtained in 4 above and collecting it from the culture. it can. “Culture” means any of culture supernatant, cultured cells or cultured cells, or disrupted cells or cells. The method for culturing the transformant of the present invention is carried out according to a usual method used for culturing a host.
As a medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms. As long as the medium can be used, either a natural medium or a synthetic medium may be used. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As the nitrogen source, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like are used. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

Culturing is performed under conditions suitable for the host cell.
For example, as the medium for culturing Escherichia coli, LB medium, M9 medium and the like are preferable. Agents such as isopropyl-1-thio-β-D-galactoside, 3β-indolylacrylic acid can be added to make the promoter work efficiently if desired. In the case of Escherichia coli, the culture is usually performed at about 15 to 37 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added. When the host is Bacillus subtilis, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation can be added.
Examples of the medium for culturing yeast include SD medium and YPD medium. The pH of the medium is preferably adjusted to about 5-8. The culture is usually performed at about 20 to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary. When cultivating a transformant whose host is an insect cell or an insect, examples of the medium include a grace insect medium containing bovine serum. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.
When cultivating a transformant whose host is an animal cell, for example, a MEM medium, DMEM medium, RPMI 1640 medium or the like containing about 5 to 20% fetal bovine serum is used as the medium. The pH is preferably about 6-8. The culture is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary. As described above, the novel BST1_ASV polypeptide of the present invention or a partial peptide thereof can be produced on the cell membrane or the like of the transformant. Separation and purification of the novel BST1_ASV polypeptide of the present invention or a partial peptide thereof from the culture can be performed, for example, by the following method.

  When the novel BST1_ASV polypeptide of the present invention or a partial peptide thereof is extracted from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, A method of obtaining a crude extract of a novel BST1_ASV polypeptide by centrifuging or filtering after lysozyme and / or freeze-thawing and the like is appropriately used. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. When the novel BST1_ASV polypeptide or a partial peptide thereof is secreted into the culture solution, the cells or cells are separated from the supernatant by a method known per se after the completion of the culture, and the supernatant is collected. Purification of the novel BST1_ASV polypeptide or its partial peptide contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods. As these known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation methods, dialysis methods, ultrafiltration methods, gel filtration methods, and mainly using differences in molecular weight such as SDS-PAGE. Method, method using differential charge such as ion exchange chromatography, method using specific affinity such as affinity chromatography, method using hydrophobic difference such as reversed phase high performance liquid chromatography, isoelectric point A method using the difference in isoelectric point such as electrophoresis is used.

6). Anti-BST1_ASV Antibody An anti-BST1_ASV antibody can be used in the method for detecting a BST1_ASV polypeptide according to the present invention.
“Antibodies” used in the present invention include monoepitope specific anti-BST1_ASV antibodies against BST1_ASV, polyepitope specific anti-BST1_ASV antibodies, single chain antibodies, and fragments thereof. These antibodies include, for example, monoclonal antibodies, polyclonal antibodies, humanized or human antibodies. In particular, humanized or human antibodies are preferred when the anticancer compound is conjugated to deliver the anticancer compound to the target cancer lesion.
(1) Polyclonal antibody A polyclonal antibody can be prepared, for example, by injecting a mixture of an immunogen and an adjuvant into a mammalian host animal. Usually, the immunogen and / or adjuvant is injected multiple times subcutaneously or intraperitoneally into the host animal. Immunogens include fusions with BST1_ASV and its heterologous polypeptide or fragments thereof. Examples of adjuvants include complete Freud and monophosphoryl lipid A synthesis-trehalose dicorynocolle auto (MPL-TDM). In order to enhance the immune response, the immunogen can be injected after binding to immunogenic proteins such as keyhole impet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Good.
After immunization, antiserum collection is started at the stage where the desired titer is exhibited while monitoring the titer using antiserum collected experimentally from the immunized animal. The target antibody can be purified from the collected antiserum according to a conventional method.
Alternatively, it may be prepared using chickens that produce IgY molecules (Schade et al.,
1996).
For details of antibody production methods, see, for example, Ausubel et al., 1987 or Harlow and Lane, 1988.

(2) Monoclonal antibodies Anti-BST1_ASV monoclonal antibodies are prepared using the hybridoma method (Milstein and Cuello, 1983).
This method includes the following four steps: (i) immunizing a host animal or lymphocytes from the host animal, (ii) monoclonal antibody-secreting (or potentially secreting) lymphocytes Collect (iii) fuse lymphocytes to immortalized cells; (iv) select cells that secrete the desired monoclonal antibody (anti-BST1_ASV).
A mouse, rat, guinea pig, hamster, or other suitable host animal is selected as the immunized animal and the immunogen is injected. Alternatively, lymphocytes obtained from immunized animals may be immunized in vitro. If human cells are desired, peripheral blood lymphocytes (PBLs) are generally used. However, spleen cells or lymphocytes from other mammals are more common and preferred. The immunogen typically includes a fusion of BST1_ASV and its heterologous polypeptide or fragments thereof.
After immunization, lymphocytes obtained from the host animal are fused with an immortal cell line using a fusing agent such as polyethylene glycol to establish hybridoma cells (Goding, 1996). Rodent, bovine, or human myeloma cells immortalized by transformation are used, or rat or mouse myeloma cell lines are used. After cell fusion, the cells are grown in a suitable medium containing one or more substrates that inhibit the growth or survival of unfused lymphocytes and immortalized cell lines. Conventional techniques use parent cells that lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, hypoxanthine, aminopterin and thymidine are added to a medium (HAT medium) that inhibits the growth of HGPRT-deficient cells and allows the growth of hybridomas.

For the preparation of monoclonal antibodies, the preferred immortal cell line is the mouse myeloma line available from the American Type Culture Collection (Manassas, VA). For production of human monoclonal antibodies by human myeloma and mouse-human heteromyeloma cell lines, see Kozbor et al., 1984; Schook, 1987.
Since hybridoma cells secrete antibodies outside the cells, the presence or absence of production of monoclonal antibodies against BST1_ASV can be confirmed using a culture solution. The binding specificity of the monoclonal antibody produced can be assessed by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) (Harlow and Lane, 1988; Harlow and Lane, 1999).
Anti-BST1_ASV monoclonal antibody-secreting hybridoma cells can be isolated as single clones by limiting dilution and subculture (Goding, 1996). Suitable media include Dulbecco's Modified Eagle's Medium, RPMI-1640, and optionally protein-free medium or serum-free medium (eg, Ultra DOMA PF or HL-1; Biowhittaker, Walkersville, MD). Alternatively, the hybridoma cells may be grown in the ascites of a suitable host animal.

Monoclonal antibodies are well known to those skilled in the art, such as protein A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation or affinity chromatography (Harlow and Lane, 1988; Harlow and Lane, 1999) from medium or ascites. Isolated and purified by
Monoclonal antibodies can also be produced by gene recombination techniques (US Pat. No. 4,166,552). In order to identify a gene encoding a desired monoclonal antibody polypeptide from a hybridoma cell line that secretes anti-BST1_ASV antibody, for example, an oligonucleotide probe that specifically binds to a mouse heavy chain and light chain antibody gene may be used. . As a result, when antibody heavy chain and light chain genes are obtained, the target antibody gene can be identified by determining the sequence of the genes. In order to express a monoclonal antibody, the isolated DNA fragment is introduced with an antibody gene of an appropriate expression vector, and the vector does not produce any other Ig protein, such as simin COS-7 cells, Chinese hamster ovary (CHO) cells. Or transfected into a host cell, such as a myeloma cell. Isolated DNA fragments can be obtained, for example, by replacing coding sequences for human heavy and light chain constant domains in place of homologous mouse sequences (US Pat. No. 4,816,567; Morrison et al., 1987) or non-Ig poly Modifications can be made by fusing Ig coding sequences to all or part of the peptide coding sequence. Such non-Ig polypeptides can replace the constant domain of the antibody or create a constant domain of one antigen binding site to create a chimeric bivalent antibody.

(3) Humanized and human antibodies Anti-BST1_ASV antibodies include humanized or human antibodies. Humanized forms of non-human antibodies are chimeric Igs, Ig chains or fragments (such as Fv, Fab ′, F (ab ′) ₂ or antigen binding regions of other antibodies) that contain minimal sequences derived from non-human Ig.
In general, humanized antibodies have one or more amino acid residues introduced from non-human-derived Ig. These non-human amino acid residues are often selected from variable domains. Humanized antibodies can be produced, for example, by replacing the mouse CDRs or CDR sequences with the corresponding human antibody sequences (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988). That is, a humanized antibody is typically a human antibody in which a specific CDR residue is substituted with a residue from a corresponding site in a mouse-derived antibody. Humanized antibodies include CDRs of non-human species, such as mice, rats or rabbits, that have the complementarity determining region (CDR) of the recipient (human Ig) depending on the residues with the desired specific affinity for the antigen. Human Igs (recipient antibodies) in which the residues from are substituted are included. In addition, nonhuman-derived residues may replace Fv framework residues of human Ig (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

7). Detection of Tumor Marker Any method can be used as a method for detecting the BST1_ASV gene or polypeptide of the present invention as a tumor marker as long as it can be performed based on the common general technical knowledge of those skilled in the art. For example, BST1_ASV An RT-PCR method, a FISH method, or an immunoassay method for detecting a BST1_ASV gene product, which detects a gene transcription product, can be used.
(1) RT-PCR method and FISH method The RT-PCR method can be easily performed using techniques well known to those skilled in the art.
Specifically, single-stranded DNA complementary to BST1_ASV mRNA or a fragment thereof, which is a transcription product of BST1_ASV gene in the sample to be examined, can be synthesized, and the DNA or the fragment thereof can be specifically amplified. After PCR amplification of the DNA or a fragment thereof with a primer, the amplified product is detected by electrophoresis or the like. For the RT-PCR method, see, for example, Ishikawa et al., J.
Clin. Oncol., 28: 723-728, 1998. Examples of the primer set that can be used for detecting BST1_ASV of the present invention include appropriate combinations of primers comprising nucleotide sequences described in SEQ ID NO: 5 to SEQ ID NO: 9. These primers can be easily synthesized by, for example, the phosphoramidide method.

  You may detect the transcription | transfer state of BST1_ASV of this invention by FISH (Fluorescent in situ hybridization) method. The probe that can be used in the FISH method can be any nucleic acid probe that specifically recognizes BST1_ASV. For example, a nucleic acid consisting of the sequence shown in SEQ ID NO: 7 or 8 and its complementary strand can be used.

  A buffy coat can be preferably used as a sample to be subjected to a method for detecting a transcription product of the BST1_ASV gene by RT-PCR or FISH. The buffy coat is a layer located between the lowermost red blood cell layer and the uppermost serum layer of the centrifuge tube when blood from the patient to be examined is centrifuged, and mainly contains white blood cells. Includes tumor cells present in the blood. Therefore, it can be suitably used in the inspection by RT-PCR or FISH method.

(3) Immunoassay method The expression of the BST1_ASV gene of the present invention can also be confirmed by detecting the presence of the BST1_ASV polypeptide with the aforementioned BST1_ASV-specific antibody or antiserum. Can be used. Specifically, a sandwich assay, a competitive assay using a sample to be examined, for example, serum derived from a patient with pancreatic cancer, a competitive assay, or immunohistological staining using a pathological section as a sample can be used. The antibody used in the immunoassay method may be labeled for immunohistochemical staining or sandwich assay. As the labeling substance, radioactive substances, colored particles such as gold colloid, fluorescent type, chemiluminescent substance, or enzyme can be used. As a method for labeling an antibody, any method can be adopted as long as it is a technique well known by those skilled in the art.

  In a sandwich assay or the like, it is desirable to immobilize the antibody or antiserum used in this assay on an individual support. For example, after dissolving an antibody or the like in a carbonate buffer (pH 9.6), It can be immobilized by adding it to the well of a microtiter plate and incubating for a predetermined time. The measurement by immunoassay is performed by detecting an immune complex of an antibody or antiserum and BST1_ASV. In immunostaining of a pathological tissue section or the like, a BST1_ASV-specific antibody is brought into contact with a paraffin-embedded section, and the resulting immune complex is detected by a CSA system (Wako) according to the instructions attached to the product. can do. When patient formation is used as a test sample, a BST1_ASV-specific antibody is immobilized on a well to provide a capture-side antibody, and the other antibody is labeled with a radioactive substance, colored particles, or enzyme, Used as detection antibody.

  Add the appropriately diluted sample to the well on which the capture antibody is immobilized, incubate for a predetermined time, remove the sample solution from the well, wash each well with an appropriate buffer, and add the detection antibody. Is done. After a predetermined incubation, the wells are washed and immune complexes are detected. Detection depends on the property of the labeling substance labeled on the detection side antibody, and detects the radiation dose, luminescent color, sphere altitude, and the like. If the labeling substance is an enzyme label, an appropriate substrate can be further added to the well, and the absorbance after a predetermined incubation can be detected (ELISA method). As the enzyme used in the ELIZA method, any enzyme can be used as long as it can be used in the present method. For example, in the case of labeling with horseradish peroxidase, 3,3 ′, 5,5′-tetra Methylbenzidine and the like can be used as the substrate. When labeling with alkaline phosphatase, p-nitrophenyl phosphate can be used as a substrate.

8). Pharmaceutical Composition It is expected that the BST1_ASV polypeptide of the present invention is specifically expressed in pancreatic cancer cells. Therefore, it is also possible to provide a medicament for treating pancreatic cancer using BST1_ASV, which is a pancreatic cancer-specific tumor marker, as a target molecule. For example, a composition containing an agent that binds specifically to BST1_ASV (such as a small molecule or a peptide) or a BST1_ASV-specific antibody or a compound that has an anticancer activity or a substance conjugated with a cytotoxin is used for treating pancreatic cancer. Can be used as a pharmaceutical composition. The composition can be used as a therapeutic agent together with a pharmaceutically acceptable carrier in the form of a pharmaceutical composition that does not adversely affect the living body.
“Pharmaceutically acceptable carrier” refers to those suitable for pharmaceutical administration, including solvents, dispersion media, coating agents, antibacterial and antifungal agents, agents that act isotonically to delay adsorption and the like. Yes (Gennaro et al., 2000). Examples of the carrier and preferred for diluting the carrier include, but are not limited to, water, saline, finger solution, dextrose solution, and 5% human serum albumin. Non-aqueous media such as liposomes and non-volatile oils are also used.

(1) Preparation of pharmaceutical composition The pharmaceutical composition of the present invention is formulated to be compatible with a therapeutically appropriate route of administration, including intravenous, oral administration, and direct administration to the stomach. Solutions or suspensions used for intravenous administration or direct administration to the stomach include, but are not limited to, sterile diluents such as water for injection, saline solutions, non-volatile oils, polyethylene glycols , Glycerin, propylene glycol, or other synthetic solvents, preservatives such as benzyl alcohol or other methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, soothing agents such as benzalkonium chloride, procaine hydrochloride, ethylenediaminetetra Chelating agents such as acetic acid (EDTA), buffers such as acetate, citrate, or phosphate, and agents for osmotic pressure adjustment such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Non-caliber preparations are contained in ampoules, glass or plastic disposable syringes or multidose vials.

(2) Injectable preparations Pharmaceutical compositions suitable for injection include sterile injectable solutions or dispersion media that are sterile aqueous solutions (water soluble) or dispersion media to be prepared at the time of use and are sterilized. Powders (including lyophilized proteins, nucleic acids, etc.) are included. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL ^™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). When used as an injection, the composition must be sterile and must be fluid enough to be administered with a syringe. The composition must be stable to chemical changes, corrosion, and the like during preparation and storage, and should not cause contamination from microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures. For example, using a coating agent such as lectin, maintaining a required particle size in the dispersion medium, and maintaining a proper fluidity by using a surfactant. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal can be used to prevent microbial contamination. In addition, polyalcohols such as sugar, mannitol, sorbitol, and agents that maintain isotonicity such as sodium chloride may be included in the composition. Compositions that can delay adsorption include agents such as aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by adding the required ingredients alone or in combination with other ingredients, then adding the required amount of active compound in a suitable solvent and sterilizing. Generally, a dispersion medium is prepared by incorporating the active compound into a sterile medium that contains a basic dispersion medium and the other necessary ingredients described above. Sterile powder preparation methods for the preparation of sterile injectable solutions include vacuum drying and lyophilization to prepare a powder containing the active ingredient and any desired ingredients derived from the sterile solution. It is.

(3) Oral composition Usually, the oral composition contains an inert diluent or a carrier that does not cause harm when taken into the body. Oral compositions are, for example, contained in gelatin capsules or compressed into tablets. For oral treatment, the active compound is incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a flowable carrier, and the composition in the flowable carrier is applied orally. In addition, pharmaceutically compatible binding agents, and / or adjuvant materials may be included.
Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds with similar properties: excipients such as microcrystalline cellulose, gum arabic, tragacanth or gelatin Binders such as starch or lactose, alginic acid, PRIMOGEL, or corn starch; lubricants such as magnesium stearate or STRROTES; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or peppermint, methyl salicylic acid Or a fragrance additive such as orange flavor.

(5) Systemic administration Systemic administration can be performed transmucosally or transdermally. For transmucosal or transdermal administration, penetrants that can penetrate the target barrier are selected. Transmucosal penetrants include surfactants, bile salts, and fusidic acid derivatives. Nasal sprays or suppositories can be used for transmucosal administration. For transmucosal administration, the active compounds are formulated in ointments, ointments, gels or creams.
The compounds can also be prepared in the form of suppositories (eg, with bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
(6) Carrier The BST1_ASV polypeptide of the present invention, a partial polypeptide thereof, or an expression vector thereof can be immediately removed from the body as a controlled release formulation such as a delivery system encapsulated in implantable tablets and microcapsules. It can be prepared using a preventable carrier. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyethylene glycol, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials are available from ALZA Corporation (Mountain View, CA) and NOVA Pharmaceuticals, Inc. (Lake Elsinore, CA) and can also be readily prepared by experts in the field. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Useful liposomes are prepared as a lipid composition comprising, but not limited to, phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanol (PEG-PE) through a filter of appropriate pore size to obtain a size suitable for use, and reverse phase. Purified by evaporation. For example, Fab ′ fragments of antibodies and the like may be bound to liposomes via a disulfide exchange reaction (Martin and Papahadjopoulos, 1982). For detailed preparation methods, see for example Eppstein et al., 1985; Hwang et al., 1980.

(7) Dosage In the treatment or prevention of a specific disease using the BST1_ASV polypeptide of the present invention, a partial polypeptide thereof, or an expression vector thereof, an appropriate dosage level is the condition of the patient to be administered, the administration method, etc. However, it can be easily optimized by those skilled in the art.
In the case of injection administration, for example, it is preferable to administer about 0.1 μg / kg to 500 mg / kg per day of the patient's body weight, and it will generally be possible to administer once or in several divided doses. Preferably, the dosage level is about 0.1 μg / kg to about 250 mg / kg per day, more preferably about 0.5 to about 100 mg / kg per day. For example, when administered conjugated to an antibody to taxol as an anticancer compound, the daily dosage of the patient's body surface area 1m ² 105~135mg is appropriate.
For oral administration, the composition is preferably provided in the form of a tablet containing 1.0 to 1000 mg of active ingredient, preferably the active ingredient contained in the dosage for the patient to be treated is 1.0 , 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400 0.0, 500.0, 600.0, 750.0, 800.0, 900.0 and 1000.0 mg. The compounds are administered on a regimen of 1 to 4 times daily, preferably once or twice daily.

(8) Unit dosage A pharmaceutical composition or formulation must be composed of uniform unit dosages to ensure a certain dosage. A unit dose is a unit formulated with a pharmaceutically acceptable carrier, including a single dose effective for treating a patient. In determining the unit dosage of the present invention, the physical and chemical characteristics of the compound to be formulated (eg, BST1_ASV polypeptide), the expected therapeutic effect, and the formulation specific to the compound It is affected by matters to be noted in

9. Detection Kit The kit for detecting a tumor marker of the present invention includes primers necessary for performing RT-PCR (for example, an appropriate primer set of SEQ ID NOs: 5 to 9), an RNase H inhibitor, and reverse transcription. Enzymes, Taq DNA polymerase, etc. may be included. In addition to the monoclonal antibody and / or polyclonal antibody that specifically binds to the BST1_ASV of the present invention necessary for immunoassay, the kit for detecting the tumor marker of the present invention includes a buffer necessary for immunoassay, Blocking agents (BSA, skim milk powder, etc.), secondary labels conjugated with detection labels (fluorescent dyes, chemiluminescent substances, gold colloids, enzymes, etc.) may be included. Further, among immunoassays, a kit for use of the ELIZA method may include a capture antibody, an enzyme-labeled detection antibody, an appropriate enzyme substrate, and the like. These reagents can be contained in a suitable container and included in the kit along with instructions for use.
(1) Container Reagents contained in the kit are supplied in any kind of container in which the constituents maintain the activity effectively for a long period of time, are not adsorbed by the material of the container, and do not undergo alteration. For example, a sealed glass ampoule includes a buffer packaged under a neutral and non-reactive gas such as nitrogen gas. Ampoules are composed of glass, polycarbonate, organic polymers such as polystyrene, ceramics, metals, or any other suitable material commonly used to hold reagents. Examples of other suitable containers include simple bottles made from similar materials such as ampoules, and packaging materials that are internally lined with foil such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. The container has a sterile access port such as a bottle having a stopper that can be penetrated by a hypodermic needle.
(2) Instructions for use The instructions for use are also attached to the kit. Instructions for using the kit comprising the pharmaceutical composition are printed on paper or other material and / or floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, etc. It may be supplied as an electrically or electromagnetically readable medium. Detailed instructions for use may be actually attached to the kit, or may be posted on a website designated by the manufacturer or distributor of the kit or notified by e-mail or the like.

  Examples are shown below, but the present invention is not limited thereto.

The in silico genes and proteins of the candidate having the narrowing blood tumor marker candidate tumor markers by analysis was performed Refine by analysis in in silico. In order to select a protein that may be a candidate marker from among proteins secreted in blood, NCBI (National Center for
Using PubMed (biological and medical literature database) existing in an integrated database operated by Biotechnology Information), papers that experimentally proved proteins present in blood were collected. Regarding blood proteins, tumor marker candidate proteins were extracted from papers (Non-patent Document 2 and Non-patent Document 3) described exhaustively by the following method and listed.
All protein names described in Non-Patent Documents 2 and 3 were extracted, and each document was listed. For the list created here, GenBank (National Institutes of
Nucleic acid database created by Health), PIR (amino acid sequence database operated by Georgetown University Medical Center), SwissProt (amino acid and protein database provided by Swiss Institute of Bioinformatics), PDB (research collaboratory for structural bioinformatics) Database), RefSeq (reference sequence database provided by NCBI), SwissProt's Primary accession number and Entry name, LocusLink (integrated database provided by NCBI), LocusID, Unigene (clustered database provided by NCBI) A program for adding an ID was created. The list of tumor marker candidate proteins prepared based on literature 2 or 3 was compared, and a list of secretory proteins in the blood that could be used as final tumor marker candidate proteins was created by eliminating the overlapping portion.

Add SwissProt's Protein name, Synonym, and Gene name to each gene and protein described in the blood secretory protein list, search them with PubMed one by one, and then connect the searchable items with the logical sum condition to query It was created. Literature search by PubMed was performed using the created query, and the number of search results for each gene and protein was added to the blood secretory protein list. A series of operations from the creation of a query to the addition of the number of query search results by PubMed was automatically performed by creating a program. In addition, by applying this program, a PubMed search was performed by combining the created query and the following words and phrases, the number of search results using the combination words was added (Table 1), and a final list was created. The following words are used in combination: tumor, tumor marker, Alternative Splicing, Alternative Splicing AND
tumor, Alternative Splicing AND tumor marker, plasma, plasma AND tumor, plasma
AND tumor marker, plasma AND Alternative Splicing

Table 1: Programs used for searches using the above combined words

The following data mining was performed based on the created list.
From the PubMed Abstract, the ratio of the number of hits of each combined term (the above 9 types) to the total number of paper hits for each gene and protein was calculated (the total number of paper hits for each gene and protein for the PubMed Abstract was A And the number of paper hits to which each of the nine types of terms is added is N. The ratio can be calculated by setting N / A). All proteins with a N / A value of 0 were excluded from the candidates, and the remaining proteins were judged from the contents of the retrieved papers as to whether they were blood secreted proteins. In other words, in order to select genes and proteins that have not been searched for as tumor markers, cancer-related factors with a small total number of papers are ranked in the top of the candidate list, and those that have already been registered or may be registered as markers Ranked in the candidate lower rank. Furthermore, genes and proteins for which ASV is being searched were ranked as candidates.
Moreover, since it is possible to predict from the three-dimensional structure whether or not the ASV protein is secreted into the blood when ASV is actually expressed by using it in a bioinformatic analysis method in in silico analysis. Those whose three-dimensional structures are registered in PDB (Protein Data Bank) were preferentially ranked as candidates.
Based on the above method, 100 tumor marker candidates were narrowed down (Table 2). Among these candidates, the BST1 gene (SEQ ID NO: 3) and protein (SEQ ID NO: 4) were included.

[Table 2]

Narrowing down tumor markers by in vitro analysis Among the 100 tumor marker candidates narrowed down in Example 1, in order to narrow down organ-specific tumor marker candidates, RT-PCR was used to select several cancer cells. In addition, the samples derived from normal cells were examined. In the RT-PCR method, the 100 gene PCR primer for the exon regions at both the 5 ′ side and the 3 ′ side of the major form so that the major form of the 100 gene and its ASV can be detected simultaneously. (Sawaday Technology) was designed.
The cancer cell line-derived total RNA used in the present invention was provided by the Cancer Center (see the brief description in the drawing of FIG. 2), and the normal cell-derived total RNA was obtained from the Human Rapid Scan (Ori -gene) (see the brief description of the drawing in FIG. 1). In addition, when performing RT-PCR, a mixture of all cDNAs derived from normal cells and cancer cells was prepared as a positive control (FIG. 2, PC). Normal cell tissues (bone marrow, Brain / cerebellum, fetal brain, fetal liver, heart, kidney, lung, placenta, prostate, salivary gland, skeletal muscle, spleen, testis, thyroid, trachea, uterus, large intestine (mucosal inner surface), small intestine, spinal cord, stomach, ovary, skin , Gallbladder, adipose tissue) cDNA was purchased from Clontech.

  As a result of RT-PCR, the band size of the PCR product detected in normal cells and the band size of the PCR product detected in cancer cells are referred to the position of the band detected in the positive control (FIG. 2, PC). A comparison was made to confirm genes expressed in an organ-specific manner. From the confirmed band position of the PCR product, a band appearing at a position different from the major form is defined as an ASV-derived band, an organ-specific ASV-derived band is cut out from the agarose gel, and the base sequence is determined according to a standard method. Decided. The determined ASV base sequence is mapped to the genome sequence using the map software est2genome (EMBOSS is “The European Molecular Biology Open Software Suite”), and compared to the exon and intron structure of the major form, the ASV genome The structure was determined.

Acquisition of BST1_ASV sequence information BST1_ASV, a selective splice variant of the BST1 gene whose mRNA expression was confirmed specifically in pancreatic cancer, was identified by the method shown in Example 2.
(1) Determination of BST1_ASV base sequence In order to identify the cDNA of BST1_ASV, PCR was performed on the cDNA prepared from the total RNA of each cancer cell line donated by the Cancer Center using the following primers. It was.
BST1_h. f1: 5′-AGGAGAGAAGGGGAGTGGAGGAAGC (SEQ ID NO: 5)
BST1_t. r1: 5′-GGCTGGAGGAGGGTTAGAGCAACAC (SEQ ID NO: 6)

PCR conditions are shown below.
Enzyme used: TAKARA LA_taq
Model used:
Biometra T-GRADIENT
Reaction solution composition: 10 × LA Buffer 1.5 μl
dNTP MIX (2.5mMeach) 2.4μl
Primer (BST1_h.f1) 2.4 μl
(BST1_t.r1) 2.4 μl
LA Taq + Taq AB (*) 0.3 μl
(*) Monoclonal antibody for Hot Start PCR anti-Taq high (TOYOBO) and each Taq polymerase were mixed in equal amounts and allowed to stand at room temperature for 15 minutes.
Template DNA 0.5 μl
Distilled water 5.5μl
Total volume 15μl
The cycle condition was maintained at 95 ° C., followed by 35 cycles of 95 ° C. for 30 seconds, 68 ° C. for 2 minutes and 30 seconds, and 68 ° C. for 2 minutes, and was maintained at 4 ° C.

  For each of normal cells (FIG. 1) and cancer cells (FIG. 2), the obtained PCR product is subjected to agarose gel electrophoresis and is located in a size different from the major form and is detected specifically for pancreatic cancer Was excised and cloned and sequenced by conventional methods. For sequencing, a sample was prepared using ABI PRISM BigDye Terminator v3.1 (Applied Biosystems) gene analysis reagent, and analysis was performed using 3730 DNA Analyzer (Applied Biosystems). The base sequence of the identified BST1_ASV is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2. The base sequence of BST1 major form is shown as SEQ ID NO: 3. FIG. 3 shows the alignment between the major form and the ASV.

(2) Examination of pancreatic cancer specificity of BST1_ASV The fact that BST1_ASV is specifically expressed in pancreatic cancer was confirmed again using a primer different from the above primers (SEQ ID NOs: 5 and 6).
The base sequence encoding BST1_ASV lacks the 15th to 188th sequences from the start codon A in the base sequence encoding the major form. Thus, primers designed for this region of ASV can anneal only to ASV without annealing to the major form, and specifically amplify only ASV (see FIG. 4). Therefore, PCR was performed using such a primer in the forward direction (SEQ ID NO: 8) or the reverse direction (SEQ ID NO: 7).

Of the primers shown below, BST1_h. f1 (SEQ ID NO: 5) and BST1_sv1. The primer set shown by r1 (SEQ ID NO: 7) is a set for specifically amplifying BST1_ASV by setting a primer (SEQ ID NO: 7) specific to ASV in the reverse direction, and BST1_sv1. f1 (SEQ ID NO: 8) and BST1_e2. The primer set represented by r1 (SEQ ID NO: 9) is a set for specifically amplifying BST1_ASV by setting a primer specific to ASV in the forward direction (see FIG. 4).
5'-side BST1_ASV-specific detection primer BST1_h. f1: 5′-AGGAGAGAAGGGGAGTGGAGGAAGC-3 ′ (SEQ ID NO: 5)
BST1_sv1. r1: 5′-GCTGTGCAGTTCTTGTTCCCCTGG-3 ′ (SEQ ID NO: 7)
3 ′ side BST1_ASV-specific detection primer BST1_sv1. f1: 5′-CGATGGCGGCCCAGGGGAACA-3 ′ (SEQ ID NO: 8)
BST1_e2. r1: 5′-TGAGGGCAGCACGGAGCAGGGGAT-3 ′ (SEQ ID NO: 9)

PCR conditions are shown below.
Enzyme used: TAKARA LA_taq
Model used:
Biometra T-GRADIENT
Reaction solution composition: 10 × LA Buffer 1.0 μl
dNTP MIX (2.5mMeach) 1.6μl
Primer (BST1_h.f1) 1.6 μl
(BST1_t.r1) 1.6 μl
LA Taq + Taq AB (*) 0.4 μl
(*) Monoclonal antibody for Hot Start PCR anti-Taq high (TOYOBO) and each Taq polymerase were mixed in equal amounts and allowed to stand at room temperature for 15 minutes.
Template DNA 0.5 μl
Distilled water 5.5μl
Total volume 10μl
Cycle conditions were maintained at 96 ° C., followed by 35 cycles of 96 ° C. for 30 seconds, 62 ° C. for 30 seconds, 72 ° C. for 1 minute and 30 seconds, and held at 72 ° C. for 2 minutes and 4 ° C.
The results of the confirmation experiment using the primers of SEQ ID NOs: 5 and 7 are shown in FIGS. 5 and 6, and the results of the confirmation experiment using the primers of SEQ ID NOs: 8 and 9 are shown in FIGS.

From the above results, BST1 major form is expressed in most normal cells examined including pancreas (FIG. 1), and in cancer cells, 4 of pancreatic cancer cells (FIG. 2, lanes 11 to 13, 15). The expression was confirmed in one of the species and squamous cell carcinoma cells (cell line derived from oral cancer, FIG. 2, lane 34) (FIG. 2). On the other hand, BST1_ASV has been shown to be expressed in bone marrow for normal cells (FIG. 1, the band indicated by ND in lane 22), but this band is not detected by an ASV-specific primer ( FIG. 5 and FIG. 7, lane 22) are considered to be non-specific amplification products by primers. Expression in other normal cells was not detected (FIGS. 1, 5, and 7). On the other hand, in cancer cells, BST1_ASV is expressed in pancreatic cancer cells (cell lines derived from pancreatic ductal pancreatic cancer) (FIGS. 2, 6, 8, lanes 12, 13, 15) and squamous cell carcinoma cells (cell lines derived from oral cancer). ) (FIGS. 2, 6, 8 and lane 34), and it was not detectable in cancer cells derived from other tissues.
That is, BST1_ASV is not expressed in normal pancreas, and since expression is recognized in the pancreas only after canceration, it can be determined that it can be used as a tumor marker specific to pancreatic cancer.

References
Adkins et al., Mol Cell Proteomics. 12: 947-955,
2002
Anderson and Anderson, Mol Cell Proteomics.
11: 845-867, 2002
Ausubel et al., Current protocols in molecular biology.John Wiley & Sons, New
York. 1987
Becker et al., Methods. Enzymol., 194: 182-184,
1990
Bondanszky et al., Peptide Synthesis, Interscience Publishers, New York 1996
Chen and Okayama, BioTechniques. 6: 632-638, 1988
Cohen et al., Proc. Natl. Acad. Sci., USA, 69: 2110-2114, 1972
Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA 87: 6743-6747, 1990
Eppstein et al., Proc Natl Acad Sci USA. 82: 3688-6892, 1985
Gennaro et al: The science and practice of pharmacy. Lippincott, Williams &
Wilkins, Philadelphia, PA. 2000
Goding et al., Academic Press, San Diego. 492 pp. 1996
Harlow and Lane, Antibodies: A laboratory manual. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor. 726 pp, 1988
Harlow and Lane, Using antibodies: A laboratory manual. Cold Spring Harbor
Laboratory PRess, Cold Spring Harbor, New York. 1999
Hinnen et al., Proc. Natl. Acad. Sci. USA, 75: 1929-1933, 1978
Hirata et al., FEBS Lett. 19: 356: 244-248, 1994
Hwang et al., Proc Natl Acad Sci USA. 77: 4030-4034, 1980
Itoh et al., J. Bacteriol., 153: 163-168, 1983
Ishihara et al., Int. Immunol. 8: 1395-1404, 1996
Ishikawa et al., J. Clin. Oncol., 28: 723-728, 1998
Jones et al., Nature. 321: 522-525, 1986
Kaisho et al., Proc. Natl. Acad. Sci. USA 91: 5325-5329, 1994
Kozbor et al., J Immunol. 133: 3001-3005, 1984
Martin and Papahadjopoulos, J Biol Chem. 257: 286-288, 1982
Milstein et al., Nature. 305: 537-540, 1983
Morrison et al., Genetically engineered antibody molecules and their application.
Ann NY Acad Sci. 507: 187-198, 1987
Presta et al., Curr Opin Biotechnol. 3: 394-398, 1992
Riechmann et al., Nature. 332: 323-327, 1988
Sambrook, J. Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor. 1989
Schade et al., The production of avian (egg yold) antibodies: IgY. The report and
recommendations of ECVAM workshop. Alternatives to Laboratory Animals (ATLA).
24: 925-934, 1996
Schook, LB 1987. Monoclonal antibody production techniques and
applications. Marcel Dekker, Inc., New York. 336 pp.
Schroeder et al., The peptide, Academic Press, New York 1965
Shigekawa and Dower BioTechniques. 6: 742-751, 1988
Verhoeyen and Winter Science 239: 1534-1536,
1988

  According to the present invention, it is possible to provide a specific detection marker in screening for pancreatic cancer, which was difficult to detect at an early stage among cancers. In addition, by using the tumor marker of the present invention, it becomes possible to deliver an anticancer compound and the like specifically for pancreatic cancer, and a new therapeutic method for pancreatic cancer can also be provided.

FIG. 1 shows the results of confirming the expression of BST1 and BST1_ASV in normal cells. PCR was performed on cDNA prepared from each normal cell using specific primers that amplify BST1 and BST1_ASV, and the synthesized PCR product was electrophoresed. The origin of normal cells corresponding to each lane is shown below; Brain, 2. 2. heart Kidney, 4. 4. spleen, Liver, 6; 6. large intestine (colon); Lung, 8. 8. small intestine, Muscle, 10. Stomach, 11. Testis, 12. Placenta, 13. Salivary glands, 14. Thyroid, 15. Adrenal gland, 16. Pancreas, 17. Ovary, 18. Uterus, 19. Prostate, 20. Skin, 21. Peripheral blood T lymphocytes, 22. Bone marrow, 23. Fetal brain, 24. Fetal liver FIG. 2 shows the results of confirming the expression of BST1 and BST1_ASV in cancer cells. The origin of the cancer cells corresponding to each lane is shown below; HepG2,2. Alexander, 3. Li7,4. Kim-1, 5. KYN-2 (hepatocellular carcinoma from lanes 1 to 5), 6. CaC0-2,7. HT-29,8. HCT-15,9. SW-48,10. SW-480 (Colon cancer up to lanes 6 to 10 (colon cancer)), 11. MIAPaCa, 12. CFPAC, 13. Capan-2, 14. HIPAF-II, 15. Panc-1 (pancreatic cancer up to lanes 11-15), 16. OV-90, 17. OMC-3, 18. MCAS, 19. RMG-1 (ovarian cancer up to lanes 16-19), 20. JEG-3, 21. JAR, 22. BeWo, 23. NJG (choriocarcinoma up to lanes 20-23), 24. Lu130, 25. Lu134-A, 26. Lu134B, 27. Lu135, 28. Lu139 (small cell lung cancer up to lanes 24-28), 29. PC-3, 30. MDA-Pca-2b, 31.22Rv-1 (prostate cancer up to lanes 29 to 31), 32. HSC-2, 33. SAS, 34. HO-1-N-1, 35. HO-1-U-1 (squamous cell carcinoma up to lanes 32-35), 36. Lu65, 37. HeLa, 38. MKN-28, 39. PlatE, 40. KIT32 (controls lanes 36-40) FIG. 3 shows the alignment of BST1 major form and BST1_ASV. FIG. 4 shows the positions of PCR primers used for confirmation of expression. The italicized part is the part deleted in ASV. Therefore, the arrow part excluding the broken line part indicates the set primer position. FIG. 5 shows the result of PCR performed on cDNA prepared from normal cells using ASV-specific primers. Each lane is the same as in FIG. FIG. 6 shows the results of PCR using cDNA prepared from cancer cells using ASV-specific primers. Each lane is the same as FIG. FIG. 7 shows the results of PCR using cDNA prepared from normal cells using ASV-specific primers. Each lane is the same as in FIG. FIG. 8 shows the results of PCR using cDNA prepared from cancer cells using ASV-specific primers. Each lane is the same as FIG.

Claims (12)

A BST1_ASV polypeptide or a salt thereof comprising the following polypeptide (a) or (b):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence represented by SEQ ID NO: 2 and specifically present in pancreatic cancer cells   A polynucleotide comprising a polynucleotide comprising the base sequence represented by SEQ ID NO: 1.   A gene encoding the BST1_ASV polypeptide.   A method for detecting pancreatic cancer cells comprising the step of detecting the BST1_ASV polypeptide.   The method according to claim 4, wherein the step of detecting the BST1_ASV polypeptide uses an immunoassay method using an antibody that specifically binds to the BST1_ASV polypeptide.   The method according to claim 5, wherein serum or a pathological tissue section derived from a patient with pancreatic cancer is used as a sample.   A method for detecting pancreatic cancer cells comprising the step of detecting the polynucleotide according to claim 2 or the gene according to claim 3.   8. The expression of the polynucleotide or the gene is detected by RT-PCR using a probe that specifically amplifies the polynucleotide or a transcript of the gene or a fragment thereof. The method described.   The method according to claim 8, wherein a buffy coat derived from a patient with pancreatic cancer is used as a sample.   A kit for carrying out the method according to claim 4, wherein the kit comprises a monoclonal antibody and / or a polyclonal antibody against the polypeptide according to claim 1.   A kit for carrying out the method according to claim 8, wherein the kit comprises at least one nucleic acid selected from the group consisting of nucleic acids having the nucleotide sequences of SEQ ID NOs: 5 to 9. .   A pharmaceutical composition comprising the antibody obtained by conjugating an anticancer compound to an antibody that specifically binds to the BST1_ASV polypeptide.