JP2010046057A - Agent for enhancing therapeutic sensitivity of cancer cell, method for determining therapeutic sensitivity of cancer cell and kit for determining therapeutic sensitivity of cancer cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means useful for diagnosis of cancers and determination of therapeutic effect and prognosis by exploring a marker (gene) contributing promotion of an order-made medical treatment of chemotherapy, radiotherapy and their combination chemoradiotherapy (CRT) for a treatment of cancers (especially esophagus squamous carcinoma). <P>SOLUTION: There is provided a method for determining therapeutic sensitivity of cancer cells. The method comprises: a step to determine expression quantity (A1) of REG1A gene or a gene functionally equivalent thereto or REG1B gene or a gene functionally equivalent thereto in a test tissue or test cell; a step to compare the expression quantity (A1) with a prescribed expression quantity (A0); and a step to estimate the sensitivity of the test tissue or test cell to radiation or an anticancer agent by determining whether the expression quantity (A1) is significantly larger than the expression quantity (A0) or not. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a therapeutic sensitivity enhancer for cancer cells, a method for determining therapeutic sensitivity of cancer cells, a therapeutic sensitivity determination kit for cancer cells, and the like. More specifically, the present invention relates to the enhancer using the REG1 gene (specifically, the REG1A gene and the REG1B gene), and the determination method / determination kit using the expression level of the gene as an index.

  Treatment for cancer (malignant tumor) is roughly divided into surgical therapy, chemotherapy, immunotherapy, hyperthermia, and radiation therapy. Of these, chemoradiotherapy (CRT), which is a combination of chemotherapy and radiation therapy, is known to show good therapeutic results for certain types of cancer. Chemotherapy, which is one of the treatment methods used in CRT, involves administering a chemotherapeutic agent (anticancer agent) to a patient. However, as a general problem in all drugs, there are individual differences in the expression of effects / side effects. There is a problem that exists.

  In recent years, with the advancement of the human genome project, research on pharmacogenomics (pharmacogenomics) that can be applied to drug development by understanding individual differences in drug sensitivity at the gene level has been actively conducted. If individual differences in drug susceptibility can be determined at the genetic level, it is possible to administer drugs with strong side effects only to patients who are truly expected to have a medicinal effect, and without initial trial treatment without trial and error medication. Therefore, it is possible to perform so-called custom-made medical care, in which appropriate medication is performed. As a result, the patient is not burdened with unnecessary physical burdens, and also contributes to the reduction of medical costs that tend to increase in recent years. Among them, anticancer agents often have very strong side effects, and application of tailor-made medicine is strongly desired.

  For example, if the sensitivity of a certain type of cancer cell to a certain type of anticancer drug can be enhanced by controlling gene expression, a true customized medicine at the gene level will be realized.

  On the other hand, it is known that there are individual differences in sensitivity to radiation therapy, which is the other treatment method used in CRT. The existence of such individual differences in radiosensitivity is also strongly presumed to be caused by differences in the genetic level of each human. Therefore, it is considered that the purpose and philosophy of pharmacogenomics can be applied to the elucidation of individual differences in radiosensitivity as well, and can contribute to the determination of a more appropriate treatment policy.

  By the way, squamous cell carcinoma of the esophagus is one of highly malignant cancers that have a very poor prognosis and follow a rapid progression. However, in recent years, it has been reported that the above-mentioned chemoradiotherapy (CRT) shows good treatment results for squamous cell carcinoma of the esophagus, and the effects comparable to surgical operations are effective for patients who have been successful in chemoradiotherapy (CRT). And improved prognosis. On the other hand, patients with low sensitivity to chemoradiotherapy (CRT) have the problem of not only obtaining a therapeutic effect but also causing serious side effects, which may miss the possibility of radical surgery. It was. Thus, for example, even if attention is paid only to esophageal squamous cell carcinoma, for example, the progress of research on individual treatment for selecting treatment suitable for individual patients is desired at present.

  In order to contribute to the realization of such tailor-made medicine, various markers for diagnosing a lesion, determining the therapeutic effect on the lesion, and prognosis have been proposed. For example, a method for determining the concentration of B-type natriuretic peptide for acute coronary syndrome has been proposed (see Patent Document 1). In addition, a technique for diagnosing a tumor by detecting chemokine CXCL16 and evaluating the anticancer action of an anticancer drug has been proposed (see Patent Document 2).

  The human REG (Regenerating gene) gene was found from the search for genes responsible for the regenerative proliferation of pancreatic islets of Langerhans in relation to diabetes, and the rat and mouse reg genes correspond to this gene. . The base sequence of the human REG gene is reported in Patent Document 3, for example. The human REG gene is known to have a multigene family classified into four subclasses of REG1, REG2, REG3, and REG4 according to the primary structure of the encoded protein. Here, the REG1 gene was discovered as a gene encoding pancreatic β-cell regenerative growth factor (REG1 protein), which was discovered as a gene specifically expressed in regenerated islets of Langerhans in rats administered with nicotinamide after 90% pancreatic resection Is. The REG1 gene includes a REG1A gene and a REG1B gene, which code for proteins secreted from the exocrine pancreatic glands and are located on the short arm of chromosome 2 (2p12). More specifically, the REG1A gene is a gene consisting of 821 bp (Refseq Accession No. NM_002909, SEQ ID NO: 1) and encodes a protein consisting of 166 amino acids (REG1A protein; RefSeq Accession No. AAH05350, SEQ ID NO: 2). is doing. The REG1B gene is a gene consisting of 812 bp (Refseq Accession No. NM — 006507, SEQ ID NO: 3) and encodes a protein consisting of 166 amino acids (REG1B protein; RefSeq Accession No. AAH27895, SEQ ID NO: 4). .

JP 2006-234823 A JP 2008-35836 A JP-A-1-132388

  The present invention relates to a marker (gene) that can contribute to the promotion of custom-made medical treatment of chemoradiotherapy (CRT) combined with chemotherapy and radiotherapy used in the treatment of cancer (particularly esophageal squamous cell carcinoma). It is an object of the present invention to provide a useful means for cancer diagnosis and therapeutic effect and prognosis determination.

  In order to solve the above-mentioned problems, the present inventors have intensively studied. In the process, we found that certain human esophageal squamous cell carcinoma cell lines were significantly more sensitive to anticancer drugs and radiation than other cell lines. In addition, the REG1 gene (REG1A gene and REG1B gene) is identified as a gene that is significantly highly expressed in the former cell line, and the expression of the gene contributes to high sensitivity to cancer treatment (chemotherapy and radiation therapy). Confirmed that. Furthermore, it has been found that if the gene is forcibly introduced to induce high expression, sensitivity to cancer treatment can be enhanced, and the present invention has been completed.

  That is, according to the first aspect of the present invention, a method for determining the therapeutic sensitivity of cancer cells is provided. The determination method comprises a step of measuring an expression level (A1) of a REG1A gene or a gene functionally equivalent thereto or a REG1B gene or a gene functionally equivalent thereto in a test tissue or a test cell; By comparing the expression level (A1) with the predetermined expression level (A0), and by comparing whether the expression level (A1) is significantly higher than the expression level (A0) Predicting therapeutic sensitivity of the test tissue or the test cell.

  At this time, the expression level (A1) can be measured, for example, by measuring the amount of mRNA expressed from the REG1 gene by Northern blotting, or by measuring the amount of protein expressed from the gene by Western blotting. . Further, the test tissue / test cell is preferably a tissue / cell derived from squamous cell carcinoma such as esophageal squamous cell carcinoma.

According to the second aspect of the present invention, in the above determination method, when measuring the amount of mRNA expressed from the REG1 gene, a nucleic acid used as a nucleic acid probe or PCR primer for measuring the amount of mRNA is also provided. . In this case, the nucleic acid is the following (a) or (b):
(A) a nucleic acid having a part of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 or a base sequence complementary thereto;
(B) A nucleic acid that hybridizes with a nucleic acid comprising the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 under highly stringent conditions or a nucleic acid having a complementary base sequence thereto.

  According to the third aspect of the present invention, when the amount of protein expressed from the REG1 gene is measured in the determination method, an antibody used for measuring the amount of protein is also provided. The antibody is an antibody against a protein comprising the same or substantially the same amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, a partial peptide thereof, or a pharmaceutically acceptable salt thereof.

  According to the 4th form of this invention, the kit for determining the therapeutic sensitivity of a cancer cell is provided. The kit includes an expression measuring means for measuring the expression of the REG1A gene or a gene functionally equivalent thereto, or a REG1B gene or a gene functionally equivalent thereto, and an expression detecting means for detecting the expression of the gene. . In the kit, the expression measuring means can be, for example, a microarray or a PCR primer.

  According to the fifth aspect of the present invention, there is provided a method for evaluating a compound effective for improving the therapeutic sensitivity of cancer cells. The evaluation method comprises a step of administering or contacting a test compound to a test animal or a test cell, and a REG1A gene or a functionally equivalent gene in the test animal or test cell, or The step of measuring the expression level (B1) of the REG1B gene or a gene functionally equivalent thereto in vitro, and comparing the expression level (B1) with the expression level (B0) before administration or contact of the test compound The effectiveness of the test compound for improving the therapeutic sensitivity of cancer cells by determining whether the expression level (B1) is significantly higher than the expression level (B0) as a result of comparison with the step And a step of evaluating.

According to the sixth aspect of the present invention, treatment of cancer cells (preferably cells of squamous cell carcinoma such as esophageal squamous cell carcinoma) comprising at least one of the following (a) to (c) as an active ingredient: Sensitivity (eg, sensitivity to radiation and / or anticancer agents) enhancers are provided;
(A) a nucleic acid having the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3;
(B) a nucleic acid encoding a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or a partial peptide thereof;
(C) a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or a partial peptide thereof, or a pharmaceutically acceptable salt thereof.

  According to the present invention, the REG1 gene is provided as a marker (gene) that can contribute to the promotion of custom-made medical treatment for cancer (particularly esophageal squamous cell carcinoma). By using the REG1 gene as a marker, various means useful for diagnosis of cancer, determination of therapeutic effect and prognosis are provided.

In Example 1, it is a figure which shows the result which confirmed the expression of REG1 (alpha) mRNA in each cell type | system | group by RT-PCR method. It is a graph which shows the result of having exposed each cell line to the single environment which assumed radiotherapy (Example 1). It is a graph which shows the result of having exposed each cell line to the single environment which assumed chemotherapy (Example 1). It is a graph which shows the result of having exposed each cell line to the combined environment assumed chemoradiotherapy (CRT) (Example 1). In Example 1, it is a graph which shows the result of having performed the caspase-3 assay. It is a graph showing the change of each survival rate of the REG1 (alpha) positive patient which performed chemoradiotherapy, and the REG1 (alpha) negative patient which performed chemoradiotherapy (Example 3). The vertical axis represents the overall survival rate with respect to the total of all causes of death (total cause of death), and the horizontal axis represents the number of months (Time (months)). It is a graph showing the change of each survival rate of the REG1 (alpha) positive patient which performed chemoradiotherapy, and the REG1 (alpha) negative patient which performed chemoradiotherapy (Example 3). The vertical axis is the survival rate (Esophageal cancer-specific survival rate) only when the cause of death is squamous cell carcinoma of the esophagus, and the horizontal axis is the number of months.

  Hereinafter, although the form for implementing this invention is demonstrated in detail, the technical scope of this invention is not limited only to the following form.

  The protein used in the present invention (also referred to as “protein of the present invention”) is a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. The protein of the present invention is used for cells of humans and other warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits, pigs, sheep, cows, monkeys, etc.) [eg, hepatocytes, spleen cells, neurons, glia. Cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages) , T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts Breast cells or stromal cells, or precursor cells of these cells, stem cells or cancer cells, etc.] or any tissue in which these cells are present [eg, brain, brain regions (eg, olfactory bulb, amygdaloid nucleus, large cell) Basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle (eg, smooth muscle, Skeletal muscle), lungs, gastrointestinal tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (eg, White adipose tissue, brown adipose tissue, etc.)] and the like. Alternatively, it may be a protein synthesized chemically or biochemically in a cell-free translation system, or a recombinant produced from a transformant introduced with a nucleic acid having a base sequence encoding the amino acid sequence. It may be a protein.

  The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 is about 50% or more, preferably about 50% or more of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. Amino acid sequences having a homology (homology) of 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more. It is done. As used herein, “homology” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm uses a sequence of sequences for optimal alignment). The percentage of identical and similar amino acid residues relative to all overlapping amino acid residues in which one or both of the gaps can be considered). “Similar amino acids” mean amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn). ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

  The homology of the amino acid sequences in this specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Local Alignment Search Tool), and the following conditions (expectation value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF). Other algorithms for determining amino acid sequence homology include, for example, Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997)) ] Needleman et al., J .; Mol. Biol. 48: 444-453 (1970) [the algorithm is incorporated into the GAP program in the GCG software package], Myers and Miller, CABIOS, 4: 11-17 (1988) [ The algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package], Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is incorporated in the FASTA program in the GCG software package] and the like, and these can be preferably used as well.

  More preferably, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 is about 50% or more of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. Preferably about 60% or more, more preferably about 70% or more, even more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more. Amino acid sequence.

  The protein used in the present invention contains an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, and the amino acid represented by SEQ ID NO: 2 or SEQ ID NO: 4. A protein having substantially the same activity as a protein containing a sequence.

  Examples of substantially the same activity include ligand binding activity and signal information transmission action. Here, “substantially the same quality” indicates that these properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Accordingly, it is preferable that the activities of the protein of the present invention are equivalent, but the degree of these activities (eg, about 0.01 to about 100 times, preferably about 0.1 to about 10 times, more preferably about 0.1 times). Quantitative factors such as 5 to 2 times) and the molecular weight of the protein may be different. The measurement of activities such as ligand binding activity and signal information transmission can be performed according to a method known per se.

  Examples of the protein used in the present invention include (i) one or more (for example, about 1 to 50, preferably about 1 to 50 in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, An amino acid sequence in which about 1 to 30, more preferably about 1 to 10, more preferably (1 to 5, 4, 3 or 2) amino acids are deleted; (ii) SEQ ID NO: 2 or sequence 1 or 2 or more (for example, about 1 to 50, preferably about 1 to 30, more preferably about 1 to 10, more preferably a number (1 to 5) in the amino acid sequence represented by No. 4: 4, 3 or 2) amino acid sequence added, (iii) one or more (for example, about 1 to 50) in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 , Preferably 1 An amino acid sequence in which about 0, more preferably about 1 to 10, more preferably a number (1 to 5, 4, 3 or 2) amino acids are inserted; (iv) SEQ ID NO: 2 or SEQ ID NO: 1 or 2 or more (for example, about 1 to 50, preferably about 1 to 30, more preferably about 1 to 10, more preferably a number (1 to 5, Also included are so-called muteins such as amino acid sequences in which 4, 3 or 2) amino acids are replaced with other amino acids, or (v) amino acid sequences combining them.

  When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.

  Preferred examples of the protein used in the present invention include, for example, human REG1A (Refseq Accession No. AAH05350) containing the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 4. Human REG1B (Refseq Accession No. AAH27895) etc. are mentioned.

In the present specification, proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. The protein used in the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, has a C-terminal carboxyl group (—COOH), carboxylate (—COO ⁻ ), amide Either (—CONH ₂ ) or ester (—COOR) may be used.

Here, as R in the case where the C-terminal is an ester (—COOR), for example, C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, C ₃ such as cyclopentyl, cyclohexyl, etc. _-8 cycloalkyl groups, C _6-12 aryl groups such as phenyl and α-naphthyl, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl C _7-14 aralkyl group such as, pivaloyloxymethyl group and the like are used.

  When the protein used in the present invention has a carboxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.

Furthermore, the protein used in the present invention, the amino acid residue (eg, methionine residue) of the N-terminal amino group protecting group (e.g., formyl group, such as C _1-6 alkanoyl such as acetyl group C _{1- 6} acyl group, etc., an N-terminal glutamine residue produced by cleavage in vivo is pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (eg, —OH, — SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected with an appropriate protecting group (for example, C _1-6 acyl group such as C _1-6 alkanoyl group such as formyl group, acetyl group, etc.) Or a complex protein such as a so-called glycoprotein having a sugar chain bound thereto.

  The partial peptide of the protein used in the present invention is any peptide as long as it is a peptide having the partial amino acid sequence of the protein used in the present invention and has substantially the same activity as the protein. Also good. Here, “substantially the same quality of activity” has the same meaning as described above. In addition, the “substantially the same quality of activity” can be measured in the same manner as the protein used in the present invention.

  For example, the amino acid sequence of the protein used in the present invention has at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 150 or more amino acid sequences. Peptides are used.

  The partial peptide used in the present invention is one or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1 to 5, 4) in the amino acid sequence. 3 or 2) amino acids are deleted, or one or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1) in the amino acid sequence. -5, 4, 3 or 2) amino acids), or one or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably about the amino acid sequence) A number (1-5, 4, 3 or 2) amino acids are inserted, or one or more (preferably about 1-20, more preferably about 1-10) in the amino acid sequence. Even more preferred Ku is the amino acid number (1～5,4,3 or 2) pieces) may be replaced with other amino acids.

In the partial peptide used in the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). Here, examples of R in the case where the C-terminus is an ester (—COOR) include the same as those described above for the protein used in the present invention. When the partial peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the same ester as the C-terminal ester is used.

  Furthermore, in the partial peptide used in the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected with a protecting group, similar to the protein used in the present invention described above, A glutamine residue produced by cleavage of the N-terminal side in vivo is pyroglutamine oxidized, a substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, or a sugar chain is bound Complex peptides such as so-called glycopeptides are also included.

  The protein or its partial peptide used in the present invention may be a free form or a salt (the same applies hereinafter unless otherwise specified). Such salts include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts), and particularly physiologically acceptable acid addition salts. Is preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The protein used in the present invention can be produced by purifying from the aforementioned human or other warm-blooded animal cells or tissues using a known protein purification method. Specifically, for example, mammalian tissues or cells are homogenized in the presence of a surfactant, and the resulting crude extract fraction of tissues is subjected to chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like. The protein used in the present invention can be prepared by subjecting to a graph or the like.

  The protein or its partial peptide used in the present invention can also be produced according to a known peptide synthesis method.

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 partial peptide or amino acid that can constitute the protein used in the present invention or a partial peptide thereof is condensed with the remaining part, and when the product has a protective group, the target protein (peptide ) Can be manufactured. Examples of known condensation methods and protecting group removal include the methods described in the following (i) to (v).
(I) M.M. Bodanszky and M.M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(Ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(Iii) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(Iv) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
(V) Supervised by Haruaki Yajima, Development of follow-up medicine, Volume 14, Peptide synthesis, Hirokawa Shoten The protein (peptide) thus obtained can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.

  When the protein (peptide) obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the protein (peptide) is obtained as a salt It can be converted into a free form or other salt by a known method or a method analogous thereto.

  For the synthesis of the protein used in the present invention or a partial peptide thereof, or an amide thereof, a commercially available resin for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein or partial peptide is excised from the resin, and at the same time, various protecting groups are removed, and further, intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or their amides. To do.

  For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide, and the like. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (eg, HOBt, HOBt), or the protected amino acid is activated in advance as a symmetric acid anhydride, HOBt ester or HOBt ester. It can later be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be used for protein condensation reactions. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range known to be usable for protein bond formation reaction, and is usually in the range of about -20 to about 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even if the reaction is repeated, acetylation of the unreacted amino acid with acetic anhydride or acetylimidazole can prevent the subsequent reaction from being affected.

  The protection of the functional group that should not be involved in the reaction of the raw material, the protecting group, the elimination of the protecting group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.

  Examples of the protective group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.

  The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower (C _1-6 ) alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Used. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.

Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like.

  Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.

  Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to about 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1, Addition of a cation scavenger such as 4-butanedithiol and 1,2-ethanedithiol is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

  Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.

  As another method for obtaining an amide form of a protein or partial peptide, for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide (protein) chain is formed on the amino group side to the desired chain length. After the extension, a protein or partial peptide from which only the N-terminal α-amino group protecting group of the peptide chain is removed and a protein or partial peptide from which only the C-terminal carboxyl group protecting group has been removed are produced, and these The protein or peptide is condensed in a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude protein or peptide. This crude protein or peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain an amide form of the desired protein or peptide.

  In order to obtain a protein or peptide ester, for example, the α-carboxyl group of the carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired protein or peptide amide is obtained in the same manner as the protein or peptide amide. An ester of a peptide can be obtained.

  The partial peptide of the protein used in the present invention can be produced by cleaving the protein used in the present invention with an appropriate peptidase.

  Furthermore, the protein used in the present invention or a partial peptide thereof is produced by culturing a transformant containing a polynucleotide encoding the protein or separating and purifying the protein or the partial peptide from the obtained culture. You can also. The polynucleotide encoding the protein or its partial peptide used in the present invention may be DNA or RNA, or may be a DNA / RNA chimera. Preferably, DNA is used. The polynucleotide may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).

  Examples of the polynucleotide encoding the protein or its partial peptide used in the present invention include genomic DNA, genomic DNA library, mammal (eg, human, cow, monkey, horse, pig, sheep, goat, dog, cat, guinea pig , Rats, mice, rabbits, hamsters, etc.) [eg hepatocytes, spleen cells, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, Endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils Spheres, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells or stromal cells, or this Progenitor cells, stem cells, cancer cells, etc.] or any tissue in which these cells exist [eg, brain, brain parts (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebrum) Cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, Spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, adipose tissue (eg, brown adipose tissue, white adipose tissue), skeletal muscle, etc.] It is done. The genomic DNA and cDNA encoding the protein or its partial peptide used in the present invention are the polymerase chain reaction (Polymerase Chain) using the genomic DNA fraction and total RNA or mRNA fraction prepared from the cells / tissues described above as templates, respectively. Direct amplification can also be carried out by RT-PCR using Reaction (hereinafter also referred to as “PCR method”) and reverse transcriptase (Reverse Transcriptase). Alternatively, genomic DNA and cDNA encoding the protein used in the present invention or a partial peptide thereof are prepared by inserting genomic DNA and total RNA or mRNA fragments prepared from the cells and tissues described above into an appropriate vector. It can also be cloned from a genomic DNA library and a cDNA library by colony or plaque hybridization method or PCR method, respectively. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.

  Examples of the DNA encoding the protein used in the present invention include a base sequence that hybridizes under high stringency conditions with a DNA containing the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3. Any DNA encoding a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 may be used.

  Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 under highly stringent conditions include, for example, about 50 from the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3. % Or more, preferably about 60% or more, more preferably about 70% or more, even more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more homology (homology). DNA containing a base sequence having If the homology is smaller than the lower limit of the above range, there is a high possibility that it does not exhibit the same or similar function as the corresponding gene. However, even if the homology is less than the lower limit of the above range, a domain having a function specific to the corresponding gene and the same or similar function when the homology between the base sequence corresponding to the domain is high May have. Such a gene (DNA) can be suitably used even if the base sequence homology is outside the above range.

  The homology of the base sequence in the present specification uses, for example, the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Local Alignment Search Tool), and the following conditions (expectation value = 10; allow gap; filtering = ON Match score = 1; mismatch score = -3). As another algorithm for determining the homology of a base sequence, the above-mentioned algorithm for calculating homology of amino acid sequences is also preferably exemplified.

  Hybridization is a method known per se or a method analogous thereto, for example, Molecular Cloning 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. More preferably, it can be carried out according to highly stringent conditions.

  High stringent conditions mean, for example, conditions in which the sodium concentration is about 19 to about 40 mM, preferably about 19 to about 20 mM, and the temperature is about 50 to about 70 ° C., preferably about 60 to about 65 ° C. . In particular, it is preferable that the sodium salt concentration is about 19 mM and the temperature is about 65 ° C. A person skilled in the art may appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc. Thus, the desired stringency can be easily adjusted.

  The DNA encoding the protein used in the present invention preferably contains a DNA (Refseq Accession No. NM_002909) containing a base sequence represented by SEQ ID NO: 1, and a base sequence represented by SEQ ID NO: 3. DNA (Refseq Accession No. NM_006507) etc. are mentioned.

  The polynucleotide (eg, DNA) encoding the partial peptide of the protein used in the present invention is any as long as it contains a base sequence encoding the partial peptide of the protein used in the present invention described above. Also good. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used.

  Examples of the DNA encoding the partial peptide of the protein used in the present invention include a partial base sequence of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, or represented by SEQ ID NO: 1 or SEQ ID NO: 3. DNA encoding a peptide that contains a base sequence that hybridizes with a polynucleotide containing the base sequence to be used under highly stringent conditions and that has substantially the same activity as the protein used in the present invention is used. It is done. The DNA capable of hybridizing with the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 has the same meaning as described above. As the hybridization method and highly stringent conditions, the same ones as described above are used.

  Means for cloning of DNA encoding the protein used in the present invention and its partial peptide (hereinafter, in the description of cloning and expression of DNA encoding these, they may be simply referred to as “protein of the present invention”) As described above, a synthetic DNA primer having a part of the base sequence encoding the protein of the present invention is used to amplify by PCR method, or a DNA incorporated into an appropriate vector is used for a part or all of the region of the protein of the present invention. Selection can be performed by hybridization with a DNA fragment to be encoded or labeled with a synthetic DNA. Hybridization methods are described in, for example, Molecular Cloning 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.

  The DNA base sequence is converted by PCR, a known kit such as Mutan ™ -super Express Km (Takara Shuzo), Mutan ™ -K (Takara Shuzo), etc., using the ODA-LA PCR method, Gapped duplex. The method can be carried out according to a method known per se such as the Kunkel method, or a method analogous thereto.

  The DNA encoding the cloned protein can be used as it is or after digestion with a restriction enzyme or addition of a linker if desired. The DNA 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 expression vector for the protein of the present invention can be produced, for example, by excising the target DNA fragment from the DNA encoding the protein of the present invention and ligating the DNA fragment downstream of the promoter in an appropriate expression vector. it can.

  Examples of the vector include plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), plasmids derived from yeast (eg, pSH19, pSH15), λ phage, and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used.

  The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when animal cells are used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.

  Of these, the CMV (cytomegalovirus) promoter, SRα promoter and the like are preferably used. When the host is Escherichia, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter, etc., and when the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

  In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter also referred to as “SV40ori”) and the like can be used as desired. Examples of selection markers include dihydrofolate reductase (hereinafter also referred to as “dhfr”) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter also referred to as “Ampr”), neomycin resistance gene (hereinafter referred to as “Neor”). And G418 resistance). In particular, when a dhfr gene-deficient Chinese hamster cell is used and the dhfr gene is used as a selection marker, the target gene can also be selected by a medium not containing thymidine.

  If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is Escherichia, PhoA signal sequence, OmpA, signal sequence, etc., and when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc. are used in yeast. In some cases, MFα • signal sequence, SUC2 • signal sequence and the like can be used, and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence and the like can be used.

  A transformant can be produced using a vector containing the DNA encoding the protein of the present invention thus constructed. As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.

  Specific examples of the genus Escherichia include, for example, Escherichia coli K12 · DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal of Molecular 41 Bio (45). 1969)], C600 [Genetics, 39, 440 (1954)] and the like.

  Examples of the Bacillus bacterium include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)].

  Examples of the yeast include, for example, Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCI KM71 or the like is used.

  As insect cells, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, and Fig cells derived from eggs of Trichoplusia ni , Cells derived from Mamestra brassicae or cells derived from Estigmena acrea. When the virus is BmNPV, sputum-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells [above, Vaughn, J. et al. L. et al. , In Vivo, 13, 213-217 (1977)].

  Examples of insects include silkworm larvae [Maeda et al. , Nature, 315, 592 (1985)].

Examples of animal cells include monkey cells COS-1, COS-3, COS-7, Vero, Chinese hamster ovary cells (hereinafter also referred to as “CHO cells”), dhfr gene-deficient CHO cells (hereinafter referred to as CHO (dhfr ⁻ )). Mouse L cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells, human 293 cells, human HeLa cells, and the like.

  For transforming Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972) and Gene, 17, 107 (1982).

  To transform Bacillus, for example, Mol. Gen. Genet. 168, 111 (1979) and the like.

  To transform yeast, see, for example, Meth. Enzymol. , 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

  Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).

  In order to transform animal cells, for example, cell engineering supplement 8 New Cell Engineering Experimental Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973) can be used. it can.

  In this way, a transformant transformed with an expression vector containing DNA encoding the protein can be obtained.

  When cultivating a transformant whose host is an Escherichia bacterium or Bacillus genus, a liquid medium is suitable as a medium used for the culture, including a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, and potato extract. Examples of inorganic or organic substances and inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to about 8.

  As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acids [Miller, Journal of Experts in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New 72] is preferred. In order to make the promoter work efficiently as necessary, a drug such as 3β-indolylacrylic acid can be added.

  When the host is an Escherichia bacterium, the culture is usually performed at about 15 to about 43 ° C. for about 3 to about 24 hours, and if necessary, aeration or agitation can be added.

  When the host is a genus Bacillus, the culture is usually performed at about 30 to about 40 ° C. for about 6 to about 24 hours, and if necessary, aeration or agitation can be added.

  When cultivating a transformant whose host is yeast, examples of the medium include a Burkholder minimum medium [Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5 to about 8. The culture is usually performed at about 20 to about 35 ° C. for about 24 to about 72 hours, and aeration and agitation are added as necessary.

When cultivating a transformant whose host is an insect cell, the medium is appropriately added with additives such as 10% bovine serum deactivated in Grace's Insect Medium (Nature, 195,788 (1962)) Etc. are used. The pH of the medium is preferably adjusted to about 6.2 to about 6.4. Culture is usually performed at about 27 ° C. for about 3 to about 5 days, and aeration (for example, 5% CO ₂ ) or agitation is added as necessary.

  When culturing a transformant whose host is an animal cell, examples of the medium include MEM medium [Science, 122,501 (1952)] containing about 5 to about 20% fetal calf serum, DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [J. Am. Med. Assoc. 199, 519 (1967)], 199 medium [Proc. Soc. Biol. Med. , 73, 1 (1950)]. The pH is preferably from about 6 to about 8. Culture is usually performed at about 30 to about 40 ° C. for about 15 to about 60 hours, and aeration and agitation are added as necessary.

  As described above, the protein of the present invention can be produced inside the cell, the cell membrane, or extracellularly of the transformant.

  Separation and purification of the protein of the present invention from the culture can be performed, for example, by the following method.

  When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thaw, etc. For example, a method of obtaining a crude protein extract by centrifugation or filtration after destroying cells or cells by the method 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 protein is secreted into the culture solution, after completion of the culture, the cells or cells and the supernatant are separated by a method known per se, and the supernatant is collected.

  Purification of the protein of the present invention contained in the thus obtained culture supernatant or extract can be performed by appropriately combining known separation / purification methods. These known separation and purification methods include mainly molecular weights such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method using difference in charge, method using difference in charge such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and difference in hydrophobicity such as reverse phase high performance liquid chromatography A method using a difference in isoelectric point, such as a method, isoelectric focusing method, or the like is used.

  When the protein of the present invention thus obtained is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when obtained in a salt, It can be converted into a free form or other salt by a method known per se or a method analogous thereto.

  The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by allowing an appropriate protein modifying enzyme to act on the protein before or after purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.

  The presence of the protein of the present invention thus produced can be measured by enzyme immunoassay or Western blotting using a specific antibody.

  Furthermore, the protein used in the present invention or a partial peptide thereof is produced in vitro using a cell-free protein translation system comprising a rabbit reticulocyte lysate, a wheat germ lysate, an E. coli lysate, etc., using an RNA corresponding to the DNA encoding it as a template. It can also be synthesized by translating. Alternatively, a cell-free transcription / translation system further containing RNA polymerase can be used to synthesize calmodulin or a DNA encoding a partial peptide thereof as a template. A commercially available cell-free protein (transcription /) translation system can be used, or a method known per se, specifically, an Escherichia coli extract can be obtained from Pratt J. et al. M.M. Et al., “Transscript and Translation”, Hames B. et al. D. And HigginsS. J. et al. Hen, IRL Press, Oxford 179-209 (1984). Commercially available cell lysates include those derived from E. coli. Examples include E. coli S30 extract system (Promega) and RTS 500 Rapid Translation System (Roche), and those derived from rabbit reticulocytes are those of Rabbit Reticulocyte Lysate System (Promega). PROTEIOSTM (made by TOYOBO) etc. are mentioned. Of these, those using wheat germ lysate are preferred. As a method for producing wheat germ lysate, for example, Johnston F. et al. B. Et al., Nature, 179, 160-161 (1957) or Erickson A. et al. H. Et al., Meth. Enzymol. 96, 38-50 (1996) or the like.

  As a system or apparatus for protein synthesis, there are a batch method (Pratt, JM et al. (1984) mentioned above), a continuous cell-free protein synthesis system (amino acid, energy source, etc.) that continuously supplies a reaction system ( Spirin AS et al., Science, 242, 1162-1164 (1988)), dialysis method (Kikawa et al., 21st Japan Molecular Biology Society, WID6), or multi-layer method (PROTEIOSTMWheat germ cell synthesis core kit handling Manual: manufactured by TOYOBO). Furthermore, a method of supplying template RNA, amino acid, energy source and the like to the synthesis reaction system when necessary, and discharging a synthesized product or a decomposed product when necessary (Japanese Patent Laid-Open No. 2000-333673) can be used.

  An antibody to the protein used in the present invention or a partial peptide thereof (hereinafter also referred to as “the antibody of the present invention”) can be a polyclonal antibody or a monoclonal antibody as long as it can recognize the protein used in the present invention or a partial peptide thereof. Any of these may be used. The isotype of the antibody is not particularly limited, but is preferably IgG, IgM or IgA, and particularly preferably IgG.

The antibody of the present invention is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding a target antigen. For example, Fab, Fab ′ other than a complete antibody molecule , F (ab ′) _{2 and} other fragments, scFv, scFv-Fc, conjugation molecules prepared by genetic engineering such as minibodies and diabodies, or molecules having protein stabilizing action such as polyethylene glycol (PEG) The derivative | guide_body modified by these etc. may be sufficient.

  An antibody against a protein used in the present invention or a partial peptide thereof (hereinafter, also referred to as “protein of the present invention” in the description of the antibody) is produced according to a known method for producing an antibody or antiserum. Can do.

  Below, the immunogen preparation method of the antibody of this invention and the manufacturing method of the said antibody are demonstrated.

(1) Preparation of antigen As an antigen used for preparing the antibody of the present invention, the above-described protein of the present invention or a partial peptide thereof, or one or more of the same antigenic determinants (synthesis) ) Any peptide or the like can be used (hereinafter, these are also simply referred to as “antigens of the present invention”).

  As described above, the protein of the present invention or a partial peptide thereof is prepared using, for example, a known method or a method analogous thereto from tissues or cells of warm-blooded animals such as (a) humans, monkeys, rats, mice, chickens, (B) chemically synthesized by a known peptide synthesis method using a peptide synthesizer or the like, (c) cultivating a transformant containing DNA encoding the protein of the present invention or a partial peptide thereof, or (d) this It is produced by biochemical synthesis using a cell-free transcription / translation system using a nucleic acid encoding the protein of the invention or a partial peptide thereof as a template.

  (A) When the protein of the present invention is prepared from a tissue or cell of a warm-blooded animal, after homogenizing the tissue or cell, the crude fraction (for example, a membrane fraction or a soluble fraction) is used as an antigen as it is. You can also. Alternatively, extraction with acid, surfactant, alcohol, etc. is performed, and the resulting extract is purified by combining chromatography such as salting out, dialysis, gel filtration, reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc. It can also be isolated. The obtained protein of the present invention can be used as an immunogen as it is, or a partial peptide can be prepared by limited degradation using a peptidase or the like and used as an immunogen.

  (B) When the antigen of the present invention is chemically prepared, the synthetic peptide includes, for example, a peptide having the same structure as that of the protein of the present invention purified from a natural material using the method (a) described above. Specifically, a peptide or the like containing one or more amino acid sequences identical to the amino acid sequence of an arbitrary position consisting of 3 or more, preferably 6 or more amino acids in the amino acid sequence of the protein is used.

  (C) When the antigen of the present invention is produced using a transformant containing DNA, the DNA can be obtained by a known cloning method [for example, Molecular Cloning 2nd ed. (Methods described in J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989)]. As the cloning method, (1) a DNA probe designed based on the gene sequence encoding the protein of the present invention is used and DNA encoding the antigen is isolated from a cDNA library by a hybridization method, or (2) A DNA primer designed based on the gene sequence encoding the protein of the present invention, a DNA encoding the antigen by a PCR method using cDNA as a template, and inserting the DNA into an expression vector suitable for the host, etc. Can be mentioned. A desired antigen can be obtained by culturing a transformant obtained by transforming a host with the expression vector in an appropriate medium.

  (D) When a cell-free transcription / translation system is used, an expression vector inserted with a DNA encoding an antigen prepared by the same method as in (c) above (for example, the DNA is under the control of a T7, SP6 promoter, etc. MRNA is synthesized using a transcription reaction solution containing RNA polymerase compatible with the promoter and a substrate (NTPs) using a placed expression vector or the like as a template, and then a known cell-free translation system (for example, And a method of performing a translation reaction using an extract of Escherichia coli, rabbit reticulocytes, wheat germ, etc.). By appropriately adjusting the salt concentration and the like, the transcription reaction and the translation reaction can be carried out collectively in the same reaction solution.

  As the immunogen, a complete molecule of the protein of the present invention or a peptide having a partial amino acid sequence thereof can be used. Examples of the partial amino acid sequence include those consisting of 3 or more consecutive amino acid residues, preferably 4 or more, more preferably 5 or more, and even more preferably 6 or more consecutive amino acid residues. It is done. Alternatively, the amino acid sequence includes, for example, 20 or less consecutive amino acid residues, preferably 18 or less, more preferably 15 or less, and even more preferably 12 or less consecutive amino acid residues. Is mentioned. Some of these amino acid residues (for example, 1 to several) may be substituted with a substitutable group (for example, Cys residue, hydroxyl group, etc.). A peptide used as an immunogen has an amino acid sequence containing one to several such partial amino acid sequences.

  Alternatively, the warm-blooded animal cell itself that expresses the protein of the present invention can be directly used as the antigen of the present invention. As the warm-blooded animal cell, a natural cell as described in the above section (a), a cell transformed by the method as described in the above section (c), and the like can be used. The host used for transformation may be any cell collected from humans, monkeys, rats, mice, hamsters, chickens, and the like. HEK293, COS7, CHO-K1, NIH3T3, Balb3T3, FM3A, L929, SP2 / 0, P3U1, B16, P388 or the like is preferably used. Natural warm-blooded animal cells or transformed warm-blooded animal cells that express the protein of the present invention may be cultured in a medium (eg, RPMI 1640) or buffer used in tissue culture (eg, Hanks' Balanced Salt Solution). Immunized animals can be injected in suspension in HBSS)). As the immunization method, any method can be used as long as it can promote antibody production, and intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, and the like are preferably used.

  The antigen of the present invention can be directly immunized if it has immunogenicity, but it has a low molecular weight (for example, a molecular weight of about 3) having only one to several antigenic determinants in the molecule. , 1,000 or less) antigens (that is, partial peptides of the protein of the present invention), since these antigens are usually hapten molecules with low immunogenicity, they are bound to or adsorbed to a suitable carrier (carrier). It can be immunized as a complex. A natural or synthetic polymer can be used as the carrier. Examples of natural polymers include serum albumin of mammals such as cows, rabbits and humans, and thyroglobulin of mammals such as cows and rabbits, such as chicken ovalbumin, such as mammals such as cows, rabbits, humans and sheep. Hemoglobin, keyhole limpet hemocyanin (KLH) and the like are used. Examples of the synthetic polymer include various latexes such as polymers or copolymers such as polyamino acids, polystyrenes, polyacryls, polyvinyls, and polypropylenes.

  The mixing ratio of the carrier and the hapten is such that any antibody can be bound or adsorbed at any ratio as long as an antibody against the antigen bound or adsorbed to the carrier is efficiently produced. The above-mentioned natural or synthetic polymer carrier, which is commonly used in the production of the above, can be used by binding or adsorbing the hapten 1 at a ratio of 0.1 to 100 by weight.

  Various condensing agents can be used for coupling the hapten and the carrier. For example, diazonium compounds such as bisdiazotized benzidine that crosslinks tyrosine, histidine, and tryptophan, dialdehyde compounds such as glutaraldehyde that crosslink amino groups, diisocyanate compounds such as toluene-2,4-diisocyanate, and thiol groups A dimaleimide compound such as N, N′-o-phenylene dimaleimide, a maleimide active ester compound that crosslinks an amino group and a thiol group, a carbodiimide compound that crosslinks an amino group and a carboxyl group, and the like are advantageously used. Moreover, when cross-linking amino groups, an active ester reagent having a dithiopyridyl group on one amino group (for example, N-succinimidyl (SPDP) 3- (2-pyridyldithio) propionate) was reacted. It is also possible to introduce a thiol group by post-reduction, introduce a maleimide group into the other amino group with a maleimide active ester reagent, and then react both.

(2) Production of monoclonal antibody (a) Production of monoclonal antibody-producing cell The antigen of the present invention is administered to a warm-blooded animal by, for example, intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, etc. It is administered to a site where production is possible by itself or together with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 1 to 6 weeks, about 2 to 10 times in total. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, hamsters, sheep, goats, donkeys, and chickens. In order to avoid the problem of anti-Ig antibody production, it is preferable to use a mammal of the same species as that of the administration target, but mice and rats are generally preferably used for the production of monoclonal antibodies.

  Since human immunization to humans is ethically difficult, when the antibody of the present invention is to be administered to humans, (i) a human antibody-producing animal (for example, mouse) prepared according to the method described later ) To obtain a human antibody, (ii) preparing a chimeric antibody, humanized antibody or fully human antibody according to the method described below, or (iii) in vitro immunization and cell immortalization by virus, human-human (or It is preferable to obtain a human antibody by combining a mouse) hybridoma production technique, a phage display method and the like. The in vitro immunization method can acquire an antigen against an antigen whose antibody production is suppressed by normal immunization, can obtain an antibody with an antigen amount on the order of ng to μg, Since it can be completed in several days, it can be preferably used also when preparing an antibody derived from a non-human animal as a method for obtaining an antibody against an unstable antigen that is difficult to prepare in large quantities.

Examples of animal cells used for in vitro immunization include lymphocytes isolated from the peripheral blood, spleen, lymph nodes, etc. of humans and warm-blooded animals (preferably mice and rats) described above, preferably B lymphocytes and the like. . For example, in the case of mouse cells or rat cells, the spleen is removed from an animal of about 4 to 12 weeks of age, and the spleen cells are separated, and an appropriate medium (for example, Dulbecco's modified Eagle medium (DMEM), RPMI 1640 medium, Ham F12 medium, etc.) And then suspended in a medium supplemented with fetal calf serum (FCS; about 5 to 20%) containing the antigen and cultured for about 4 to 10 days using a CO ₂ incubator or the like. Examples of the antigen concentration include, but are not limited to, 0.05 to 5 μg. It is preferable to prepare a thymocyte culture supernatant of an animal of the same strain (preferably about 1 to 2 weeks of age) according to a conventional method and add it to the medium.

  In vitro immunization of human cells, it is difficult to obtain a thymocyte culture supernatant, so that several cytokines such as IL-2, IL-4, IL-5, IL-6 and optionally an adjuvant substance (for example, , Muramyl dipeptide, etc.) is preferably added to the medium together with the antigen to perform immunization.

  When producing a monoclonal antibody, select an individual or a cell population in which an antibody titer is increased from a warm-blooded animal (eg, mouse, rat) or animal cell (eg, human, mouse, rat) immunized with an antigen. The spleen or lymph nodes are collected 2 to 5 days after the final immunization, or cultured for 4 to 10 days after in vitro immunization, and then cells are collected to isolate antibody-producing cells, and these are fused with myeloma cells to produce antibodies Production hybridomas can be prepared. The antibody titer in serum can be measured, for example, by reacting a labeled antigen with antiserum and then measuring the activity of the labeling agent bound to the antibody.

  The myeloma cell is not particularly limited as long as it can produce a hybridoma that secretes a large amount of antibody, but it preferably does not produce or secrete an antibody itself, and more preferably has high cell fusion efficiency. In order to facilitate selection of hybridomas, it is preferable to use a HAT (hypoxanthine, aminopterin, thymidine) sensitive strain. For example, NS-1, P3U1, SP2 / 0, AP-1 etc. are used as mouse myeloma cells, and R210. RCY3, Y3-Ag1.2.3 and the like, and examples of human myeloma cells include SKO-007, GM1500-6TG-2, LICR-LON-HMy2, UC729-6 and the like.

  The fusion operation can be carried out according to known methods, for example the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but preferably PEG is used. The molecular weight of PEG is not particularly limited, but PEG 1000 to PEG 6000 having low toxicity and relatively low viscosity is preferable. Examples of the PEG concentration include about 10 to 80%, preferably about 30 to 50%. As the PEG dilution solution, various buffer solutions such as serum-free medium (for example, RPMI 1640), complete medium containing about 5 to 20% serum, phosphate buffered saline (PBS), Tris buffer, and the like can be used. it can. If desired, DMSO (for example, about 10 to 20%) can be added. The pH of the fusion solution is, for example, about 4 to 10, preferably about 6 to 8.

The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of bone marrow cells is usually about 1: 1 to 20: 1, and is usually incubated at 20 to 40 ° C., preferably 30 to 37 ° C. for usually 1 to 10 minutes ( For example, cell fusion can be carried out efficiently by incubating with CO ₂ .

  Antibody-producing cell lines can also be obtained by infecting antibody-producing cells with a virus capable of transforming lymphocytes to immortalize the cells. Examples of such viruses include Epstein-Barr (EB) virus. Most people have immunity because they have been infected with this virus as an asymptomatic infection of infectious mononucleosis, but when normal EB virus is used, virus particles are also produced. Appropriate purification should be performed. Recombinant EB virus that retains the ability to immortalize B lymphocytes but lacks the ability to replicate viral particles (for example, switching from a latent infection state to a lytic infection state) as an EB system without the possibility of viral contamination It is also preferable to use a deficiency in the gene.

Since B95-8 cells derived from marmoset secrete EB virus, B lymphocytes can be easily transformed using the culture supernatant. The cells are cultured, for example, in a serum and penicillin / streptomycin (P / S) -added medium (for example, RPMI 1640) or a serum-free medium to which a cell growth factor is added, and then the culture supernatant is separated by filtration or centrifugation. The antibody-producing B lymphocytes are suspended at an appropriate concentration (for example, about 10 ⁷ cells / mL) and incubated at 20 to 40 ° C., preferably 30 to 37 ° C. for usually about 0.5 to 2 hours. Antibody-producing B cell lines can be obtained. When human antibody-producing cells are provided as mixed lymphocytes, most people have T lymphocytes that are toxic to EB virus-infected cells, so to increase the frequency of transformation For example, it is preferable to remove T lymphocytes in advance by forming E rosette with sheep erythrocytes or the like. Alternatively, sheep erythrocytes bound with a soluble antigen can be mixed with antibody-producing B lymphocytes, and a lymphocyte specific for the target antigen can be selected by separating rosettes using a density gradient such as Percoll. Furthermore, by adding a large excess of antigen, antigen-specific B lymphocytes are capped and no longer present IgG on the surface, so when mixed with sheep erythrocytes bound with anti-IgG antibodies, antigen-specific B lymphocytes Only form a rosette. Therefore, antigen-specific B lymphocytes can be selected by collecting a non-rosette-forming layer using a density gradient such as Percoll from this mixture.

  Human antibody-secreting cells that have acquired unlimited proliferative ability by transformation can be back-fused with mouse or human myeloma cells in order to stably maintain the antibody-secreting ability. As the myeloma cells, the same ones as described above can be used.

Hybridoma screening and breeding are usually performed in animal cell culture medium (for example, RPMI 1640) containing 5-20% FCS or serum-free medium supplemented with cell growth factor, with addition of HAT (hypoxanthine, aminopterin, thymidine). Is called. Examples of the concentrations of hypoxanthine, aminopterin, and thymidine include about 0.1 mM, about 0.4 μM, and about 0.016 mM, respectively. For selection of human-mouse hybridomas, ouabain resistance can be used. Since human cell lines are more sensitive to ouabain than mouse cell lines, unfused human cells can be eliminated by adding them to the medium at about 10 ⁻⁷ to 10 ⁻³ M.

  For selection of hybridomas, it is preferable to use feeder cells or certain cell culture supernatants. Feeder cells can be irradiated by irradiation with cells that can produce large amounts of growth factors that are useful for the appearance of hybridomas. Those having reduced proliferative power are used. For example, mouse feeder cells include spleen cells, macrophages, blood, thymocytes, and the like, and human feeder cells include peripheral blood mononuclear cells and the like. Examples of the cell culture supernatant include primary culture supernatants of the above-mentioned various cells and culture supernatants of various cell lines.

  Hybridomas can also be selected by separating the cells that bind to the antigen using a fluorescence activated cell sorter (FACS) after the antigen is fluorescently labeled and reacted with the fused cells. In this case, since a hybridoma that produces an antibody against the target antigen can be selected directly, the cloning effort can be greatly reduced.

  Various methods can be used for cloning of hybridomas producing monoclonal antibodies against the target antigen.

  Since aminopterin inhibits many cell functions, it is preferably removed from the medium as soon as possible. In the case of mice and rats, since most myeloma cells die within 10 to 14 days, aminopterin can be removed after 2 weeks of fusion. However, human hybridomas are usually maintained in an aminopterin-added medium for about 4 to 6 weeks after fusion. It is desirable to remove hypoxanthine and thymidine at least 1 week after removal of aminopterin. That is, in the case of mouse cells, for example, a complete medium supplemented with hypoxanthine and thymidine (HT) (for example, RPMI 1640 supplemented with 10% FCS) is added or exchanged 7 to 10 days after the fusion. Visible clones appear about 8 to 14 days after the fusion. If the diameter of the clone is about 1 mm, the amount of antibody in the culture supernatant can be measured.

The amount of antibody can be measured, for example, by hybridoma culture supernatant on a solid phase (for example, a microplate) on which a target antigen or a derivative thereof or a partial peptide thereof (including a partial amino acid sequence used as an antigenic determinant) is adsorbed directly or together with a carrier. And then radioactive materials (eg ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C), enzymes (eg β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase), fluorescent substances (Eg, fluorescamine, fluorescein isothiocyanate), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin), etc. labeled with an anti-immunoglobulin (IgG) antibody (same as the animal from which the original antibody-producing cells are derived) Anti-IgG against a kind of animal-derived IgG Or a method for detecting an antibody against a target antigen (antigenic determinant) bound to a solid phase, adding a hybridoma culture supernatant to a solid phase adsorbed with an anti-IgG antibody or protein A, A target antigen labeled with the same labeling agent as described above or a derivative thereof or a partial peptide thereof is added, and a method of detecting an antibody against the target antigen (antigenic determinant) bound to the solid phase can be performed.

  As a cloning method, a limiting dilution method is usually used, but cloning using soft agar or cloning using FACS (described above) is also possible. Cloning by the limiting dilution method can be performed, for example, by the following procedure, but is not limited thereto.

  The positive well is selected by measuring the amount of antibody as described above. Appropriate feeder cells are selected and added to a 96 well plate. Cells are aspirated from antibody-positive wells, suspended in complete medium (for example, RMPI 1640 supplemented with 10% FCS and P / S) to a density of 30 cells / mL, and 0.1 mL ( 3 cells / well), dilute the remaining cell suspension to 10 cells / mL and sprinkle in another well (1 cell / well), and further dilute the remaining cell suspension to 3 cells / mL To another well (0.3 cells / well). Incubate for about 2 to 3 weeks until a visible clone appears, measure the amount of antibody, select a positive well, and clone again. Since cloning is relatively difficult for human cells, a 10 cell / well plate is also prepared. Usually, monoclonal antibody-producing hybridomas can be obtained by subcloning twice, but it is desirable to repeat recloning periodically for several months in order to confirm the stability.

Hybridomas can be cultured in vitro or in vivo. As an in vitro culture method, the monoclonal antibody-producing hybridoma obtained as described above is used in a well plate while maintaining the cell density at, for example, about 10 ⁵ to 10 ⁶ cells / mL and gradually decreasing the FCS concentration. There is a method of gradually increasing the scale.

As an in vivo culture method, for example, a mouse in which plasma oil (MOPC) was induced by injecting mineral oil into the abdominal cavity (mice compatible with the hybridoma parent strain) was observed after 10 ⁶ to 10 ⁶ Examples include a method in which a hybridoma of about 10 ⁷ cells is injected intraperitoneally, and ascites is collected under anesthesia 2 to 5 weeks later.

(B) Purification of monoclonal antibody Separation and purification of the monoclonal antibody can be performed by a method known per se, for example, separation and purification of immunoglobulin [Example: salting out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion Absorbing and desorbing using an exchanger (eg, DEAE, QEAE), ultracentrifugation, gel filtration, antigen-binding solid phase, or using an active adsorbent such as protein A or protein G, and removing the antibody to dissociate the antibody Can be carried out according to a specific purification method etc.

  As described above, a monoclonal antibody can be produced by culturing a hybridoma in vivo or ex vivo of a warm-blooded animal and collecting the antibody from the body fluid or culture.

  When the antibody of the present invention is used for the prevention / treatment of cancer, the antibody must have antitumor activity, so it is necessary to examine the degree of antitumor activity of the obtained monoclonal antibody. Antitumor activity can be measured by comparing cancer cell proliferation, cell adhesion, apoptosis induction, etc. in the presence and absence of antibodies.

  In a preferred embodiment, the antibody of the present invention is used as a pharmaceutical for human administration. Therefore, the antibody of the present invention (preferably a monoclonal antibody) is an antibody (specifically, a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, etc.) with reduced risk of showing antigenicity when administered to humans. And particularly preferably a fully human antibody. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to the methods described below. In addition, a fully human antibody can be produced from the above-mentioned human-human (or mouse) hybridoma, but in order to provide a large amount of antibody stably and at low cost, a human antibody-producing animal (described later) For example, it is desirable to produce using mouse or phage display method.

(I) Production of Chimeric Antibody As used herein, a “chimeric antibody” is derived from a mammalian species having sequences of heavy and light chain variable regions (V _H and V _L ), and comprises constant regions (C _H and An antibody whose sequence of C _L ) is derived from another mammalian species. The sequence of the variable region is preferably derived from an animal species that can easily produce a hybridoma such as a mouse, and the sequence of the constant region is preferably derived from the mammalian species to be administered.

Examples of the method for producing a chimeric antibody include the method described in US Pat. No. 6,331,415 or a method obtained by partially modifying it. Specifically, first, mRNA or total RNA is prepared from a monoclonal antibody-producing hybridoma (for example, mouse-mouse hybridoma) obtained as described above according to a conventional method, and cDNA is synthesized. Then the cDNA as a template, appropriate primers (e.g., oligo DNA, the N-terminal sequence of the _{C H} and _{C L} as an antisense primer containing a nucleotide sequence encoding each N-terminal sequences of the _{V H} and _{V L} as the sense primer A DNA encoding _VH and _VL is amplified and purified by PCR according to a conventional method using an oligo DNA that hybridizes with a nucleotide sequence encoding (see, for example, Bio / Technology, 9: 88-89, 1991). By the same method, other mammals (e.g., human) DNA encoding a _{C H} and _{C L} amplified and purified by RT-PCR method from RNA was prepared from lymphocytes or the like. Using a conventional method, V _H and C _H , _VL and C _L were ligated, and the obtained chimeric H chain DNA and chimeric L chain DNA were respectively combined with appropriate expression vectors (for example, CHO cells, COS cells, mice). It is inserted into a promoter (eg, a vector containing a CMV promoter, SV40 promoter, etc.) having transcriptional activity in myeloma cells or the like. DNAs encoding both strands may be inserted into separate vectors or tandemly inserted into one vector. Host cells are transformed with the obtained chimeric H chain and chimeric L chain expression vectors. Examples of host cells include animal cells such as mouse myeloma cells, Chinese hamster ovary (CHO) cells, monkey-derived COS-7 cells, Vero cells, rat-derived GHS cells, and the like. For transformation, any method applicable to animal cells may be used, and preferred examples include electroporation. After culturing in a medium suitable for host cells for a certain period, the culture supernatant is recovered and purified by the same method as described above, whereby a chimeric monoclonal antibody can be isolated. Alternatively, transgenic animals such as cattle, goats, and chickens have been established as host cells, and germline cells of animals that have accumulated extensive breeding know-how as livestock (poultry) are used to produce transgenic animals by conventional methods. By doing so, a chimeric monoclonal antibody can also be obtained easily and in large quantities from the milk or egg of the animal obtained. In addition, transgenic cells such as corn, rice, wheat, soybean, tobacco, etc. have been established, and plant cells grown in large quantities as main crops are used as host cells for microinjection, electroporation, and intact cells into protoplasts. It is also possible to produce a transgenic plant using the particle gun method, Ti vector method, etc., and obtain a chimeric monoclonal antibody in large quantities from the seeds and leaves obtained.

If the obtained chimeric monoclonal antibody is degraded with papain, Fab is obtained, and if it is degraded with pepsin, F (ab ′) ₂ is obtained.

In addition, DNA encoding mouse _VH and _VL is converted into a suitable linker, for example, a peptide comprising 1 to 40 amino acids, preferably 3 to 30 amino acids, more preferably 5 to 20 amino acids (eg, [Ser- (Gly) m ] Or [(Gly) m-Ser] n (m is an integer of 0 to 10, n is an integer of 1 to 5) etc. or a minibody monomer by ligating the DNA encoding the C _H3 via a suitable linker may also be a scFv-Fc by ligating a DNA encoding a _{C H} full length through a suitable linker. DNA encoding such genetically modified (conjugated) antibody molecules can be expressed in microorganisms such as Escherichia coli and yeast by placing them under the control of an appropriate promoter, producing a large amount of antibody molecules. can do.

When DNA encoding mouse _VH and _VL is inserted in tandem downstream of one promoter and introduced into Escherichia coli, a dimer called Fv is formed by monocistronic gene expression. Moreover, when an appropriate amino acid in FR of _VH and _VL is substituted with Cys using molecular modeling, a dimer called dsFv is formed by an intermolecular disulfide bond between both chains.

(Ii) Humanized antibody As used herein, the term “humanized antibody” refers to all regions other than the complementarity determining region (CDR) existing in the variable region (that is, the framework region (FR) in the constant region and the variable region. )) Sequences are derived from humans, and only CDR sequences are derived from other mammalian species. As other mammalian species, for example, an animal species that can easily produce a hybridoma such as a mouse is preferable.

Examples of methods for producing humanized antibodies include the methods described in US Pat. Nos. 5,225,539, 5,585,089, 5,693,761 and 5,693,762, or those The method etc. which modified | changed partially are mentioned. Specifically, in the same manner as in the case of the chimeric antibody, DNA encoding V _H and V _L derived from a mammal species other than human (eg, mouse) is isolated, and then an automatic DNA sequencer ( For example, the sequence is performed using Applied Biosystems, etc., and the obtained nucleotide sequence or the amino acid sequence deduced therefrom is used as a known antibody sequence database [for example, Kabat database (Kabat et al., “Sequences of Proteins of Immunological Institute”). , US Department of Health and Human Services, Public Health Service, NIH edition, 5th edition, 1991)) and the like. To determine the fine-FR. Nucleotide sequences encoding L and H chains of human antibodies having an FR sequence similar to the determined FR sequence [eg, human κ-type L chain subgroup I and human H chain subgroup II or III (Kabat et al., 1991 (See above))] is designed by replacing the CDR coding region with a nucleotide sequence encoding the determined heterologous CDR, and the nucleotide sequence is divided into fragments of about 20 to 40 nucleotides. A sequence complementary to the sequence is divided into fragments of about 20 to 40 bases so as to alternately overlap with the fragments. Each fragment was synthesized using a DNA synthesizer, hybridized according to a conventional method, and ligated to obtain DNAs encoding V _H and V _L having FRs derived from human and CDRs derived from other mammalian species. Can be built. In order to transplant CDRs derived from other mammalian species into human-derived _VH and _VL more rapidly and efficiently, it is preferable to use site-directed mutagenesis by PCR. Examples of such a method include a sequential CDR transplantation method described in JP-A-5-227970. The DNA encoding the V _H and V _L obtained in this way, the introduction and respectively connected with DNA encoding a C _H and C _L from humans into a suitable host cell in the same manner as with the chimeric antibody By doing so, cells or transgenic animals and plants that produce humanized antibodies can be obtained.

  Humanized antibodies can also be modified to scFv, scFv-Fc, minibody, dsFv, Fv, etc. using genetic engineering techniques in the same way as chimeric antibodies, and can be produced in microorganisms such as Escherichia coli and yeast by using appropriate promoters. Can be made.

  The humanized antibody production technique can be applied to, for example, production of a monoclonal antibody that can be preferably administered to other animal species for which hybridoma production techniques have not been established. For example, animals that are widely bred as domestic animals (poultry) such as cattle, pigs, sheep, goats, chickens, and pet animals such as dogs and cats can be mentioned.

(Iii) Production of fully human antibody using human antibody-producing animal A functional human Ig gene is introduced into a non-human warm-blooded animal knocked out (KO) of an endogenous immunoglobulin (Ig) gene and immunized with an antigen. Thus, human antibodies are produced instead of the animal-derived antibodies. Therefore, using an animal with established hybridoma production technology, such as a mouse, it is possible to obtain a fully human monoclonal antibody by the same method as the production of a conventional mouse monoclonal antibody. First, a mouse in which a human Ig H chain and L chain minigene was introduced using a normal transgenic (Tg) technique and a mouse in which an endogenous mouse Ig gene was inactivated using a normal KO technique. Some of the human monoclonal antibodies produced using human antibody-producing mice (see Immunol. Today, 17, 391-397 (1996)) obtained by mating have already been in the clinical stage. Production of human Ig human antibodies (HAHA) has not been reported.

  Thereafter, Abgenix [trade name: XenoMouse (Nat. Genet., 15, 146-156 (1997); see US Pat. No. 5,939,598, etc.)] and Medarex [trade name: Hu-Mab Mouse ( Nat. Biotechnol., 14, 845-851 (1996); see US Pat. No. 5,545,806, etc.)] using a yeast artificial chromosome (YAC) vector to introduce a larger human Ig gene. It has been possible to produce human antibodies richer in repertoire. However, human Ig genes, for example, in the case of heavy chains, have VDJ exons in which various combinations of about 80 V fragments, about 30 D fragments, and 6 J fragments encode antigen binding sites. Since the diversity is realized, the total length of the H chain reaches about 1.5 Mb (Chromosome 14), the κ L chain reaches about 2 Mb (Chromosome 2), and the λ L chain reaches about 1 Mb (Chromosome 22). In order to reproduce various antibody repertoires similar to those in humans with other animal species, it is desirable to introduce the full length of each Ig gene, but conventional gene transfer vectors (plasmids, cosmids, BACs, YACs, etc.) The DNA that can be inserted into is usually several kb to several hundred kb, and it has been difficult to introduce the full length by the conventional transgenic animal production technique in which the cloned DNA is injected into a fertilized egg.

  Tomizuka et al. (Nat. Genet., 16, 133-143 (1997)) introduced a natural fragment (hCF) of a human chromosome carrying an Ig gene into a mouse (chromosome transfer (TC) mouse) to produce a full-length human. Mice carrying the Ig gene were produced. That is, first, a human-mouse hybrid cell having a human chromosome obtained by labeling the chromosome 14 containing the H chain gene and the chromosome 2 containing the κ L chain gene with, for example, a drug resistance marker or the like is used for a spindle thread formation inhibitor (for example, , Colcemid) to prepare microcells in which one to several chromosomes or fragments thereof are encapsulated in the nuclear membrane, and introduce the chromosomes into mouse ES cells by the micronucleus fusion method. A hybrid ES cell retaining a chromosome having a human Ig gene or a fragment thereof is selected using a medium containing a drug, and microinjected into a mouse embryo by a method similar to that for producing a normal KO mouse. A germline chimera is selected from the resulting chimeric mice using the coat color as an index, and the TC mouse strain that transmits the human chromosome 14 fragment (TC (hCF14)) and the TC mouse strain that transmits the human chromosome 2 fragment. (TC (hCF2)) is established. A mouse (KO (IgH) and KO (Igκ)) in which an endogenous heavy chain gene and a κ light chain gene are KOed by a conventional method is prepared, and all four kinds of gene modifications are carried out by repeating crossing of these four strains. A mouse strain (double TC / KO) can be established.

  An antigen-specific human monoclonal antibody-producing hybridoma can be produced by applying the same method as that for producing a normal mouse monoclonal antibody to the double TC / KO mouse produced as described above. However, since hCF2 containing a κ light chain gene is unstable in mouse cells, there is a drawback that the efficiency of obtaining a hybridoma is lower than that in a normal mouse.

  On the other hand, the Hu-Mab Mouse contains about 50% of the κ light chain gene, but has a structure in which the variable region cluster is doubled, and thus exhibits the same diversity of κ chains as the full length (on the other hand, the H chain gene). Since only about 10% is contained, the diversity of the heavy chain is low and the responsiveness to the antigen is insufficient), and since it is inserted into the mouse chromosome by the YAC vector (Igκ-YAC), It is kept stable. Taking advantage of this advantage, a TC (hCF14) mouse and a Hu-Mab Mouse are crossed to produce a mouse (trade name: KM mouse) that stably holds hCF14 and Igκ-YAC. The efficiency of obtaining the hybridoma and the antigen affinity of the antibody can be obtained.

  Furthermore, in order to more fully reproduce various antibody repertoires in humans, human antibody-producing animals into which a λL chain gene has been further introduced can be produced. Such an animal is prepared by producing a TC mouse (TC (hCF22)) into which human chromosome 22 carrying a λL chain gene or a fragment thereof is introduced in the same manner as described above, and the double TC / KO mouse or KM mouse. Or by constructing a human artificial chromosome (HAC) containing, for example, an H chain locus and a λ L chain locus and introducing it into mouse cells (Nat. Biotechnol., 18, 1086-1090 (2000)).

  When the antibody of the present invention is used as a pharmaceutical, it is preferably a monoclonal antibody, but may be a polyclonal antibody. When the antibody of the present invention is a polyclonal antibody, the use of a hybridoma is not required. Therefore, a hybridoma production technique is not established, but an animal species for which a transgenic technique is established, preferably an ungulate such as a cow is selected. If a human antibody-producing animal is produced by the same method as described above, a larger amount of human antibody can be produced at a low cost (for example, Nat. Biotechnol., 20, 889-894 (2002)). reference). The resulting human polyclonal antibody can be purified by collecting blood, ascites, milk, eggs, etc., preferably milk and eggs of human antibody-producing animals, and combining the same purification techniques as described above.

(Iv) Production of fully human antibodies using phage display human antibody library Another approach for producing fully human antibodies is to use phage display. In this method, mutations caused by PCR may be introduced in addition to CDRs. Therefore, there are a few reports of HAHA production in the clinical stage, but there is no risk of infection with heterologous viruses derived from host animals. And the specificity of the antibody is infinite (antibodies against prohibited clones, sugar chains, etc. can be easily produced).

  Examples of the method for preparing a phage display human antibody library include, but are not limited to, the following.

  The phage to be used is not particularly limited, but usually filamentous phage (Ff bacteriophage) is preferably used. Examples of a method for displaying a foreign protein on the phage surface include a method of expressing and displaying on a coat protein as a fusion protein with any of the coat proteins of g3p and g6p to g9p, and g3p or g8p is often used. This is a method of fusing to the N-terminal side. The phage display vector includes 1) a foreign protein fused to the coat protein gene of the phage genome, and all coat proteins displayed on the phage surface are displayed as fusion proteins with the foreign protein. E. coli having a gene encoding a fusion protein inserted separately from the wild type coat protein gene and expressing the fusion protein and the wild type coat protein simultaneously, and 3) E. coli having a phagemid vector having the gene encoding the fusion protein Infecting a helper phage having a wild-type coat protein gene to produce phage particles that simultaneously express the fusion protein and the wild-type coat protein. Since dyeing ability is lost, for the production of antibody libraries 2) or 3) of types used.

  Specific examples of the vector include those described in Holt et al. (Curr. Opin. Biotechnol., 11, 45-449 (2000)). For example, pCES1 (see J. Biol. Chem., 274, 18218-18230 (1999)) is a DNA encoding a kappa light chain constant region and a g3p signal downstream of a g3p signal peptide under the control of one lactose promoter. This is a Fab expression type phagemid vector in which DNA encoding CH3, His-tag, c-myc tag, and g3p coding sequence are arranged via an amber stop codon (TAG) downstream of the peptide. When introduced into Escherichia coli having an amber mutation, Fab is displayed on the g3p coat protein, but when expressed in HB2151 strain or the like not having an amber mutation, a soluble Fab antibody is produced. Further, as the scFv expression type phagemid vector, for example, pHEN1 (J. Mol. Biol., 222, 581-597 (1991)) or the like is used.

  On the other hand, examples of the helper phage include M13-K07, VCSM13 and the like.

  In addition, as another phage display vector, a sequence containing a codon encoding cysteine is linked to the 3 ′ end of the antibody gene and the 5 ′ end of the coat protein gene, and both genes are separated separately (not as a fusion protein). Examples are those that are expressed and designed so that antibodies can be displayed on the coat protein on the phage surface via S—S bonds between the introduced cysteine residues (Morphosys CysDisplay ™ technology).

  Examples of human antibody libraries include naive / non-immunized libraries, synthetic libraries, immune libraries, and the like.

  A naive / non-immunized library is a library obtained by obtaining VH and VL genes possessed by normal humans by the RT-PCR method and randomly cloning them into the above phage display vector. Normally, mRNA derived from lymphocytes such as peripheral blood, bone marrow, and tonsils of normal persons is used as a template. In order to eliminate V gene bias such as disease history, an amplification of only IgM-derived mRNA that has not undergone class switching due to antigen sensitization is called a naive library. Representative examples include a CAT library (J. Mol. Biol., 222, 581-597 (1991); Nat. Biotechnol., 14, 309-314 (see 1996)), an MRC library. (See Annu. Rev. Immunol., 12, 433-455 (1994)), Dyax's library (J. Biol. Chem., 1999 (supra); Proc. Natl. Acad. Sci. USA, 14, 7969-7974 (2000)).

  A synthetic library selects a specific functional antibody gene in human B cells, and replaces a portion of an antigen-binding region such as CDR3 of a V gene fragment with a DNA encoding a random amino acid sequence of an appropriate length. It is a library. Since a library can be constructed from a combination of VH and VL genes that produce functional scFv and Fab from the beginning, it is said that the antibody expression efficiency and stability are excellent. Typical examples include Morphosys' HuCAL library (see J. Mol. Biol., 296, 57-86 (2000)), BioInvent's library (Nat. Biotechnol., 18, 852 (2000)). And a library of Crucell (see Proc. Natl. Acad. Sci. USA, 92, 3938 (1995); J. Immunol. Methods, 272, 219-233 (2003)).

An immunized library is a lymphocyte collected from a human who has an increased blood antibody titer against a target antigen, such as a patient with cancer, autoimmune disease or infection, or a person who has been vaccinated, or the above in vitro immunization method. MRNA is prepared from human lymphocytes and the like artificially immunized with the target antigen in the same manner as in the above naive / non-immunized library, and _VH and _VL genes are amplified by RT-PCR method to form a library It is a thing. Since the target antibody gene is contained in the library from the beginning, the target antibody can be obtained even from a relatively small size library.

The greater the diversity of the library, the better. However, in reality, the number of phages that can be handled by the following panning operations (1011 to 1013 phages) and the number of phages necessary for isolation and amplification of clones by normal panning (100 to 1) In this case, about 10 ⁸ to 10 ¹¹ clones are appropriate, and an antibody having a Kd value of usually about 10 ⁻⁹ can be screened with a library of about 10 ⁸ clones.

  The process of selecting antibodies against the target antigen by the phage display method is called panning. Specifically, for example, a carrier on which an antigen is immobilized is contacted with a phage library, and after washing away unbound phage, the bound phage is eluted from the carrier, and the phage is propagated by infecting E. coli. The phages displaying antigen-specific antibodies are concentrated by repeating a series of operations 3 to 5 times. As a carrier for immobilizing an antigen, various carriers used in normal antigen-antibody reaction and affinity chromatography, for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass, Examples thereof include microplates, tubes, membranes, columns, beads, and the like made of metal, and surface plasmon resonance (SPR) sensor chips. For the immobilization of the antigen, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. For example, a biotin- (strept) avidin system is preferably used. When the endogenous ligand that is the target antigen is a small molecule such as a peptide, special care must be taken so that the moiety used as the antigenic determinant is not covered by binding to the carrier. For washing non-binding phages, a blocking solution such as a BSA solution (1-2 times), PBS containing a surfactant such as Tween (3-5 times), and the like can be sequentially used. There is also a report that the use of a citrate buffer (pH 5) or the like is preferable. For elution of specific phage, an acid (for example, 0.1 M hydrochloric acid) is usually used, but cleavage with a specific protease (for example, a gene encoding a trypsin cleavage site at the junction between an antibody gene and a coat protein gene) (In this case, since the wild type coat protein is displayed on the surface of the eluted phage, it is possible to infect and proliferate E. coli even if all of the coat protein is expressed as a fusion protein) Competitive elution with soluble antigen or reduction of S—S bond (for example, in CysDisplayTM described above, after panning, antigen-specific phages are recovered by dissociating the antibody and coat protein using an appropriate reducing agent) Elution) is also possible. In the case of elution with acid, after neutralization with Tris or the like, the eluted phage is infected with E. coli, and after culturing, the phage is recovered by a conventional method.

  When the phages displaying antigen-specific antibodies are concentrated by panning, they are infected with E. coli and then seeded on a plate for cloning. The phages are collected again, and the antigen binding activity is confirmed by the above-described antibody titer measurement method (for example, ELISA, RIA, FIA, etc.) or measurement using FACS or SPR.

Isolation and purification of antibodies from phage clones displaying selected antigen-specific antibodies can be achieved, for example, when using a vector in which an amber stop codon is introduced at the junction between the antibody gene and the coat protein gene as a phage display vector. When the phage is infected with E. coli without an amber mutation (for example, HB2151 strain), soluble antibody molecules are produced and secreted into the periplasm or medium. Can be recovered and purified using the same purification technique as described above. If a His-tag or c-myc tag is introduced, it can be easily purified using an IMAC or anti-c-myc antibody column. In addition, when utilizing cleavage by a specific protease during panning, the antibody molecule is separated from the phage surface when the protease is acted on, so the target antibody is purified by carrying out the same purification operation. be able to.
Fully human antibody production techniques using human antibody-producing animals and phage display human antibody libraries can also be applied to produce monoclonal antibodies of other animal species. For example, animals that are widely bred as domestic animals (poultry) such as cattle, pigs, sheep, goats, chickens, and pet animals such as dogs and cats can be mentioned. In non-human animals, the use of an immune library is more effective because there are few ethical problems with respect to artificial immunity of the target antigen.

(3) Production of polyclonal antibody The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, an immune antigen (protein or peptide antigen) itself or a complex of it and a carrier protein is prepared, and a warm-blooded animal is immunized in the same manner as the above monoclonal antibody production method. It can be produced by collecting the antibody-containing material and separating and purifying the antibody.

  Regarding the complex of immunizing antigen and carrier protein used to immunize warm-blooded animals, the type of carrier protein and the mixing ratio of carrier protein and hapten are determined based on the antibody against the hapten immunized by cross-linking to carrier protein. Any one can be crosslinked at any ratio as long as it is efficient. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are about 0.1 to about 20, preferably about 0.1 to about 20 by weight with respect to hapten 1. A method of coupling at a ratio of about 1 to about 5 is used.

  In addition, various condensing agents can be used for coupling of the hapten and the carrier protein, but active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, dithiobilidyl group, and the like are used.

  The condensation product is administered to a warm-blooded animal as it is, or together with a carrier and a diluent, at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually performed once every about 1 to about 6 weeks, about 2 to about 10 times in total.

  Polyclonal antibodies can be collected from blood, ascites, etc., preferably from blood of warm-blooded animals immunized by the above method.

  The polyclonal antibody titer in the antiserum can be measured in the same manner as the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

  Hereinafter, the protein of the present invention or a partial peptide thereof (hereinafter also simply referred to as “the protein of the present invention”), a nucleic acid encoding the protein of the present invention or a partial peptide thereof (hereinafter also simply referred to as “the nucleic acid of the present invention”). The use of the above-described antibody of the present invention will be described. All of the following uses were found for the first time by the present inventors that "expression of the REG1 gene (REG1A gene and REG1B gene) contributes to high sensitivity to cancer treatment (chemotherapy and radiotherapy)". Based on knowledge. Note that the mechanism by which the expression of the REG1 gene contributes to high sensitivity to cancer treatment has not been fully clarified. However, death of cancer cells by irradiation or administration of a chemotherapeutic agent (anticancer agent) is known to be due to activation of the apoptotic pathway in the cancer cells, and from this, the REG1 gene is in cancer cells. It is strongly speculated that it contributes to high therapeutic sensitivity by affecting the activation of the apoptotic pathway.

(1) Method for Determining Treatment Sensitivity of Cancer Cells First, the treatment sensitivity of cancer cells can be determined using the REG1 gene as a marker. That is, according to the first aspect of the present invention, a method for determining the therapeutic sensitivity of cancer cells is provided. In the present application, “therapeutic sensitivity” means sensitivity to any treatment (for example, irradiation of radiation or administration of a chemotherapeutic agent (anticancer agent)) performed on cancer cells for the purpose of treating cancer. . More specifically, the sensitivity to radiation (radiation sensitivity) means how much cancer cells are killed by radiation. Similarly, sensitivity (chemotherapy sensitivity) to administration of a chemotherapeutic agent (anticancer agent) means how much cancer cells are killed by administration of the chemotherapeutic agent (anticancer agent). In the present application, “therapeutic sensitivity” may be sensitivity to only one type of treatment (treatment) or may be sensitivity to two or more types of treatment (treatment). In the present specification, the term “cancer cell” is used as a concept including cells that are oriented to become cancerous in the future.

  The “method for determining the therapeutic sensitivity of cancer cells” of the present embodiment is the REG1A gene or a functionally equivalent gene (hereinafter also simply referred to as “REG1A gene”) or REG1B gene or Measuring the expression level (A1) of a functionally equivalent gene (hereinafter also simply referred to as “REG1B gene”) (all of these genes are also collectively referred to as “REG1 gene”), and the expression level The step of comparing (A1) with a predetermined expression level (A0) and, as a result of comparison, determining whether the expression level (A1) is significantly greater than the expression level (A0), Predicting therapeutic sensitivity of the test tissue or the test cell. Hereinafter, it demonstrates in detail in order of a process.

  In the “method for determining therapeutic sensitivity of cancer cells” of this embodiment, first, the REG1A gene or a gene functionally equivalent thereto, or the REG1B gene or a gene functionally equivalent thereto in a test tissue or test cell. The expression level (A1) is measured.

  In measuring the gene expression level, a test tissue / test cell to be measured is collected from a living body (for example, a human living body). There is no restriction | limiting in particular about a concrete method, A conventionally well-known knowledge can be referred suitably.

  Here, the “test tissue” is a tissue that can be extracted from a living body that is a target of treatment sensitivity determination, and the type thereof is not particularly limited as long as it is a cancer tissue that needs to be determined for treatment sensitivity. Examples of such tissues include, for example, squamous cell carcinoma (such as esophageal squamous cell carcinoma), neuroblastoma, retinoblastoma, brain tumor, head and neck cancer, pituitary adenoma, glioma, auditory schwannoma, oral cancer, Pharyngeal cancer, laryngeal cancer, thyroid cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, biliary tract cancer, penile cancer, vulvar cancer, renal pelvis and ureter Cancer, kidney cancer, testicular cancer, prostate cancer, bladder cancer, uterine cancer, villous disease, vaginal cancer, ovarian cancer, fallopian tube cancer, ovarian germ cell tumor, skin cancer, mycosis fungoides, malignant melanoma, soft tissue sarcoma , Bone tumor, malignant lymphoma, leukemia, myelodysplastic syndrome, multiple myeloma, tissue derived from lymphedema. Among these, a tissue derived from squamous cell carcinoma is preferable, and a tissue derived from esophageal squamous cell carcinoma is particularly preferable.

  Similarly, the “test cell” in the present invention is a tissue that can be extracted from a living body that is a target for determination of therapeutic sensitivity, and the type thereof is a cell derived from a cancer tissue that needs to be determined for therapeutic sensitivity. There is no particular limitation. Examples of such cells include, for example, squamous cell carcinoma (esophageal squamous cell carcinoma), neuroblastoma, retinoblastoma, brain tumor, head and neck cancer, pituitary adenoma, glioma, acoustic schwannoma, oral cancer, pharynx Cancer, laryngeal cancer, thyroid cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, biliary tract cancer, penile cancer, vulva cancer, renal pelvic cancer , Kidney cancer, testicular cancer, prostate cancer, bladder cancer, uterine cancer, villous disease, vaginal cancer, ovarian cancer, fallopian tube cancer, ovarian germ cell tumor, skin cancer, mycosis fungoides, malignant melanoma, soft tissue sarcoma, Examples include cells derived from bone tumor, malignant lymphoma, leukemia, myelodysplastic syndrome, multiple myeloma, lymphedema. Of these, cells derived from squamous cell carcinoma are preferred, and cells derived from esophageal squamous cell carcinoma are particularly preferred.

  Next, the REG1 gene that is a gene to be examined in the present invention will be described.

  The REG gene is known to have a multigene family classified into four subclasses of REG1, REG2, REG3, and REG4 according to the primary structure of the encoded protein. The REG1 gene includes a REG1A gene and a REG1B gene, which code for proteins secreted from the exocrine pancreatic glands and are located on the short arm of chromosome 2 (2p12). More specifically, the REG1A gene is a gene consisting of 821 bp (Refseq Accession No. NM_002909, SEQ ID NO: 1) and encodes a protein consisting of 166 amino acids (REG1A protein; RefSeq Accession No. AAH05350, SEQ ID NO: 2). is doing. The REG1B gene is a gene consisting of 812 bp (Refseq Accession No. NM — 006507, SEQ ID NO: 3) and encodes a protein consisting of 166 amino acids (REG1B protein; RefSeq Accession No. AAH27895, SEQ ID NO: 4). . The gene used in the determination method of this embodiment may be a gene functionally equivalent to the REG1 gene (REG1A gene or REG1B gene).

  Here, the “REG1 gene (REG1A gene or REG1B gene)” is a functionally equivalent gene, although the nucleotide sequence is different from that of the REG1 gene but shows a relatively high homology and is the same as the protein encoded by the REG1 gene. Or a gene encoding a protein having similar activity. Specifically, “containing a base sequence that hybridizes with DNA containing the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 under highly stringent conditions, SEQ ID NO: 2 or SEQ ID NO: The DNA described above as “DNA encoding a protein having substantially the same quality of activity as the protein containing the amino acid sequence represented by 4” can be used as “a gene functionally equivalent to the REG1 gene”.

  In this step, the expression level (A1) of the REG1 gene in the test tissue or test cell is measured. There is no particular limitation on the method for measuring the expression level of the REG1 gene. In this step, the expression level (A1) of the REG1 gene can be measured by measuring the amount of mRNA expressed from the gene. In this case, the amount of mRNA is, for example, Northern blot method or microarray method using a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 or a nucleic acid containing a part thereof as a probe, SEQ ID NO: 1 or It can be measured by a PCR method or RT-PCR method using a nucleic acid containing SEQ ID NO: 3 or a part thereof as a PCR primer, or a method analogous thereto. On the other hand, in this step, the expression level (A1) of the REG1 gene may be measured by measuring the amount of protein expressed from the gene. At this time, the amount of the protein is determined by Western blotting using the antibody that recognizes the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 or a part thereof, in a cell extract or the like. It can be measured by a method such as a method, an ELISA method, an immunohistochemical method, or a similar method. Here, the “expression amount” of a gene refers to the absolute amount or relative amount of the gene transcript, and in the case of the relative amount, it is relative to the expression amount in a comparison target (for example, normal tissue / normal cell) described later. In such a comparison, the expression level of the gene may be determined. In this step, the gene whose expression level is measured may be only one of the REG1A gene or the REG1B gene, or the expression level is measured for both genes to comprehensively determine the therapeutic sensitivity of cancer cells. Also good.

  In the “method for determining the therapeutic sensitivity of cancer cells” of this embodiment, the expression level (A1) measured in the above step is then compared with a predetermined expression level (A0). Here, the “predetermined expression level (A0)” is an expression level that serves as a reference when determining the treatment sensitivity of a test tissue or a test cell, and is a value that can be arbitrarily set in advance. In setting the “predetermined expression level (A0)” in advance, the “REG1 gene expression level, which was first discovered by the present inventors, is sensitive to the treatment of cancer cells (for example, radiation irradiation and chemotherapeutic agents ( Considering the fact that it shows a positive correlation with the administration of the anticancer agent)). That is, in the process described later, as a standard for determining the presence / absence / highness of the therapeutic sensitivity of the test tissue / test cell, “if this level of expression is' therapeutic sensitivity” or “therapeutic sensitivity” The expression level determined to be “high” ”can be set in advance as a predetermined expression level (A0). More specifically, a correlation between the expression level of the REG1 gene and treatment sensitivity (radiosensitivity and / or chemotherapeutic agent sensitivity) is determined in advance for a specific tissue or a cell derived from the tissue, and based on this. The desired “predetermined expression level (A0)” can be set.

  In the “method for determining therapeutic sensitivity of cancer cells” of this embodiment, subsequently, as a result of the comparison performed above, it is determined whether or not the expression level (A1) is significantly higher than the expression level (A0). There is no particular limitation on the method for determining the presence or absence of a significant difference, and statistical methods well known to those skilled in the art can be used. As an example, statistically “significantly high” often means that the null hypothesis H0 is rejected at a significance level of 0.05 (5%). And based on the result of this judgment, the treatment sensitivity of the said test tissue or the said test cell is estimated. That is, when the expression level (A1) is significantly larger than the expression level (A0), the test tissue / test cell that is the target of determination is “therapeutic sensitivity” or “therapeutic sensitivity is high”. It is predicted. On the other hand, if the expression level (A1) is not significantly greater than the expression level (A0), the test tissue / test cell that is the target of determination is “not therapeutically sensitive” or It is predicted that “therapeutic sensitivity is not high”. Based on such determination, determination of a more appropriate treatment policy can be promoted. For example, for a patient having a cancer lesion derived from a tissue / cell determined to be “having (high)” sensitivity to chemotherapy and / or radiation therapy by the determination method of the present embodiment, chemotherapy and / or A treatment policy with radiation therapy as the first choice is determined. This makes it possible to avoid surgical excision with physical and mental burdens for such patients, and to provide effective treatment with less burden and risk of side effects for the patients. Is possible. On the other hand, for a patient having a cancer lesion derived from a tissue / cell that is determined to be “not (low)” sensitive to chemotherapy and / or radiotherapy by the determination method of the present embodiment, chemotherapy / radiotherapy It is possible to avoid unnecessary medication / radiation that causes inconvenience because it is not effective by performing treatment with other options (for example, surgical resection).

(2) Nucleic acid probe / PCR primer The nucleic acid of the present invention can be used as a nucleic acid probe or a PCR primer for measuring the amount of mRNA in the above-mentioned “method for determining the therapeutic sensitivity of cancer cells”. That is, according to the second aspect of the present invention, there is provided the following nucleic acid (a) or (b) used as a nucleic acid probe or a PCR primer for measuring the amount of mRNA in the above-described determination method:
(A) a nucleic acid having a part of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 or a base sequence complementary thereto;
(B) A nucleic acid that hybridizes with a nucleic acid comprising the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 under highly stringent conditions or a nucleic acid having a complementary base sequence thereto.

  Here, the nucleic acid (a) or (b) can be used when the expression level of the REG1 gene is measured by measuring the amount of mRNA expressed from the gene in the determination method described above. The nucleic acid can be used as a nucleic acid probe in, for example, a Northern blot method or a microarray method. In this case, the nucleic acid may be labeled with a radioisotope. The nucleic acid can also be used as a PCR primer in a PCR method or RT-PCR method. These techniques for measuring the expression level of the REG1 gene can be performed by referring to knowledge well known in the art.

(3) Antibody The antibody against the protein of the present invention can be used to measure the amount of protein in the above-described “method for determining therapeutic sensitivity of cancer cells”. That is, according to the third aspect of the present invention, the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 is used to measure the amount of protein in the determination method described above. An antibody against a protein comprising the amino acid sequence or a partial peptide thereof or a pharmaceutically acceptable salt thereof is provided.

  Here, the antibody can be used when the expression level of the REG1 gene is measured by measuring the amount of protein expressed from the gene in the determination method described above. There is no particular limitation on a specific method for measuring the expression level of the REG1 gene using the above-mentioned antibody, and well-known knowledge in this technical field can be referred to as appropriate. Examples include, for example, Western blotting, ELISA, radioimmunoassay, “sandwich” immunoassay, immunohistochemistry, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutination assay And various competitive and non-competitive assay methods such as a method for complementation, a complement binding assay, an immunoradiation assay, a fluorescence immunoassay, and a protein A immunoassay.

(4) Cancer Cell Therapy Sensitivity Determination Kit According to the present invention, a cancer cell therapy sensitivity determination kit (also referred to as “kit of the present invention”) for carrying out the determination method provided as the first aspect described above. ) Can also be provided (fourth form). The therapeutic sensitivity determination kit according to the fourth aspect of the present invention comprises an expression measuring means for measuring the expression of a REG1A gene or a functionally equivalent gene thereof, or a REG1B gene or a functionally equivalent gene thereof, and the gene Expression detecting means for detecting the expression of.

  In the kit of the present invention, the expression measuring means for measuring the expression of the predetermined gene is not particularly limited. For example, the nucleic acid probe used in the Northern blot method for measuring the expression of the predetermined gene, the predetermined gene Examples include PCR primers for amplification or a microarray on which the predetermined gene is plotted. The expression detection means for detecting the expression of the predetermined gene is not particularly limited, and examples thereof include a fluorescent labeling reagent and a radioisotope (RI) labeling reagent for visualizing a nucleic acid probe for Northern blot. . In addition, knowledge known in this technical field can be referred to as appropriate.

  By using such a kit, cancer cells can be easily and quickly passed through the process of determining the therapeutic sensitivity of cancer cells by extracting genomic DNA from the test tissue or test cells and confirming the nucleotide sequence. It becomes possible to determine the treatment sensitivity of the.

(5) Method for evaluating compound effective in improving treatment sensitivity of cancer cell As described above, the present inventors have described that "REG1 gene (REG1A gene and REG1B gene) expression is cancer treatment (chemotherapy and radiation therapy). For the first time has been found. Based on such knowledge, according to the present invention, a method for evaluating a compound effective for improving the therapeutic sensitivity of cancer cells can also be provided. That is, the method for evaluating a compound effective for improving the therapeutic sensitivity of cancer cells according to the fifth aspect of the present invention comprises the step of administering or contacting a test compound to a test animal or test cell. Measuring in vitro the expression level (B1) of the REG1A gene or a functionally equivalent gene thereof, or the REG1B gene or a functionally equivalent gene in the test animal or test cell, and the expression As a result of comparing the amount (B1) with the expression level (B0) before administration or contact of the test compound, is the expression level (B1) significantly higher than the expression level (B0)? And determining the effectiveness of the test compound for improving the therapeutic sensitivity of cancer cells by judging whether or not. Hereinafter, it demonstrates in detail in order of a process.

  First, a test compound is administered to or contacted with a test animal or a test cell. Here, as a test compound, as long as it is a candidate compound of a drug for the purpose of treatment, prevention or diagnosis of cancer, its structure and properties are not particularly limited, and the compound type is not limited. Moreover, there is no restriction | limiting in particular in the method of administering a test compound to a test animal, What is necessary is just to select a suitable administration method each time. For example, oral administration and parenteral administration (for example, transdermal administration, intramuscular injection, intravenous injection, subcutaneous injection) can be mentioned. Further, there is no particular limitation on the method of contacting the test compound with the test cell, for example, the cell and the test compound are mixed in a solution such as a culture solution or a buffer solution (for example, phosphate buffer solution), The method of making both contact is mentioned.

  Subsequently, the expression level (B1) of the REG1A gene or a gene functionally equivalent thereto, or the REG1B gene or a gene functionally equivalent thereto in the test animal or test cell is measured in vitro. The method for measuring the expression level of the gene is not particularly limited, and the above-described embodiment in the above-mentioned column “(1) Method for determining therapeutic sensitivity of cancer cells” can be similarly employed.

  Thereafter, the expression level (B1) after administration / contact measured above is compared with the expression level (B0) before administration / contact of the test compound. That is, in this step, it is determined whether the expression level after administration / contact (B1) is significantly higher than the expression level (B0) before administration / contact with respect to the expression level of the gene to be measured. There is no particular limitation on the method for determining the presence or absence of a significant difference, and statistical methods well known to those skilled in the art can be used. And based on the result of this judgment, the effectiveness of the said test compound with respect to the improvement of the treatment sensitivity of a cancer cell is evaluated. That is, when the expression level (B1) is significantly higher than the expression level (B0), it is predicted that the test compound to be determined is “effective in improving the therapeutic sensitivity of cancer cells”. . On the other hand, if the expression level (B1) is not significantly higher than the expression level (B0), the test compound that is the subject of determination is said to be “effective in improving the therapeutic sensitivity of cancer cells. Not expected ". Based on such determination, it becomes possible to screen for a compound effective for improving the therapeutic sensitivity of cancer cells by a simple and rapid technique.

(6) Agent for enhancing therapeutic sensitivity of cancer cells In the above (1) to (5), a technique using the REG1 gene as a marker is provided for the purpose of determining the therapeutic sensitivity of a test tissue / test cell.

  Here, the present inventors have further developed the finding that “expression of the REG1 gene (REG1A gene and REG1B gene) contributes to high sensitivity to cancer treatment (chemotherapy and radiotherapy)”, and REG1 An attempt was made to induce high expression by forcibly introducing a gene into a test tissue / test cell. It was also found that the sensitivity of a test tissue / test cell to cancer treatment can be enhanced by induction of such high expression. Therefore, in the following, the use of the protein of the present invention or the nucleic acid of the present invention as a therapeutic sensitivity enhancer provided based on such knowledge will be described in detail.

According to the sixth aspect of the present invention, treatment of cancer cells (preferably cells of squamous cell carcinoma such as esophageal squamous cell carcinoma) comprising at least one of the following (a) to (c) as an active ingredient: Sensitivity (eg, sensitivity to radiation and / or anticancer agents) enhancers are provided;
(A) a nucleic acid having the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3;
(B) a nucleic acid encoding a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or a partial peptide thereof;
(C) a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or a partial peptide thereof, or a pharmaceutically acceptable salt thereof.

  Since the specific forms of the above (a) to (c) contained in these therapeutic sensitivity enhancers as active ingredients are as described above, detailed description thereof is omitted here.

  When using the therapeutic sensitivity-enhancing agent of this form, a composition containing at least one of the above (a) to (c) is treated with a cancer treatment (radiotherapy and / or chemotherapy) for a patient in need of treatment. ) Prior to administration of an effective amount (ie, an appropriate dose). At this time, the nucleic acid having the base sequence represented by SEQ ID NO: 1 and the nucleic acid having the base sequence represented by SEQ ID NO: 3 may each be contained alone in the pharmaceutical composition and administered. It may be contained in a pharmaceutical composition and administered as a combination. Similarly, a nucleic acid encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof, and the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 4 Each nucleic acid encoding a protein containing the same amino acid sequence or a partial peptide thereof may be contained alone in a pharmaceutical composition or administered as a mixture of both. May be. Furthermore, a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a pharmaceutically acceptable salt thereof, and an amino acid sequence represented by SEQ ID NO: 4 A protein comprising the same or substantially the same amino acid sequence or a partial peptide thereof or a pharmaceutically acceptable salt thereof may be contained alone in a pharmaceutical composition and administered as a combination of both. It may be contained in a pharmaceutical composition for administration.

  When the nucleic acid of the present invention is used as a radiation sensitivity enhancer for the above purpose, the nucleic acid is introduced into a vector used for gene delivery and expressed in the body of a patient requiring treatment as a transgene by any expression promoter. You can do it. At this time, the nucleic acid having the base sequence represented by SEQ ID NO: 1 and the nucleic acid having the base sequence represented by SEQ ID NO: 3 may be introduced individually into the vector, or both may be introduced simultaneously. Also good. The vector into which the nucleic acid is introduced is preferably prepared based on a DNA or RNA virus. Although the kind of such viral vector is not specifically limited, MoMLV vector, herpes virus vector, adenovirus vector, AAV vector, HIV vector, SIV vector, Sendai virus vector, etc. are mentioned.

  Also, a pseudo-type type in which one or more of the constituent proteins of the viral vector are replaced with a constituent protein of a heterologous virus, or a part of the nucleic acid sequence constituting the genetic information is replaced with a nucleic acid sequence of a heterologous virus. These viral vectors can also be used. For example, a pseudo-type virus vector in which Env protein, which is an HIV coat protein, is replaced with VSV-G protein, which is a coat protein of Vesicular stomatitis virus (VSV) can be used (Naldini L et al .: Science). 272 263- (1996)).

  Furthermore, viruses having a host range other than humans can be used as virus vectors as long as they enable gene transfer. As vectors not derived from viruses, calcium phosphate and nucleic acid complexes, liposomes, cationic lipid complexes, Sendai virus liposomes, polymer carriers having a polycation as the main chain, and the like can be used. Furthermore, electroporation, a gene gun, etc. can be used as a gene introduction system.

  In order to introduce the nucleic acid of the present invention into the vector as described above to cause gene expression, an expression cassette equipped with an expression promoter is preferably used. The expression cassette used is not particularly limited as long as the gene can be expressed in the target cell. A person skilled in the art can easily select such an expression cassette, but is preferably an expression cassette capable of gene expression in animal-derived cells, more preferably a gene in mammalian-derived cells. An expression cassette capable of expression is selected, and an expression cassette capable of gene expression in a human-derived cell is particularly preferably selected. In addition to the nucleic acid of the present invention, the expression cassette includes a promoter and enhancer for transcription of a gene, a poly A signal, a marker gene for labeling and / or selection of a cell into which the gene has been introduced, and a genomic DNA sequence of the cell Any sequence such as a virus-derived gene sequence for efficiently inserting the gene into the cell, a signal sequence for secreting the protein produced by gene expression outside the cell and / or retaining it locally in the cell can be used. sell.

  Examples of promoters used in the expression cassette include adenovirus, cytomegalovirus, human immunodeficiency virus, simian virus 40, rous sarcoma virus, herpes simplex virus, murine leukemia virus, simbis virus, hepatitis A virus, hepatitis B Virus, hepatitis C virus, papilloma virus, human T cell leukemia virus, influenza virus, Japanese encephalitis virus, JC virus, parvovirus B19, poliovirus and other promoters derived from viruses, albumin, SRα, heat shock protein, elongation factor And the like, promoters derived from mammals, chimeric promoters such as CAG promoter, promoters whose expression is induced by tetracycline, steroids, and the like.

  The nucleic acids and proteins of the present invention can be configured in the form of a suitable pharmaceutical composition when used as the therapeutic sensitivity enhancer described above. Therefore, the nucleic acid and the like can be formulated according to the following preparation method, a preferable administration route can be set, and the dose can be determined so as to achieve a desired therapeutic sensitivity enhancement effect.

  The pharmaceutical composition comprising the nucleic acid or protein of the present invention is not particularly limited, but is encapsulated in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated food and beverage, retrovirus or other vector parts. It can be built into pharmaceutical products.

  More specifically, for example, a composition containing the nucleic acid of the present invention by dissolving the recombinant viral vector containing the nucleic acid of the present invention in an appropriate solvent such as water, physiological saline, or isotonic buffer. Can be prepared. In addition, a composition containing the protein of the present invention can be prepared by dissolving the protein of the present invention in a suitable solvent such as water, physiological saline or isotonic buffer. At this time, polyethylene glycol, glucose, various amino acids, collagen, albumin or the like may be added as a protective material to prepare a composition. In addition, the composition optionally includes a pharmaceutically acceptable carrier, diluent, excipient, stabilizer, etc., which is often preferred. These formulation aids are described in detail in Remington's Pharmaceutical Sciences, 16 (1980) (Mack publisher).

  The pharmaceutical composition containing the nucleic acid and / or protein of the present invention as an active ingredient provides a desired therapeutic line sensitivity enhancing effect when administered to a patient by setting an appropriate administration route. There is no particular limitation on the administration method when administering the pharmaceutical composition to the living body. However, it is preferable to administer by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal injection. Or other administration methods, such as injection | pouring, may be preferably employ | adopted so that it can administer continuously in a bloodstream. A particularly preferred mode of administration is intravenous administration. The dosage of the pharmaceutical composition of the present invention varies depending on the administration route, the condition of the patient receiving the administration, age, weight, sex, etc., but the treating physician can determine the optimal dosage for the patient. It is. For example, when administered by injection, it is preferable to administer a daily dose of about 0.1 μg / kg to 1000 mg / kg, more preferably a daily dose of about 1 μg / kg to 100 mg / kg.

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
This shows the base sequence of DNA (CDS) encoding REG1A protein.
[SEQ ID NO: 2]
The amino acid sequence of REG1A protein is shown.
[SEQ ID NO: 3]
This shows the base sequence of DNA (CDS) encoding REG1B protein.
[SEQ ID NO: 4]
The amino acid sequence of REG1B protein is shown.
[SEQ ID NO: 5]
The base sequence of the forward primer used for amplification of the human REG1α gene is shown.
[SEQ ID NO: 6]
The base sequence of the reverse primer used for amplification of the human REG1α gene is shown.
[SEQ ID NO: 7]
The base sequence of the forward primer used for amplification of the human GAPDH gene is shown.
[SEQ ID NO: 8]
The base sequence of the reverse primer used for amplification of the human GAPDH gene is shown.

  EXAMPLES Hereinafter, although an Example and a reference example demonstrate this invention more concretely, the technical scope of this invention is not limited to these.

Example 1: Basic study in vitro As a basic study in vitro, four types of cell lines of esophageal squamous cell carcinoma cell lines, TE-4, TE-5, TE-9, and TE-12, were used. The presence or absence of a correlation between the expression of the REG1α gene and the survival rate after chemotherapy and / or radiation therapy was examined. All of these cell lines were obtained from Tohoku University Institute of Aging Medicine, Medical Cell Resource Center.

  In this example, the expression level of REG1α mRNA in each cell line is measured by RT-PCR (reverse transcriptase-polymerase chain reaction method), and the expression level of REG1α protein in each cell line is determined by immunohistochemistry. It was measured. The specific method is as follows.

(RT-PCR method)
Total RNA was isolated from each cell line using ISOGEN (Nippon Gene Co., Ltd.). Subsequently, cDNA was reverse-transcribed from a 1 μg sample of the total RNA using SuperScript III reverse transcriptase kit (Invitrogen). Thereafter, PCR was performed using Platinum Taq DNA polymerase (Invitrogen). The base sequences of the primers used for PCR are as follows:
(Human REG1α)
Forward: 5′-AACATGAATTCGGGCAACC-3 ′ (SEQ ID NO: 5)
Reverse: 5′-AGGAGAACTTGTCTTCACAA-3 ′ (SEQ ID NO: 6)
(Human GAPDH)
Forward: 5'-CGGAGTCAACGGATTTTGGTCGTAT-3 '(SEQ ID NO: 7)
Reverse: 5′-AGCCTCTCTCCATGGTGGTGAAGAC-3 ′ (SEQ ID NO: 8)
The amplified product was subjected to 1.5% agarose gel electrophoresis and stained with ethidium bromide to confirm the expression of REG1α mRNA in each cell line. The result is shown in FIG. As shown in FIG. 1, expression of REG1α mRNA was confirmed only with TE-12 except for AGS as a positive control.

(Immunohistochemistry)
For the purpose of confirming the expression of REG1α protein in each cell line of TE-5, TE-9, and TE-12, and AGS (positive control), immunohistochemistry was performed using Dako Cytomation Environment + system-HRP (manufactured by Dako). Staining was performed. Specifically, each of these cell lines was cultured for 48 hours in 24 plates and then fixed with TBS containing 4% paraformaldehyde for 10 minutes. Incubate the cells in 3% hydrogen peroxide solution for 10 minutes to block the activity of endogenous peroxidase, and further incubate the cells in 1% BSA for 60 minutes to block nonspecific protein binding. did. The cells were then incubated overnight at 4 ° C. with anti-human REG1 monoclonal antibody (diluted 1: 100 with TBS, 17.2 μg / mL) and then incubated with Envision Plus reagent (Dako) for 60 minutes. The signal was then developed by incubating the cells in a substrate-chromogen solution for 10 minutes. Finally, the mounting medium was applied for several seconds (VectaMount, manufactured by Vector Laboratories). The obtained immune complex was observed using a confocal laser scanning microscope (MRC-2000, manufactured by Bio-Rad). As a result, expression of REG1 protein was confirmed only in TE-12 except AGS which is a positive control.

(Chemotherapeutic agent / radiation treatment)
The TE-5, TE-9, and TE-12 cell lines classified above were exposed to an environment simulating chemotherapy and / or radiation therapy, and their sensitivity was assessed. Specifically, first, cells were seeded in a 96-well plate at a concentration of 1 × 10 ³ cells / well and incubated at 37 ° C. for 24 hours. Then, 5 Gy or 10 Gy of radiation was irradiated as a single environment assuming radiotherapy. On the other hand, cisplatin (CDDP), which is a chemotherapeutic agent (anticancer agent), was added at a concentration of 1 μM or 10 μM as a single environment assuming chemotherapy. Furthermore, as a combined environment assuming chemoradiotherapy (CRT), radiation was irradiated at 5 Gy, and CDDP 1 μM or 10 μM was added simultaneously with the irradiation.

(Evaluation of cell proliferation; MTT assay)
Cell proliferation (viability) was assessed by MTT assay for each of the cell lines treated above and the controls that did not undergo the treatment for each.

  Specifically, after further incubation for 72 hours after the above treatment, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide at a concentration of 5.5 mg / mL was added to the medium. 10 μL) and incubated at 37 ° C. for 4 hours. Thereafter, 90 μL of extraction solution (40% N, N-dimethylformamide, 2% acetic acid, 20% SDS, and 0.03N HCl) was added and incubated at room temperature for 2 hours. Then, the proliferation (survival rate) of the cells was evaluated by measuring the absorbance at a wavelength of 570 nm with a microplate reader (Model 550, manufactured by Bio-Rad).

(result)
The results of evaluation by the above method are shown in FIGS. FIG. 2 is a graph showing the results of exposing each cell line to a single environment assuming radiation therapy. As shown in FIG. 2, TE-12, which is a REG1α-positive cell, is significantly different from TE-5 and TE-9, which are the same negative cells, at both 5 Gy and 10 Gy at a significance level of 1%. High sensitivity was shown.

  FIG. 3 is a graph showing the results of exposing each cell line to a single environment assuming chemotherapy. As shown in FIG. 3, REG1α-positive cells, TE-12, were significantly significant at a significance level of 5% when the CDDP concentration was 1 μM with respect to TE-5 and TE-9, which were the same negative cells. Was highly sensitive.

  FIG. 4 is a graph showing the results of exposing each cell line to a combined environment assuming chemoradiotherapy (CRT). As shown in FIG. 4, REG1α-positive cells, TE-12, were significantly significant at a significance level of 5% when the CDDP concentration was 1 μM with respect to TE-5 and TE-9, which were the same negative cells. Was highly sensitive.

  Thus, REG1α-positive cells show a higher survival rate compared to REG1α-negative cells in each of the single environment of radiation therapy and chemotherapy, and the combined environment of these, and treatment sensitivity is high. It was confirmed to be high.

(Evaluation of apoptotic activity by radiation treatment; caspase-3 assay)
The caspase-3 assay was performed by the following method for the purpose of evaluating the degree of activation of apoptosis by 5 Gy irradiation treatment for the four cell lines described above.

  Using a kit modified from CPP32 Colorimetric Assay Kit (Clontech), the activity of CPP32 protease (caspase-3), which is an important early event in the apoptotic pathway, was measured. Cells were sonicated in lysis buffer (5 mL / 100 mg liver), incubated on ice for 10 minutes, then centrifuged at 150 × g for 3 minutes at 4 ° C. The supernatant was incubated and mixed with 50 μL of reaction buffer containing 10 mM dithiothreitol and 5 μM substrate and incubated at 37 ° C. for 1 hour. The optical density of the obtained sample was measured by spectrophotometry at 405 nm. The result is shown in FIG. As shown in FIG. 4, in REG1α-positive cells, TE-12, caspase-3 significantly decreased at a significance level of 0.5% compared to TE-5 and TE-9, which are the negative cells. It was shown that the activity was enhanced. From this, it is presumed that the high therapeutic sensitivity exhibited by REG1α-positive cells is due to the enhancement of apoptotic activity.

Example 2: Introduction of REG1α gene in vitro The REG1α gene was introduced (transfected) into the same TE-5 and TE-9 cell lines as described above, and the expression of the gene was forcibly induced. Specifically, a cDNA fragment having the base sequence represented by SEQ ID NO: 1 was inserted into the XhoI / XBaI site of a pCI-neo mammalian expression vector (Promega) to construct an expression vector. Next, the expression vector constructed above or a control vector containing no inserted cDNA was introduced into the cell line by electroporation, and then incubated for 2 weeks to obtain a cell line stably expressing the REG1α gene.

  The obtained REG1α-positive cells and control cells were evaluated for cell proliferation (viability) using the MTT assay by the same method as in Example 1 described above.

  As a result, REG1α-positive cells in which the REG1α gene was forcibly introduced were significantly higher than the control cells in each of the single environment of radiation therapy and chemotherapy, and both of these combined environments. The survival rate was shown. From this, it was shown that forced expression of the REG1α gene leads to enhanced therapeutic sensitivity of cancer cells.

Example 3: In vivo clinical study In this example, an in vivo clinical study was performed. This study was approved and carried out by the Akita University School of Medicine Ethics Committee.

  Among the 62 patients who received continuous chemoradiotherapy at Akita University Hospital between 2003 and 2005, the target patients received concurrent chemotherapy and 45 Gy (75% of the total dose) or more. There are 42 patients who obtained paraffin specimens in good condition from biopsy specimens. All patients were diagnosed with squamous cell esophageal squamous cell carcinoma based on endoscopic biopsy tissue. For all patients, the stage and treatment strategy for esophageal squamous cell carcinoma was determined by a review panel of radiation researchers, physicians, and surgeons. In addition, the classification of the stage was in accordance with the tumor node metastasis (TNM) classification of the UICC international anti-cancer association of Maligant Tumors (6th edition).

  First, in preparation of paraffin specimens, endoscopic tissue removal was performed at a pre-treatment stage. Subsequently, the obtained esophageal squamous cell carcinoma deparaffin specimen was autoclaved at 121 ° C. for 15 minutes. Subsequently, it was blocked for 30 minutes at room temperature using a methanol solution containing 0.3% hydrogen peroxide, and further for 30 minutes at room temperature using 10% BSA / TBS. It was then incubated overnight at 4 ° C. with anti-human REG1α polyclonal antibody (1: 400 dilution, 2.5 μg / mL, Biovendor Laboratory Medicine, Czech) in PBS. Thereafter, the mixture was incubated with Envision (Dako Corporation, Denmark) for 30 minutes and reacted with DAB (manufactured by Dojin Research Institute) for 2 minutes. Finally, the sections were counterstained with Mayer's hematoxylin for 30 seconds. As a control, a sample that was not treated with anti-human REG1α polyclonal antibody was prepared. As a result of staining, samples having a staining area of 10% or more in visual observation were classified as REG1α positive, and samples less than 10% were classified as REG1α negative. The determination of this stained area was made by one pathologist and two surgeons.

  Table 1 below shows the correlation between the expression of REG1α and clinical findings in cases where chemoradiotherapy (CRT) was performed. As shown in Table 1, as a result of the evaluation by immunohistochemistry, 23 out of 42 patients were REG1α positive (REG positive) and 19 were REG1α negative (REG negative). In addition, various pathological parameters (from age (Age (years, Averages ± SD)) to additional chemotherapy (Additional Chemotherapy)) were compared between these two groups (REG1α positive and REG1α negative). P value) was 0.7907 to 0.0554, both of which were larger than 0.05 and were judged to have no significant difference at a significance level of 5%.

  On the other hand, for those chemoradiation sensitivity (Clinical response for CRT), the significance value (P value) is 0.0268, which is less than 0.05, so the null hypothesis Was rejected and judged to have a significant difference at a significance level of 5%. That is, it has been shown that the probability of successful chemoradiotherapy is significantly higher for REG1α-positive patients.

  As is clear from Table 1, 8 (35%) of 23 REG1α-positive patients were completely cured (Complete Response (CR)) by radical chemoradiotherapy. On the other hand, in 19 REG1α-negative patients, only 1 (5%) reached complete cure (CR).

  FIG. 5 and FIG. 6 show graphs showing changes in survival rates of REG1α-positive patients who received chemoradiotherapy and REG1α-negative patients who received chemoradiotherapy. In FIG. 5, the vertical axis represents the overall survival rate with respect to the total of all causes of death (total cause of death), and the horizontal axis represents the number of months (Time (months)). In FIG. 6, the vertical axis represents the survival rate (Esophageal cancer-specific survival rate) only when the cause of death is esophageal squamous cell carcinoma, and the horizontal axis represents the number of months.

  Comparing FIG. 5 and FIG. 6, the survival rate of REG1α-positive patients was significantly higher than the survival rate of REG1α-negative patients in both the survival rate for the overall cause of death and the survival rate for the cause of death of only esophageal squamous cell carcinoma. It was. In fact, most (95%) REG1α-negative patients died within 12 months after diagnosis, while 10 (43%) of 23 REG1α-positive patients were alive for at least 2 years after diagnosis.

  Furthermore, in Table 1, these findings, age, tumor location (Tumor location (upper vs middle or lower)), depth of invasion (Depth of invasion (T2-3 vs T4)), lymph node metastasis (Lymph node) metastasis (N1 vs N0)), distant metastasis (Distant metastasis (M0 vs M1)), tumor differentiation (Tumor differentiation (low differentiation vs non-low differentiation (medium / high differentiation))), clinical stage (stage) II-III vs. IV)), and REG1α expression (positive vs. negative) consistent with univariate and multivariate analysis. These results indicate that REG1α expression is an important prognostic factor in patients with esophageal squamous cell carcinoma who have undergone chemoradiotherapy.

Reference Example: Relationship between surgical operation and REG1α expression Although not directly described in Table 1 and FIGS. 5 to 6, 76 patients with squamous cell carcinoma of the esophagus who were surgically removed were used as subjects. The relationship between surgical prognosis and REG1α expression was examined. Of the 76 patients described above, 21 (28%) had undergone only surgical removal, and patients who received additional chemotherapy or additional chemotherapy for cancer recurrence There were 55 people (72%).

  When these 76 patients were classified by the same immunohistochemical method as described above, there were 54 (71%) REG1α-positive patients and 22 (29%) REG1α-negative patients. There were no significant differences in clinical pathology parameters between these two groups. In addition, there was no significant difference between REG1α-positive patients and REG1α-negative patients from the overall cause of death or the survival rate due to death from esophageal squamous cell carcinoma alone.

Claims (19)

Measuring the expression level (A1) of the REG1A gene or a functionally equivalent gene thereof or the REG1B gene or a functionally equivalent gene thereof in a test tissue or a test cell;
Comparing the expression level (A1) with a predetermined expression level (A0);
As a result of comparison, predicting the therapeutic sensitivity of the test tissue or the test cell by determining whether the expression level (A1) is significantly greater than the expression level (A0);
A method for determining the therapeutic sensitivity of cancer cells, comprising:   The determination method according to claim 1, wherein the expression level (A1) is measured by measuring the amount of mRNA expressed from the gene.   The determination method according to claim 2, wherein the mRNA amount is measured by Northern blotting.   The determination method according to claim 1, wherein the expression level (A1) is measured by measuring the amount of protein expressed from the gene.   The determination method according to claim 4, wherein the protein amount is measured by Western blotting.   The determination method according to claim 1, wherein the treatment sensitivity is sensitivity to radiation and / or a chemotherapeutic agent.   The determination method according to claim 1, wherein the cancer is squamous cell carcinoma.   The determination method according to claim 7, wherein the squamous cell carcinoma is esophageal squamous cell carcinoma. The following nucleic acid (a) or (b) used as a nucleic acid probe or PCR primer for measuring the amount of mRNA according to claim 2 or 3:
(A) a nucleic acid having a part of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 or a base sequence complementary thereto;
(B) A nucleic acid that hybridizes with a nucleic acid comprising the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 under highly stringent conditions or a nucleic acid having a complementary base sequence thereto.   A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or a partial peptide thereof, used for measuring the amount of protein according to claim 4 or 5 Alternatively, an antibody against a pharmaceutically acceptable salt thereof. An expression measuring means for measuring the expression of the REG1A gene or a gene functionally equivalent thereto or the REG1B gene or a gene functionally equivalent thereto;
An expression detection means for detecting expression of the gene;
A therapeutic sensitivity determination kit for cancer cells, comprising:   The kit according to claim 11, wherein the expression measuring means is a microarray or a primer for PCR. Administering or contacting a test compound to a test animal or test cell;
Measuring in vitro the expression level (B1) of the REG1A gene or a gene functionally equivalent thereto or the REG1B gene or a gene functionally equivalent thereto in the subject animal or cell;
Comparing the expression level (B1) with the expression level (B0) before administration or contact of the test compound;
As a result of the comparison, the effectiveness of the test compound for improving the therapeutic sensitivity of cancer cells is evaluated by determining whether the expression level (B1) is significantly higher than the expression level (B0). Process,
And a method for evaluating a compound effective for improving the therapeutic sensitivity of cancer cells.   A therapeutic sensitivity enhancer for cancer cells, comprising as an active ingredient a nucleic acid having the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.   A therapeutic enhancer for cancer cells, comprising as an active ingredient a nucleic acid encoding a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or a partial peptide thereof.   A cancer cell comprising, as an active ingredient, a protein comprising the same or substantially the same amino acid sequence as SEQ ID NO: 2 or SEQ ID NO: 4, or a partial peptide thereof, or a pharmaceutically acceptable salt thereof Treatment sensitivity enhancer.   The therapeutic sensitivity enhancer according to any one of claims 14 to 16, wherein the therapeutic sensitivity is sensitivity to radiation and / or a chemotherapeutic agent.   The therapeutic sensitivity enhancer according to any one of claims 14 to 17, wherein the cancer is squamous cell carcinoma.   The therapeutic sensitivity enhancer according to claim 18, wherein the squamous cell carcinoma is esophageal squamous cell carcinoma.