Patent Application
20100239501
The present invention relates to identification of genes related to bone and joint diseases such as osteoarthritis and of polymorphisms in the gene that correlate with the diseases, prophylaxis or treatment of bone and joint diseases based thereon, diagnosis of genetic susceptibility to the diseases, and the like.
Although lifestyle-related diseases of bone and joints have direct effects on life in only a few cases, they represent the major cause of deterioration of the QOL of the elderly because they interfere with the activities of daily living (ADL) due to pain, gait disturbance and the like. Also, because these diseases are characterized by rapid rises in incidence with aging and by a chronic course, they impose a great burden on national medical economics, posing an important problem to be overcome in aging society as a whole. The World Health Organization (WHO) positioned the first 10 years of the 21 century as “The Bone and Joint Decade (BJD)” and begun efforts to conquer bone and joint diseases.
Osteoarthritis (hereinafter to be sometimes referred to as “OA”), a bone and joint disease accompanied by chronic arthritis, is characterized by cartilage destruction and proliferative changes in bone and cartilage due to regressive degeneration of cartilage. The number of patients suffering from this disease is as many as 5 million to 7 million in Japan alone; it is reported that not less than 80% of the people aged 60 years or more exhibit symptoms of osteoarthritis in their knees, elbows, hip joints, and spine, and this disease is a representative common disease. At present, no fundamental therapeutic approach for OA is available, with symptomatic therapies for pain relief, such as administration of non-steroidal anti-inflammatory analgesics, hyaluronic acid, or steroid agents, forming the mainstream. For advanced cases, surgeries such as arthroscopic surgery, osteotomy, or prosthetic replacement are indicated; however, because of the limited durability of joint prosthesis, it is thought to be desirable to avoid this surgery until about 55 years of age. Accordingly, there is a demand for the development of a therapeutic method for suppressing retrograde changes of joints.
OA is a multifactor disease involved by environmental factors and genetic factors. Elucidation of a genetic factor leads to the presentation of a drug discovery target. The present inventors have found to date that polymorphs in the CALM1 gene, which is one of the genes that encode calmodulin, and the gene for asporin, which is one of the extracellular substrate proteins that have been reported to be highly expressed in OA cartilage, correlate with OA, and have reported that abnormalities in these genes are associated with the onset and/or progression of OA (Patent Document 1 and Non-patent Documents 1 and 2).
About half the existing pharmaceuticals exhibit their actions via receptors on cell membrane, and G protein coupled receptors (GPCRs), in particular, account for a major percentage; therefore, GPCRs are the most promising drug discovery target. However, no GPCR associated with the onset or progression of OA has been reported.
Lysophosphatidic acid (LPA) is a bioactive lipid that controls a wide variety of biological processes; as receptors thereof, 5 kinds of GPCRs (LPA1-LPA5) have been reported to date. Of them, LPA1 is also called EDG2 or VZG-1 (Non-patent Documents 3 and 4) (hereinafter, denoted as “EDG2” herein). Because LPA has algesic action (Non-patent Documents 5 and 6), and also because it is suggested that early intervention at the LPA receptor level may be effective in suppressing the progression or mitigating the severity of neurogenic pain (NP). (Non-patent Document 7), EDG2 antagonists are expected to serve as candidate therapeutics for neurogenic pain (NP) (Non-patent Document 8). However, there has been no report of the relationship between EDG2 and bone/joint diseases, including OA.
It is an object of the present invention to identify genes involved in the onset and/or progression of bone/joint diseases, including OA, particularly GPCR genes, and elucidate the functions thereof, to thereby provide novel and radical means for the prevention/treatment of the diseases. It is another object of the present invention to provide a convenient and highly reliable diagnostic method for genetic susceptibility to bone and joint diseases.
The present inventors, to accomplish the above-described objects, focused on the GPCR gene family, which is promising as a drug discovery target, to find candidate genes, and performed correlation analyses between a knee OA (KOA) group and a control group using already reported polymorphs (marker SNPs). As a result, the inventors found that polymorphs on the EDG2 gene exhibit a high correlation in two independent populations (general population and cohort population). Furthermore, the inventors identified a plurality of SNPs correlating with KOA by linkage disequilibrium (LD) mapping. One of them (i-EDG2-09) was present in the promoter region of the EDG2 gene; as a result of gel shift analysis, the existence of intranuclear factors that bind to disease-susceptible alleles more firmly was revealed. As a result of reporter assay, it was found that in disease-susceptible alleles, EDG2 transcription activity is elevated.
As a result of expression analysis in major organs and tissues, EDG2 was found to be highly expressed in the synovial membrane and spinal cord. Hence, to analyze the role of EDG2 in synovial cells, OA synovial cells were stimulated with LPA; the expression of inflammatory cytokines rose transiently, and 1 to 2 hours after the peak was reached, the expression of MMP-1, 3, and 13 rose. Meanwhile, since EDG2 is also highly expressed in the spinal cord, and LPA reportedly has algesic action, a correlation analysis was performed on a population with complaint of pain as a subjective symptom in a cohort population; a significant correlation was observed in i-EDG2-09.
These results show that polymorphs of the promoter region of the EDG2 gene alter the amount of EDG2 expressed, and are involved in the onset/progression of KOA, and strongly suggest that EDG2 antagonists suppress the induction of inflammatory cytokines to suppress cartilage destruction and lessen joint pain that accompanies KOA.
The present inventors conducted further investigations based on these findings, and have developed the present invention.
Accordingly, the present invention provides:
Because EDG2 induces inflammatory cytokines in synovial cells and raises the expression of cartilage substrate decomposing enzyme, increased expression of the EDG2 gene leads to cartilage destruction. Therefore, by inhibiting the expression and/or activity of EDG2 or LPA, which is a ligand thereof, inflammatory cytokine induction and/or cartilage destruction can be suppressed. Because the LPA-EDG2 signal is also involved in pain, by inhibiting the expression and/or activity of EDG2 or LPA, pain in a bone/joint disease can be suppressed. Furthermore, because polymorphs in the EDG2 gene correlate with bone/joint diseases, the polymorphs can be utilized for convenient determination of genetic susceptibility to bone/joint diseases.
The protein comprising the same or substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO:2 used in the present invention (hereinafter, sometimes generically simply referred to as “EDG2”) may be a natural EDG2 protein derived from a cell [for example, hepatocyte, splenocyte, nerve cell, glial cell, pancreatic β cell, myelocyte, mesangial cell, Langerhans' cell, epidermal cell, epithelial cell, goblet cell, endothelial cell, smooth muscle cell, fibroblast, fibrocyte, myocyte, adipocyte, immune cell (e.g., macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, eosinophil, monocyte), megakaryocyte, synovial cell, chondrocyte, bone cell, osteoblast, osteoclast, mammary gland cell or interstitial cell, or corresponding precursor cell or stem cell thereof, and the like] of a human or other warm-blooded animal (for example, monkey, bovine, horse, swine, sheep, goat, rabbit, mouse, rat, guinea pig, hamster, chicken, and the like), or any tissue or organ where such cells are present [for example, brain or any portion of the brain (e.g., olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, hypophysis, stomach, pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow, adrenal gland, skin, muscle (e.g., skeletal muscle), lung, gastrointestinal tract (e.g., large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, adipose tissue (e.g., brown adipose tissue, white adipose tissue), and the like], preferably synovial membrane, whole brain, spinal cord or the like, and, as described below, may be a protein synthesized chemically or biochemically using a cell-free protein synthesis system. Alternatively, the protein may be a recombinant protein produced from a transformant incorporating a nucleic acid comprising a base sequence that encodes the above-described amino acid sequence.
Substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO:2 refers to an amino acid sequence having a homology of about 50% or more, preferably about 60% or more, more preferably about 70% or more, still more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more, to the amino acid sequence shown by SEQ ID NO:2. Here, “a homology” means a ratio (%) of identical amino acid residues and similar amino acid residues to all overlapping amino acid residues in the optimal alignment (preferably, the algorithm considers introduction of gaps on one or both sides of the sequence for the best alignment) where two amino acid sequences are aligned using a mathematical algorithm known in the technical field. “Similar amino acid” means an amino acid having similar physiochemical properties; examples thereof include amino acids classified under the same group, 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 a hydroxyl group (Ser, Thr) and amino acids having a small side-chain (Gly, Ala, Ser, Thr, Met). Substitution by such similar amino acids is expected not to change the phenotype of proteins (i.e., conservative amino acid substitution). Specific examples of the conservative amino acid substitution are known in the technical field and are described in various documents (see, for example, Bowie et al., Science, 247:1306-1310 (1990)).
Amino acid sequence homology in the present description can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectancy=10; gaps allowed; matrix=BLOSUM62; filtering=OFF). As examples of other algorithms for determination of amino acid sequence homology, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993) [the algorithm is incorporated in the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997))], the algorithm described in Needleman et al., J. Mol. Biol., 48:444-453 (1970) [the algorithm is incorporated in the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4:11-17 (1988) [the algorithm is incorporated in the ALIGN program (version 2.0), which is part of the CGC sequence alignment software package], the algorithm described in 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 can be mentioned, which can likewise be used preferably.
A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO:2 refers to a protein comprising the above-described “substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO:2”, and possessing substantially the same quality of activity as a protein comprising the amino acid sequence shown by SEQ ID NO:2. As examples of substantially the same quality of activity, LPA binding activity, LPA signal transduction activities [for example, GTP binding activity, adenylate cyclase (AC) inhibitory activity, phospholipase C (PLC) β activity enhancement activity, inflammatory cytokine (e.g., TNFα, IL-1b, IL-6 and the like) induction activity, subsequent cartilage substrate decomposing enzyme (e.g., MMP-1, MMP-3, MMP-13 and the like) induction activity, algesic action and the like] and the like can be mentioned. “Substantially the same quality” means that the activities are qualitatively (e.g., physiologically or pharmacologically) equivalent to each other. Therefore, it is preferable that the above-described quantitative factors such as the extent of activity be equivalent to each other, but the quantitative factors may be different (for example, about 0.01 to about 100 times, preferably about 0.1 to about 10 times, more preferably about 0.5 to about 2 times).
A measurement of EDG2 activity can be performed in accordance with a method known per se. For example, LPA binding activity can be measured using 3H-labeled LPA, and GTP binding activity can be measured using a 35S-labeled GTP analogue (e.g., GTPγS and the like) with RI activity as an index. A measurement of AC activity can be performed by measuring the cAMP content using an anti-cAMP antibody, or with the expression of a reporter gene under the control of cAMP responding element (CRE) as an index. A measurement of PLCβ activity can be performed by measuring the Ca2+ content in the cell using a fluorescent probe (e.g., fura-2, indo-1, fluor-3, Calcium-Green I and the like) or a Ca-sensitive luminescent protein (e.g., aequorin and the like), or by measuring the resulting inositol-1,4,5-triphosphate using 3H-labeled phosphatidylinositol-4,5-diphosphbric acid, or with the expression of a reporter gene under the control of TPA responding element (TRE) as an index. Algesic action can be evaluated by a publicly known pain test such as von Frey test.
The EDG2 used in the present invention also includes, for example, a protein comprising (1) an amino acid sequence resulting from deletion of 1 or 2 or more [preferably, 1 to about 30, more preferably 1 to about 10, still more preferably 1 to several (e.g., 5, 4, 3, 2)] amino acids in the amino acid sequence shown by SEQ ID NO:2, (2) an amino acid sequence resulting from addition of 1 or 2 or more [preferably, 1 to about 30, more preferably 1 to about 10, still more preferably 1 to several (e.g., 5, 4, 3, 2)] amino acids to the amino acid sequence shown by SEQ ID NO:2, (3) an amino acid sequence resulting from insertion of 1 or 2 or more [preferably, 1 to about 30, more preferably 1 to about 10, still more preferably 1 to several (e.g., 5, 4, 3, 2)] amino acids into the amino acid sequence shown by SEQ ID NO:2, (4) an amino acid sequence resulting from substitution of 1 or 2 or more [preferably, 1 to about 30, more preferably 1 to about 10, still more preferably 1 to several (e.g., 5, 4, 3, 2)] amino acids in the amino acid sequence shown by SEQ ID NO:2 with other amino acids, or (5) an amino acid sequence being a combination thereof, and possessing substantially the same quality of activity as a protein comprising the amino acid sequence shown by SEQ ID NO:2.
When an amino acid sequence has been inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited, as far as the activity of the protein is not interfered with.
Meanwhile, a protein comprising substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO:2, but not possessing substantially the same quality of activity as a protein consisting of the amino acid sequence shown by SEQ ID NO:2, for example, a protein incorporating a mutation (for example, deletion, insertion, substitution, modification and the like) in a region involved in the activation of a conjugated G protein a subunit (e.g., Gi/o) (G protein activating domain; GAD) (for example, Arg residue on the N-terminal side of intracellular third loop and/or RRNR sequence on the C-terminal side and the like) and the like (i.e., dominant negative mutant; DNM) is capable of blocking EDG2-mediated signal transduction from LPA by binding to LPA, so that such a protein can be useful for the purpose of suppressing the signal transduction action.
For the proteins identified by amino acid sequences herein, the left end indicates the N-terminal (amino terminal) and the right end indicates the C-terminal (carboxyl terminal), according to the common practice of peptide designation. In the EDG2 used in the present invention, including proteins comprising the amino acid sequence shown by SEQ ID NO:2, the C-terminal may be any of a carboxyl group (—COOH), a carboxylate (—COO−), an amide (—CONH2) and an ester (—COOR).
Here, as R in the ester, a C1-6 alkyl group, for example, methyl, ethyl, n-propyl, isopropyl and n-butyl, a C3-8 cycloalkyl group, for example, cyclopentyl and cyclohexyl, a C6-12 aryl group, for example, phenyl and α-naphthyl, a phenyl-C1-2 alkyl group, for example, benzyl and phenethyl, a C7-14 aralkyl group, for example, an α-naphthyl-C1-2 alkyl group, for example, α-naphthylmethyl, a pivaloyloxymethyl group; and the like can be used.
When the protein used in the present invention has a carboxyl group (or carboxylate) at a position other than the C-terminal, a protein wherein the carboxyl group is amidated or esterified is also included in the protein used in the present invention. In this case, as the ester, the above-described ester at the C terminal, and the like, for example, are used.
Furthermore, the protein used in the present invention also includes a protein wherein the amino group of the N-terminal amino acid residue (e.g., methionine residue) is protected by a protecting group (for example, C1-6 acyl groups such as C1-6 alkanoyls such as formyl group and acetyl group, and the like); a protein wherein the glutamine residue produced upon cleavage at the N terminal in vivo has been converted to pyroglutamic acid, a protein wherein a substituent (for example, —OH, —SH, amino group, imidazole group, indol group, guanidino group and the like) on a side chain of an amino acid in the molecule is protected by an appropriate protecting group (for example, C1-6 acyl groups such as C1-6 alkanoyl groups such as formyl group and acetyl group, and the like), a conjugated peptide such as what is called a glycopeptide having a sugar chain bound thereto (for example, the Asn residue shown by amino acid numbers 27 and 35 in the amino acid sequence shown by SEQ ID NO:2 is possibly an N-binding glycosylation site), and the like.
As specific examples of the protein used in the present invention, human EDG2 consisting of the amino acid sequence shown by SEQ ID NO:2 (GenBank accession No., NP—001392, NP—476500 and the like), or an ortholog thereof in another mammal and the like can be mentioned. For example, mouse EDG2 is registered with the NCBI database as GenBank accession No., NP—034466, and exhibits an amino acid identity of about 96% to human EDG2. Rat EDG2 is registered with the NCBI database as GenBank accession No. NP—446388, and exhibits an amino acid identity of about 97% to human EDG2.
The partial peptide of EDG2 used in the present invention may be any peptide comprising the same or substantially the same amino acid sequence as a partial amino acid sequence of the amino acid sequence shown by SEQ ID NO:2, as far as the aforementioned substantially the same quality of activity as EDG2 used in the present invention is possessed. Here, “substantially the same quality of activity” is defined as described above. A measurement of “substantially the same quality of activity” can be performed in the same manner as the above. Herein, the partial peptide is hereinafter referred to as “the active peptide of the present invention”.
Specifically, as the partial peptide, a peptide consisting of at least 50 or more, preferably 70 or more, more preferably 100 or more, amino acid sequences of the amino acid sequences that constitute EDG2 used in the present invention, and the like are used. The partial peptide of EDG2 used in the present invention may have (1) 1 or 2 or more [preferably, 1 to about 30, more preferably 1 to about 10, still more preferably 1 to several (e.g., 5, 4, 3, 2)] amino acids deleted in the amino acid sequence thereof, or (2) 1 or 2 or more [preferably, 1 to about 30, more preferably 1 to about 10, still more preferably 1 to several (e.g., 5, 4, 3, 2)] amino acids added to the amino acid sequence thereof, or (3) 1 or 2 or more [preferably, 1 to about 30, more preferably 1 to about 10, still more preferably 1 to several (e.g., 5, 4, 3, 2)] amino acids inserted into the amino acid sequence thereof, or (4) 1 or 2 or more [preferably 1 to about 10, more preferably 1 to several (e.g., 5, 4, 3, 2)] amino acids in the amino acid sequence thereof substituted by other amino acids, or (5) a combination thereof.
Meanwhile, peptides comprising a partial amino acid sequence of EDG2, but not possessing substantially the same quality of activity as EDG2, for example, ones having a partial amino acid sequence of the amino acid sequence shown by SEQ ID NO:2 that contains a region involved in the binding to LPA (ligand-binding domain; LBD) but does not contain a G protein activation domain (GAD), and the like are not included in “the active peptide of the present invention”. However, such peptides, like the above-described dominant negative mutant (DNM) of EDG2, are capable of blocking the EDG2-mediated signal transduction from LPA by binding to LPA, so that they can be useful for the purpose of suppressing the signal transduction action. Herein, such a partial peptide is hereinafter referred to as “the inhibitory peptide of the present invention”.
In the partial peptide of EDG2 used in the present invention (encompasses both the active peptide of the present invention and the inhibitory peptide of the present invention; hereinafter, in this case, sometimes referred to as “the partial peptide of the present invention”), the C-terminal may be any of a carboxyl group (—COOH), a carboxylate (—COO−), an amide (—CONH2) and an ester (—COOR). Here, as R in the ester, the same as those mentioned for EDG2 above can be mentioned. When the partial peptide has a carboxyl group (or a carboxylate) at a position other than the C terminal, a partial peptide wherein the carboxyl group is amidated or esterified is also included in the partial peptide of EDG2 used in the present invention. In this case, as the ester, the above-described ester at the C terminal, and the like, for example, are used. Furthermore, the partial peptide also includes a protein wherein the amino group of the N terminal amino acid residue (e.g., methionine residue) is protected by a protecting group, a protein wherein Gln, which is produced upon cleavage at the N terminal in vivo, has been converted to pyroglutamic acid, a protein wherein a substituent on a side chain of an amino acid in the molecule is protected by an appropriate protecting group, a conjugated peptide such as what is called a glycopeptide having a sugar chain bound thereto, and the like, as with the case of EDG2.
As the salt of EDG2 or a partial peptide thereof used in the present invention, physiologically acceptable salts with acid (e.g., inorganic acid, organic acid) or base (e.g., alkali metal salts) can be mentioned, and physiologically acceptable acid addition salts are preferred. Useful salts include, for example, salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or salts with organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
EDG2 or a salt thereof used in the present invention can be prepared from a cell or tissue of the aforementioned human or a warm-blooded animal by a method known per se of protein purification. Specifically, EDG2 used in the present invention can be purified or isolated by homogenizing tissue or cells of the animals, and extracting by an acid, and applying the extract to a combination of chromatographies such as reversed-phase chromatography, ion exchange chromatography, and the like.
EDG2 or its partial peptide or a salt thereof used in the present invention (hereinafter also comprehensively referred to as “EDG2s”) can also be produced according to a publicly known method of peptide synthesis.
The method of peptide synthesis may be any of, for example, a solid phase synthesis process and a liquid phase synthesis process. A desired protein can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion, and removing any protecting group the resultant product may have. Here, the condensation and the protecting group removal are conducted in accordance with methods known per se, for example, the methods indicated in (1) and (2) below:
For the synthesis of EDG2s, an ordinary commercially available resin for protein synthesis can be used. As examples of such resins, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin, 4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin and the like can be mentioned. Using such a resin, an amino acid having an appropriately protected α-amino group and side chain functional group is condensed on the resin in accordance with the sequence of the desired protein or the like according to one of various methods of condensation known per se. At the end of the reaction, the protein or a partial peptide thereof is cleaved from the resin and at the same time various protecting groups are removed, and a reaction to form an intramolecular disulfide bond is carried out in a highly diluted solution to obtain the desired protein or a partial peptide thereof or an amide thereof.
For the above-described condensation of protected amino acids, various activation reagents which can be used for protein synthesis can be used, and a carbodiimide is preferably used. As the carbodiimide, DCC, N,N′-diisopropylcarbodiimide, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide and the like are used. For the activation using these carbodiimides, the protected amino acid, along with a racemation-suppressing additive (for example, HOBt, HOOBt), may be added directly to the resin, or the protected amino acid may be activated in advance as a symmetric acid anhydride or HOBt ester or HOOBt ester and then added to the resin.
Solvents used for the activation of protected amino acids and condensation thereof with a resin can be appropriately selected from among solvents that are known to be usable for protein condensation reactions. As examples of useful solvents, acid amides such as N,N-dimethylformamide, N,N-dimethylacetamide and N-methylpyrrolidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; sulfoxides such as dimethyl sulfoxide; ethers such as pyridine dioxane and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; suitable mixtures thereof; and the like can be mentioned. Reaction temperature is appropriately selected from the range that is known to be usable for protein binding reactions, and is normally selected from the range of about −20° C. to about 50° C. An activated amino acid derivative is normally used from 1.5 to 4 times in excess. When a test using the ninhydrin reaction reveals that the condensation is insufficient, sufficient condensation can be conducted by repeating the condensation reaction without elimination of protecting groups. If the condensation is insufficient even though the reaction is repeated, unreacted amino acids may be acetylated using acetic anhydride or acetylimidazole to prevent the subsequent reaction from being influenced.
A protecting method and a protecting group for a functional group that should not be involved in the reaction of raw materials, a method of removing the protecting group, a method of activating a functional group involved in the reaction, and the like can be appropriately selected from among publicly known groups or publicly known means.
As examples of the protecting group for an amino group of the starting material, Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C1-Z, Br—Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc, and the like can be used.
A carboxyl group can be protected, for example, by alkyl esterification (for example, linear, branched or cyclic alkyl esterification with methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, and the like), aralkyl esterification (for example, benzyl esterification, 4-nitrobenzyl esterification, 4-methoxybenzyl esterification, 4-chlorobenzyl esterification, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazidation, t-butoxycarbonyl hydrazidation, trityl hydrazidation, and the like.
The hydroxyl group of serine can be protected by, for example, esterification or etherification. As examples of a group suitable for this esterification, lower (C1-6) alkanoyl groups such as an acetyl group, aroyl groups such as a benzoyl group, and groups derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group, and the like are used. As examples of a group suitable for etherification, a benzyl group, a tetrahydropyranyl group, a t-butyl group, and the like can be mentioned.
As examples of the protecting group for the phenolic hydroxyl group of tyrosine, Bzl, Cl2-Bzl, 2-nitrobenzyl, Br—Z, t-butyl, and the like can be used.
As examples of the protecting group for the imidazole of histidine, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like are used.
As examples of the method of removing (eliminating) a protecting group, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon; acid treatment by means of anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethane-sulfonic acid, trifluoroacetic acid, or a mixture solution thereof; base treatment by means of diisopropylethylamine, triethylamine, piperidine, piperazine or the like; and reduction with sodium in liquid ammonia, and the like are used. The elimination reaction by the above-described acid treatment is generally carried out at a temperature of about −20° C. to about 40° C.; the acid treatment is efficiently conducted by adding a cation scavenger, for example, anisole, phenol, thioanisole, m-cresol, p-cresol, dimethylsulfide, 1,4-butanedithiol and 1,2-ethanedithiol. Also, a 2,4-dinitrophenyl group used as a protecting group of the imidazole of histidine is removed by thiophenol treatment; a formyl group used as a protecting group of the indole of tryptophan is removed by acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol, or the like, as well as by alkali treatment with a dilute sodium hydroxide solution, dilute ammonia, or the like.
As examples of those obtained by activation of the carboxyl group in the starting material, a corresponding acid anhydride, an azide, an activated ester [an ester with an alcohol (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB, N-hydroxysuccimide, N-hydroxyphthalimide, or HOBt)] and the like are used. As examples of those obtained by activation of the amino group in the starting material, a corresponding phosphoric amide is used.
In another method of preparing an amide of a protein or a partial peptide thereof, for example, the α-carboxyl group of the carboxy terminal amino acid is first amidated and hence protected, and a peptide (protein) chain is elongated to a desired chain length toward the amino group side, thereafter a protein or a partial peptide thereof having the protecting group for the N terminal α-amino group of the peptide chain only removed and a protein or a partial peptide thereof having the protecting group for the C terminal carboxyl group only removed are prepared, and these proteins or peptides are condensed in a mixed solvent described above. For details about the condensation reaction, the same as above applies. After the protected protein or peptide obtained by the condensation is purified, all protecting groups can be removed by the above-described method to yield a desired crude protein or peptide. By purifying this crude protein or peptide using various publicly known means of purification, and freeze-drying the main fraction, a desired amide of the protein or peptide can be prepared.
In order to obtain esters of the protein or peptide, a desired ester of the protein or peptide can be prepared by, for example, condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to yield an amino acid ester, and then treating the ester in the same manner as with an amide of the protein or peptide.
The partial peptide of EDG2 or salt thereof used in the present invention can also be produced by cleaving the EDG2 obtained by any of the methods described above or below or a salt thereof with an appropriate peptidase.
EDG2s of the present invention thus obtained can be purified or isolated by a known method of purification. Here, as examples of the method of purification, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, combinations thereof and the like can be mentioned.
When thus obtained protein or partial peptide is in a free form, the free form can be converted into a suitable salt form by a known method or an analogue thereto, and on the other hand, when the protein is obtained in the form of a salt, it can be converted into the free form or in the form of a different salt by a known method or an analogue thereto.
EDG2s of the present invention can also be produced by cultivating a transformant harboring expression vectors comprising nucleic acid that encodes EDG2 or a partial peptide thereof to produce EDG2s, and separating and purifying EDG2s from the culture obtained.
The nucleic acid that encodes EDG2 or a partial peptide thereof may be any one, as long as it comprises a base sequence that encodes the amino acid sequence of the aforementioned EDG2 used in the present invention or a partial amino acid sequence thereof. Although the nucleic acid may be a DNA, an RNA, or a DNA/RNA chimera, it is preferably a DNA. Additionally, the nucleic acid may be double-stranded or single-stranded. In the case of a double-stranded nucleic acid, it may be a double-stranded DNA, a double-stranded RNA, or a DNA:RNA hybrid.
As the DNA that encodes EDG2 or a partial peptide thereof, genomic DNA, a cDNA derived from a cell [for example, hepatocyte, splenocyte, nerve cell, glial cell, pancreatic β cell, myelocyte, mesangial cell, Langerhans' cell, epidermal cell, epithelial cell, goblet cell, endothelial cell, smooth muscle cell fibroblast, fibrocyte, myocyte, adipocyte, immune cell (for example, macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, eosinophil, monocyte), megakaryocyte, synovial cell, chondrocyte, bone cell, osteoblast, osteoclast, mammary gland cell or interstitial cell, or corresponding precursor cell, stem cell or cancer cell thereof, and the like] of a human or other warm-blooded animal (for example, monkey, bovine, horse, swine, sheep, goat, rabbit, mouse, rat, guinea pig, hamster, chicken, and the like), or any tissue or organ where such cells are present [for example, brain or any portion of the brain (e.g., olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, hypophysis, stomach, pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow, adrenal gland, skin, muscle (e.g., skeletal muscle), lung, gastrointestinal tract (e.g., large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, adipose tissue (e.g., brown adipose tissue, white adipose tissue), and the like], preferably synovial membrane, whole brain, spinal cord and the like, synthetic DNA and the like can be mentioned. The genomic DNA and cDNA that encode EDG2 or a partial peptide thereof can also be directly amplified by polymerase chain reaction (hereinafter abbreviated as “PCR method”) and reverse transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”) using a genomic DNA fraction and a total RNA or mRNA fraction prepared from one of the above-described cells or tissues as the respective templates. Alternatively, the genomic DNA and cDNA that encode EDG2 or a partial peptide thereof can also be cloned from a genomic DNA library and cDNA library prepared by inserting a fragment of a genomic DNA and total RNA or mRNA, respectively, prepared from one of the above-described cells or tissues into an appropriate vector, by the colony or plaque hybridization method or the PCR method and the like. The vector used for the library may be any of a bacteriophage, a plasmid, a cosmid, a phagemid and the like.
As examples of the DNA that encodes EDG2, DNA comprising the base sequence shown by SEQ ID NO:1, DNA that comprises a base sequence hybridizing to the base sequence shown by SEQ ID NO:1 under highly stringent conditions, and that encodes a protein or peptide having substantially the same quality of activity as the aforementioned protein comprising the amino acid sequence shown by SEQ ID NO:2, and the like can be mentioned.
As examples of the DNA capable of hybridizing to the base sequence shown by SEQ ID NO:1 under highly stringent conditions, DNA that comprises a base sequence showing a homology of about 60% or more, preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, to the base sequence shown by SEQ ID NO:1, and the like are used.
Base sequence homology in the present description can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectancy=10; gap allowed; filtering=ON; match score=1; mismatch score=−3). As preferable examples of other algorithms for determining base sequence homology, the above-described amino acid sequence homology calculation algorithm can also be mentioned.
Hybridization can be conducted according to a method known per se or a method based thereon, for example, a method described in Molecular Cloning, 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) and the like. When a commercially available library is used, hybridization can be conducted according to the method described in the instruction manual attached thereto. Hybridization can preferably be conducted under highly stringent conditions.
As examples of the highly stringent conditions, conditions of a sodium salt concentration of about 19 to about 40 mM, preferably about 19 to about 20 mM, and a temperature of about 50° C. to about 70° C., preferably about 60° C. to about 65° C. (in particular, the case of a sodium salt concentration of about 19 mM and a temperature of about 65° C. is preferred), or a hybridization reaction in 6×SSC (sodium chloride/sodium citrate) at 45° C. followed by washing in 0.2×SSC/0.1% SDS at 65° C. once or more and the like can be mentioned. Those skilled in the art are able to easily obtain desired stringency by changing the salt concentration of the hybridization solution, hybridization reaction temperature, probe concentration, probe length, the number of mismatches, hybridization reaction time, the salt concentration of the washing solution, washing temperature and the like as appropriate.
The DNA that encodes EDG2 is preferably a human EDG2 cDNA comprising the base sequence shown by SEQ ID NO:1 (GenBank accession No., NM—001401) or an allele mutant thereof or an ortholog thereof in another warm-blooded animal (for example, mouse, rat, guinea pig, hamster, rabbit, sheep, goat, swine, bovine, horse, bird, cat, dog, monkey, chimpanzee and the like) and the like. For example, mouse EDG2 is registered with the NCBI database as GenBank accession No., NM—010336. Rat EDG2 is registered with the NCBI database as GenBank accession No., NM—053936.
The DNA that encodes the partial peptide of EDG2 may be any one comprising the base sequence that encodes the same or substantially the same amino acid sequence as a portion of the amino acid sequence shown by SEQ ID NO:2. The DNA may be any of genomic DNA, cDNA derived from the above-described cell or tissue, and synthetic DNA.
Specifically, as examples of the DNA that encodes the partial peptide,
As examples of the DNA capable of hybridizing to the DNA comprising the base sequence shown by SEQ ID NO:1 under highly stringent conditions, a DNA comprising a base sequence showing a homology of about 60% or more, preferably about 70% or more, more preferably about 80% or more, and particularly preferably about 90% or more, to the corresponding part in the base sequence, and the like are used.
The DNA that encodes EDG2 or a partial peptide thereof can be cloned by amplifying it by the PCR method using a synthetic DNA primer comprising a portion of the base sequence that encodes the protein or peptide, or by hybridizing DNA incorporated in an appropriate expression vector to a labeled DNA fragment or synthetic DNA that encodes a portion or the entire region of EDG2. Hybridization can be conducted according to, for example, a method described in Molecular Cloning, 2nd edition (ibidem) and the like. When a commercially available library is used, hybridization can be conducted according to the method described in the instruction manual attached to the library.
The base sequence of DNA can be converted according to a method known per se, such as the ODA-LA PCR method, the Gapped duplex method, the Kunkel method and the like, or a method based thereon, using a publicly known kit, for example, Mutan™-super Express Km (Takara Shuzo Co., Ltd.), Mutan™-K (Takara Shuzo Co., Ltd.) and the like.
The cloned DNA can be used as is, or after digestion with a restriction endonuclease or addition of a linker as desired, depending on the purpose of its use. The DNA may have the translation initiation codon ATG at the 5′ end thereof, and the translation stop codon TAA, TGA or TAG at the 3′ end thereof. These translation initiation codons and translation stop codons can be added using an appropriate synthetic DNA adapter.
By transforming a host with an expression vector comprising the above-described DNA that encodes EDG2 or a partial peptide thereof, and culturing the transformant obtained, the protein or peptide can be produced.
A expression vector comprising DNA that encodes EDG2 or a partial peptide thereof can be produced by, for example, cutting out a desired DNA fragment from the DNA that encodes EDG2, and joining the DNA fragment downstream of a promoter in an appropriate expression vector.
Useful expression vectors include plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194); plasmids derived from yeast (e.g., pSH19, pSH15); bacteriophages such as λ phage; animal viruses such as retrovirus, vaccinia virus and baculovirus; pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo, and the like.
The promoter may be any promoter, as long as it is appropriate for the host used to express the gene.
For example, when the host is an animal cell, the SRα promoter, the SV40 promoter, the LTR promoter, the CMV (cytomegalovirus) promoter, the HSV-TK promoter and the like are used. Of these, the CMV promoter, the SRα promoter and the like are preferred.
When the host is a bacterium of the genus Escherichia, the trp promoter, the lac promoter, the recA promoter, the λPL promoter, the lpp promoter, the T7 promoter and the like are preferred.
When the host is a bacterium of the genus Bacillus, the SPO1 promoter, the SPO2 promoter, the penP promoter and the like are preferred.
When the host is yeast, the PHO5 promoter, the PGK promoter, the GAP promoter, the ADH promoter and the like are preferred.
When the host is an insect cell, the polyhedrin prompter, the P10 promoter and the like are preferred.
Useful expression vectors include, in addition to the above, those optionally harboring an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter also abbreviated as SV40ori) and the like. As examples of the selection marker, the dihydrofolate reductase (hereinafter also abbreviated as dhfr) gene [methotrexate (MTX) resistance], the ampicillin resistance gene (hereinafter also abbreviated as Ampr), the neomycin resistance gene (hereinafter also abbreviated as Neor, G418 resistance) and the like can be mentioned. In particular, when a Chinese hamster cell lacking the dhfr gene is used in combination with the dhfr gene as the selection marker, a target gene can also be selected using a thymidine-free medium.
In addition, as required, a base sequence encoding a signal sequence (signal codon) that matches the host may be added to the 5′ terminal side of the DNA encoding EDG2 or a partial peptide thereof. Useful signal sequences include a PhoA signal sequence, an OmpA signal sequence and the like when the host is a bacterium of the genus Escherichia; an α-amylase signal sequence, a subtilisin signal sequence and the like when the host is a bacterium of the genus Bacillus; an MFα signal sequence, an SUC2 signal sequence and the like when the host is yeast; and an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence and the like when the host is an animal cell.
Useful hosts include, for example, a bacterium of the genus Escherichia, a bacterium of the genus Bacillus, yeast, an insect cell, an insect, an animal cell and the like.
Useful bacteria of the genus Escherichia include, for example, Escherichia coli K12 DH1 (Proc. Natl. Acad. Sci. U.S.A., Vol. 60, 160 (1968)), JM103 (Nucleic Acids Research, Vol. 9, 309 (1981)), JA221 (Journal of Molecular Biology, Vol. 120, 517 (1978)), HB101 (Journal of Molecular Biology, Vol. 41, 459 (1969)), C600 (Genetics, Vol. 39, 440 (1954)) and the like.
Useful bacteria of the genus Bacillus include, for example, Bacillus subtilis MI114 (Gene, Vol. 24, 255 (1983)), 207-21 (Journal of Biochemistry, Vol. 95, 87 (1984)) and the like.
Useful yeasts include, for example, Saccharomyces cerevisiae AH22, AH22R−, NA87-11A, DKD-5D and 20B-12, Schizosaccharomyces pombe NCYC1913 and NCYC2036, Pichia pastoris KM71, and the like.
Useful insect cells include, for example, Spodoptera frugiperda cell (Sf cell), MG1 cell derived from the mid-intestine of Trichoplusia ni, High Five™ cell derived from an egg of Trichoplusia ni, cell derived from Mamestra brassicae, cell derived from Estigmena acrea, and the like can be mentioned when the virus is AcNPV. When the virus is BmNPV, useful insect cells include Bombyx mori N cell (BmN cell) and the like. Useful Sf cells include, for example, Sf9 cell (ATCC CRL1711), Sf21 cell (both in Vaughn, J. L. et al., In Vivo, 13, 213-217 (1977) and the like.
Useful insects include, for example, a larva of Bombyx mori (Maeda et al., Nature, Vol. 315, 592 (1985)) and the like.
Useful animal cells include, for example, monkey cell COS-7, Vero, Chinese hamster cell CHO (hereafter abbreviated as CHO cell), Chinese hamster cell lacking the dhfr gene CHO (hereafter abbreviated as CHO(dhfr−) cell), mouse L cell, mouse AtT-20, mouse myeloma cell, rat GH3, human FL cell and the like.
Transformation can be carried out according to the kind of host in accordance with a publicly known method.
A bacterium of the genus Escherichia can be transformed, for example, in accordance with a method described in Proc. Natl. Acad. Sci. U.S.A., Vol. 69, 2110 (1972), Gene, Vol. 17, 107 (1982) and the like.
A bacterium of the genus Bacillus can be transformed, for example, according to a method described in Molecular and General Genetics, Vol. 168, 111 (1979) and the like.
Yeast can be transformed, for example, in accordance with a method described in Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978) and the like.
An insect cell and an insect can be transformed, for example, according to a method described in Bio/Technology, 6, 47-55 (1988) and the like.
An animal cell can be transformed, for example, in accordance with a method described in Saibo Kogaku (Cell Engineering), extra issue 8, Shin Saibo Kogaku Jikken Protocol (New Cell Engineering Experimental Protocol), 263-267 (1995), published by Shujunsha, or Virology, Vol. 52, 456 (1973).
Cultivation of a transformant can be carried out according to the kind of host in accordance with a publicly known method.
For example, when a transformant whose host is a bacterium of the genus Escherichia or the genus Bacillus is cultivated, the culture medium is preferably a liquid medium. Also, the medium preferably contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Here, as examples of the carbon source, glucose, dextrin, soluble starch, sucrose and the like can be mentioned; as examples of the nitrogen source, inorganic or organic substances such as an ammonium salt, a nitrate salt, corn steep liquor, peptone, casein, meat extract, soybean cake, potato extract and the like can be mentioned; as examples of the inorganic substance, calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like can be mentioned. In addition, the medium may be supplemented with yeast extract, vitamins, growth promoting factor and the like. Preferably, the pH of the medium is about 5 to about 8.
As an example of the medium used to cultivate a transformant whose host is a bacterium of the genus Escherichia, a M9 medium supplemented with glucose and a casamino acid (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York, 1972) can be mentioned. As required, in order to increase promoter efficiency, a chemical agent such as 3β-indolylacrylic acid may be added to the medium.
Cultivation of a transformant whose host is a bacterium of the genus Escherichia is normally carried out at about 15° C. to about 43° C. for about 3 to about 24 hours. As necessary, the culture may be aerated or agitated.
Cultivation of a transformant whose host is a bacterium of the genus Bacillus is normally carried out at about 30° C. to about 40° C. for about 6 to about 24 hours. As necessary, the culture may be aerated or agitated.
As examples of the medium for cultivating a transformant whose host is a yeast, Burkholder's minimum medium [Bostian, K. L. et al., Proc. Natl. Acad. Sci. USA, vol. 77, 4505 (1980)] and SD medium supplemented with 0.5% casamino acid [Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA, vol. 81, 5330 (1984)] can be mentioned. The medium's pH is preferably about 5 to 8. Cultivation is normally carried out at about 20° C. to about 35° C. for about 24 to about 72 hours. As necessary, the culture may be aerated or agitated.
Useful medium for cultivating a transformant whose host is an insect cell or an insect include, for example, Grace's insect medium [Grace, T. C. C., Nature, 195, 788 (1962)] supplemented with additives such as inactivated 10% bovine serum as appropriate. The medium's pH is preferably about 6.2 to 6.4. Cultivation is normally carried out at about 27° C. for about 3 to 5 days. As necessary, the culture may be aerated or agitated.
Useful medium for cultivating a transformant whose host is an animal cell include, for example, MEM medium supplemented with about 5 to 20% fetal bovine serum [Science, Vol. 122, 501(1952)], DMEM medium [Virology, Vol. 8, 396(1959)], RPMI 1640 medium [The Journal of the American Medical Association, Vol. 199, 519(1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1(1950)] and the like. The medium's pH is preferably about 6 to 8. Cultivation is normally carried out at about 30° C. to 40° C. for about 15 to 60 hours. As necessary, the culture may be aerated or agitated.
As described above, EDG2s can be produced in a cell of the transformant or outside the cell.
EDG2s can be separated and purified from the culture obtained by cultivating the aforementioned transformant according to a method known per se.
For example, when EDG2s is extracted from a cultured bacterum, a method is used as appropriate wherein bacteria or cells are collected by a known means, suspended in an appropriate buffer solution, and disrupted by means of sonication, lysozyme and/or freeze-thawing and the like, after which a crude extract of soluble protein is obtained by centrifugation or filtration. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride and a surfactant such as Triton X-100™.
Isolation and purification of EDG2s contained in the thus-obtained soluble fraction can be conducted according to a method know per se. Useful methods include methods based on solubility, such as salting-out and solvent precipitation; methods based mainly on molecular weight differences, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods based on charge differences, such as ion exchange chromatography; methods based on specific affinity, such as affinity chromatography; methods based on hydrophobicity differences, such as reversed-phase high performance liquid chromatography; and methods based on isoelectric point differences, such as isoelectric focusing. These methods can be combined as appropriate.
When the thus-obtained EDG2 or a partial peptide thereof is a free form, it can be converted to a salt by a method known per se or a method based thereon; when the protein or peptide is obtained as a salt, it can be converted to a free form or another salt by a method known per se or a method based thereon.
Note that EDG2s produced by the transformant can also be optionally modified by the action of an appropriate protein-modifying enzyme, before or after purification, or can have a polypeptide thereof removed partially. As such, useful protein-modifying enzymes include, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of the thus-obtained EDG2s can be confirmed by enzyme immunoassay, Western blotting and the like using a specific antibody.
Furthermore, EDG2 or a partial peptide thereof can also be synthesized by in vitro translation using a cell-free protein translation system comprising a rabbit reticulocyte lysate, wheat germ lysate, Escherichia coli lysate and the like, with RNA corresponding to the above-described DNA that encodes the calmodulin or a partial peptide thereof as the template. Alternatively, EDG2 or a partial peptide thereof can be synthesized using a cell-free transcription/translation system containing RNA polymerase, with the DNA that encodes EDG2 or a partial peptide thereof as the template. The cell-free protein (transcription/) translation system used may be a commercial product, and may be prepared in accordance with a method known per se; specifically, an Escherichia coli extract can be prepared in accordance with the method described in Pratt J. M. et al., Transcription and Translation, Hames B. D. and Higgins S. J. eds., IRL Press, Oxford 179-209 (1984) and the like. Commercially available cell lysates include those derived from Escherichia coli such as the E. coli S30 extract system (manufactured by Promega) and the RTS 500 Rapid Translation System (manufactured by Roche), those derived from rabbit reticulocytes such as the Rabbit Reticulocyte Lysate System (manufactured by Promega), and those derived from wheat germ such as PROTEIOS™ (manufactured by TOYOBO). Of these, one using a wheat germ lysate is suitable. For preparing a wheat germ lysate, a method described in, for example, Johnston F. B. et al., Nature, 179:160-161 (1957) or Erickson A. H. et al., Meth. Enzymol., 96:38-50 (1996), can be used.
As a system or apparatus for protein synthesis, the batch method (Pratt, J. M. et al. (1984), ibidem), a continuous cell-free protein synthesis system wherein amino acids, energy sources and the like are continuously supplied to the reaction system [Spirin A. S. et al., Science, 242:1162-1164 (1988)], the dialysis method (Kigawa et al., 21st general assembly of the Molecular Biology Society of Japan, WIDE) or the overlay method (instruction manual of the PROTEIOS™ Wheat germ cell-free protein synthesis core kit: manufactured by TOYOBO) and the like can be mentioned. Furthermore, a method wherein template RNA, amino acids, energy sources and the like are supplied to the synthetic reaction system whenever necessary, and synthesized products and decomposed products are discharged whenever necessary (JP-A-2000-333673) and the like can be used.
The nucleic acids having “the base sequence encoding the protein comprising an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO 2, or a part thereof”, or “the base sequence which is complementary to the base sequence or a part thereof” are meant to include not only above-described nucleic acids encoding EDG2 or its partial peptide, but also a base sequence having mismatched codon-frame. The nucleic acid may be a DNA, an RNA, or a DNA/RNA chimera. It is preferably a DNA. Additionally, the nucleic acid may be double-stranded or single-stranded. In the case of a double-stranded nucleic acid, it may be a double-stranded DNA, a double-stranded RNA, or a DNA:RNA hybrid.
The nucleic acid comprising a base sequence complementary to a subject region of the objective nucleic acid, i.e., the nucleic acid capable of hybridizing with the objective nucleic acid can be said to be “antisense” against the objective nucleic acid. On the other hand, the nucleic acid comprising a base sequence having homology to a subject region of the objective nucleic acid can be said to be “sense” against the objective nucleic acid. As used herein, “having homology” or “(being) complementary” means having homology or complementarity of about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more between the base sequences.
Nucleic acid comprising a base sequence which is complementary to the base sequence encoding EDG2 or a part thereof (hereinafter, also referred to as the “antisense EDG2”) can be designed and synthesized on the basis of base sequence information of cloned or sequenced nucleic acid encoding EDG2. Such nucleic acid can inhibit replication or expression of the gene encoding the protein of the present invention. That is, the antisense EDG2 can hybridize to RNA transcripted from the genes encoding EDG2 and inhibit mRNA synthesis (processing) or function (translation into protein).
The length of the subject region of the antisense EDG2 is not particularly limited as long as the antisense nucleic acid inhibits translation of EDG2 protein of as results of hybridization of the antisense nucleic acid, and may be whole sequence or partial sequence of mRNA encoding the protein, for example, about 15 bases or so in the case of a short one and full-length in the case of a long one, of mRNA or initial transcription product. Considering ease of synthesis and antigenicity, an oligonucleotide comprising about 15 to about 30 bases is preferred but not limited thereto. Specifically, for example, the 5′ end hairpin loop; 5′ end 6-base-pair repeats, 5′ end untranslated region, translation initiation codon, protein coding region, translation initiation codon, 3′ end untranslated region, 3′ end palindrome region, and 3′ end hairpin loop of nucleic acid encoding EDG2, may be selected as subject regions, though any other region maybe selected as a target in the genes encoding EDG2. For example, the subject region is also preferably intron part of the gene.
Further, the antisense EDG2 may inhibit RNA transcription by forming triple strand (triplex) by binding to the genes encoding EDG2 which is double stranded DNA as well as inhibits translation into protein by hybridizing with mRNA or initial transcription product encoding EDG2.
Examples of the antisense nucleic acid include deoxypolynucleotides containing 2-deoxy-D-ribose, ribonucleotides containing D-ribose, any other type of nucleotides which are N-glycosides of a purine or pyrimidine base, or other polymers containing non-nucleotide backbones (e.g., commercially available nucleic acid polymers specific for protein nucleic acids and synthetic sequence) or other polymers containing particular linkages (provided that the polymers contain nucleotides having such an alignment that allows base pairing or base bonding, as found in DNA or RNA), etc. It may be double-stranded DNA; single-stranded DNA, double-stranded RNA, single-stranded RNA or a DNA:RNA hybrid, and may further include unmodified polynucleotides (or unmodified oligonucleotides), those with known modifications, for example, those with labels known in the art, those with caps, those which are methylated, those with substitution of one or more naturally occurring nucleotides by their analogue, those with intramolecular modifications of nucleotides such as those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and those with charged linkages or sulfur-containing linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those having side chain groups such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.), saccharides (e.g., monosaccharides, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those with modified linkages (e.g.; alpha anomeric nucleic acids, etc.), etc. As used herein, the terms “nucleoside”, “nucleotide” and “nucleic acid” are used to refer to moieties that contain not only the purine and pyrimidine bases, but also other heterocyclic bases, which have been modified. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines and other heterocyclic rings. Modified nucleotides or modified nucleotides also include modifications on the sugar moiety, wherein, for example, one or more hydroxyl groups may optionally be substituted with a halogen atom(s), an aliphatic group(s), etc., or may be converted into the functional groups such as ethers, amines, or the like.
Preferably, the antisense nucleic acid is optionally modified RNA or DNA. Specific examples of the modified nucleic acid (RNA, DNA) are, but not limited to, sulfur and thiophosphate derivatives of nucleic acids and those resistant to degradation of polynucleoside amides or oligonucleoside amides. The antisense EDG2 can be designed preferably based on the following plan, that is by increasing the intracellular stability of the antisense nucleic acid, increasing the cell permeability of the antisense nucleic acid, increasing the affinity of the nucleic acid to the targeted sense strand to a higher level, or minimizing the toxicity, if any, of the antisense nucleic acid. Many of such modifications are known in the art, as disclosed in J. Kawakami, et al., Pharm. Tech. Japan, Vol. 8, 247, 1992; Vol. 8, 395, 1992; S. T. Crooke, et al. ed., Antisense Research and Applications, CRC Press, (1993); etc.
The antisense nucleic acids may contain sugars, bases or bonds, which are changed or modified. The antisense nucleic acids may also be provided in a specialized form such as liposomes, microspheres, or may be applied to gene therapy, or may be provided in combination with attached moieties. Such attached moieties include polycations such as polylysine that act as charge neutralizers of the phosphate group backbone, or hydrophobic moieties such as lipids (e.g., phospholipids, cholesterols, etc.) that enhance the interaction with cell membranes or increase uptake of the nucleic acid. Preferred examples of the lipids to be attached are cholesterols or derivatives thereof (e.g., cholesteryl chloroformate, cholic acid, etc.). These moieties may be attached to the nucleic acid at the 3′ or 5′ ends thereof and may also be attached thereto through a base, sugar, or intramolecular nucleoside linkage. Other groups may be capping groups specifically placed at the 3′ or 5′ ends of the nucleic acid to prevent degradation by nucleases such as exonuclease, RNase, etc. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol, tetraethylene glycol, etc.
Ribozymes which can cleave specifically mRNA or initial transcription product encoding EDG2 inside the code region (comprising intron moiety in the case of initial transcription product) can be also included in the antisense EDG2. The “ribozyme” means RNA having enzyme activity cleaving nucleic acid. However, it has been shown recently that oligo DNA having base sequence of the enzyme activity site also has nucleic acid cleavage activity similarly. Thus, in the present specification, ribozyme is meant to include DNA as long as it has sequence-specific nucleic acid cleavage activity. As most highly used ribozyme, there is self-splicing RNA found in infectious RNA such as viroid and virusoid. Hammerhead type and hairpin type, etc. are known. The hammerhead type exhibits enzyme activity at about 40 bases or so, and can specifically cleave only target mRNA by rendering several bases (about 10 bases or so in total) at the both ends which are adjacent to hammerhead structure moiety, to a sequence complementary to mRNA of the desired cleavage site. This type of ribozyme takes RNA only as a substrate, and thus has an advantage of not attacking genome DNA. When mRNA encoding EDG2 has double strand structure by itself, the target sequence can be made to be single stranded by using hybrid ribozyme ligated to RNA motif derived from virus nucleic acid which can bind specifically to RNA helicase [Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)]. Further, when ribozyme is used in the form of an expression vector comprising DNA encoding the same, it can be also made to be a hybrid ribozyme further ligated to the sequence obtained by modifying tRNA to promote transfer of the transcription product to cytoplasm [Nucleic Acids Res., 29(13): 2780-2788 (2001)].
Double stranded oligo RNA (siRNA) which comprise a base sequence complementary to a partial sequence (comprising intron part in the case of initial transcription product) in the code region of mRNA or initial transcription product encoding EDG2 can be also included in the antisense EDG2. It has been known that so-called RNA interference (RNAi), which is a phenomenon that if short double stranded RNA is introduced into cells, mRNA complementary to its RNA is degraded, occur in the nematodes, insect, plant, etc. Recently, it has been found that this phenomenon also occurs in mammal cells [Nature, 411(6836): 494-498 (2001)], which is drawing attention as an alternative technique to ribozymes.
The antisense oligonucleotide and ribozyme of the present invention can be prepared by determining a subject region of mRNA or initial transcription product on the basis of sequence information of cDNA or genome DNA encoding EDG2, and synthesizing its complementary sequence using commercially available DNA/RNA automatic synthesizer (Applied Biosystems, Beckman, etc.). siRNA having RNAi activity can be prepared by synthesizing sense strand and antisense strand with a DNA/RNA automatic synthesizer, respectively, denaturing them in a suitable annealing buffer, at about 90 to about 95° C. for about 1 minute or so, and annealing them at about 30 to about 70° C. for about 1 to about 8 hours. It can be also prepared as longer double stranded polynucleotide by synthesizing complementary oligonucleotide chains to overlap alternately, annealing them, and ligating them with ligase. Alternatively, the siRNA can be designed to be synthesized as an RNA (shRNA) by joining a sense strand and an antisense strand via a linker of an appropriate length (for example, about 3 to about 10 bases), and to be processed by an enzyme dicer (dicer) in the animal cell into which it is introduced, and the like. Furthermore, an expression vector wherein the DNAs that encode a sense strand and an antisense strand are placed under the control of respective Pol III system promoters such as U6 and H1, or an expression vector wherein the DNA that encodes an RNA strand wherein the above-described sense strand and antisense strand have been joined via a linker is placed under the control of a Pol III system promoter, may be prepared and allowed to be expressed in an animal cell to form of an siRNA.
The inhibitory activity of gene expression of the antisense EDG2 can be examined using a transformant comprising nucleic acid encoding EDG2, a gene expression system for gene encoding EDG2 in vivo and in vitro, or a translation system of EDG2 in vivo and in vitro.
The present invention also provides an antibody to EDG2 or its partial peptide or a salt thereof (hereinafter, also abbreviated as “anti-EDG2 antibody”). The antibodies may be any of polyclonal antibodies and monoclonal antibodies as long as they have specific affinity to EDG2 or its partial peptide or a salt thereof. The antibodies may be manufactured by known methods for manufacturing antibodies or antisera, using EDG2 or its partial peptide or a salt thereof as antigens.
(a) Preparation of monoclonal antibody-producing cells EDG2 or its partial peptide or a salt thereof (EDG2s) is administered to mammals either solely or together with carriers or diluents to the site where the production of antibody is possible by the administration. In order to potentiate the antibody productivity upon the administration, complete Freund's adjuvants or incomplete Freund's adjuvants may be administered. The administration is usually performed once in every 2 to 6 weeks and approximately 2 to 10 times in total. Examples of the applicable mammals are monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and the like, with mice and rats being preferred.
For example, from mammals, e.g., mice, immunized with an antigen, one wherein the antibody titer is noted is selected, then the spleen or lymph node is collected after 2 to 5 days from the final immunization and antibody-producing cells contained therein are fused with myeloma cells from same or different race of animal to give monoclonal antibody-producing hybridomas. Measurement of the antibody titer in antisera may be made, for example, by reacting labeled EDG2, which will be described later, with the antiserum followed by assaying the binding activity of the labeling agent bound to the antibody. The fusion may be operated, for example, by the known Koehler and Milstein method [Nature, vol. 256, 495 (1975)]. Examples of the fusion accelerator are polyethylene glycol (PEG), Sendai virus, etc., of which PEG is preferably employed.
Examples of the myeloma cells are mammalian myelomas such as NS-1, P3U1, SP2/0, AP-1 etc. In particular, P3U1 is preferably employed. A preferred ratio of the count of the antibody-producing cells used (spleen cells) to the count of myeloma cells is within a range of approximately 1: 1 to 20:1. When PEG (preferably, PEG 1000 to PEG 6000) is added in a concentration of approximately 10 to 80% or so followed by incubating at 20° C. to 40° C., preferably at 30° C. to 37° C. for 1 to 10 minutes, an efficient cell fusion can be performed.
A monoclonal antibody-producing hybridoma can be screen for by a method which comprises adding the culture supernatant of a hybridoma to a solid phase (e.g., microplate) adsorbed with an antigen directly or together with a carrier, adding an anti-immunoglobulin antibody (when mouse cells are used for the cell fusion, anti-mouse immunoglobulin antibody is used) labeled with a radioactive substance or an enzyme, or Protein A and detecting the monoclonal antibody bound to the solid phase; a method which comprises adding the culture supernatant of a hybridoma to a solid phase adsorbed with an anti-immunoglobulin antibody or Protein A, adding EDG2 labeled with a radioactive substance or an enzyme and detecting the monoclonal antibody bound to the solid phase; etc.
The monoclonal antibody can be selected by known methods or by analogues of these methods. In general, the selection of the monoclonal antibody can be effected in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin and thymidine). Any medium for the selection for the monoclonal antibody and growth can be employed as far as the hybridoma can grow therein. For example, RPMI 1640 medium containing 1% to 20%, preferably 10% to 20% fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1% to 10% fetal calf serum, a serum free medium for culture of a hybridoma (SFM-101, Nissui Seiyaku Co., Ltd.), etc. may be used for the selection and growth medium. The cultivation is performed generally at 20° C. to 40° C., preferably at about 37° C., for 5 days to 3 weeks, preferably 1 to 2 weeks. The cultivation may be performed normally in 5% CO2. The antibody titer of the culture supernatant of hybridomas can be determined as in the assay for the antibody titer in the antisera described above.
Separation and purification of the obtained monoclonal antibody can be performed by a method known per se, for example methods applied to separation and purification of immunoglobulins [e.g., salting-out, alcohol precipitation, isoelectric point precipitation, electrophoresis, adsorption and desorption with ion exchangers (e.g., DEAE), ultracentrifugation, gel filtration, or a specific purification method which comprises collecting only an antibody with an activated adsorbent such as an antigen-binding solid phase, Protein A, Protein G, etc. and dissociating the binding to give the antibody].
When the antibody of the present invention is utilized to inhibit the activity of EDG2, the antibody must possess EDG2 activity inhibitory activity (i.e., neutralizing activity), so that it is necessary to examine the degree of the neutralizing activity of the monoclonal antibody obtained. The neutralizing activity can be measured by performing the screening method of the present invention described below in the presence and absence of the antibody, and comparing the cell-stimulating activities mediated by EDG2, and the like.
In a preferred embodiment, because the antibody of the present invention is used as a pharmaceutical for a human recipient, the antibody (preferably monoclonal antibody) of the present invention is an antibody having a reduced risk of exhibiting antigenicity when administered to humans, specifically a fully human antibody, a humanized antibody, a mouse-human chimera antibody or the like, and particularly preferably a fully human antibody. A humanized antibody and a chimera antibody can be prepared by gene engineering according to the method described below. Although a fully human antibody can also be produced from a human-human (or mouse) hybridoma, it is desirable, for supplying a large amount of antibody stably and at low cost, that the antibody be produced using a human antibody-producing animal (e.g., mouse) or the phage display method.
As used herein, “a chimeric antibody” means an antibody wherein the sequences of the variable regions of the H chain and L chain (VH and VL) thereof are derived from a mammalian species, and wherein the sequences of the constant regions (CH and CL) are derived from another mammalian species. The sequences of the variable regions are preferably derived from, for example, an animal species permitting easy preparation of a hybridoma, such as mouse, and the sequences of the constant regions are preferably derived from the recipient mammalian species.
As examples of the method of preparing a chimeric antibody, the method described in U.S. Pat. No. 6,331,415 or a partially modified method thereof and the like can be mentioned. 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, to synthesize cDNA. DNAs that encode VH and VL are amplified and purified by PCR according to a conventional method with the cDNA as the template, using appropriate primers (for example, oligo DNAs comprising the base sequences that encode the N-terminal sequences of VH and VL, respectively, as the sense primers, and oligo DNAs that hybridize to the base sequences that encode the N-terminal sequences of CH and CL, respectively, as the antisense primer (see, for example, Bio/Technology, 9: 88-89, 1991)). In the same manner, DNAs that encode CH and CL are amplified and purified from an RNA prepared from lymphocytes and the like of another mammal (e.g., human) by RT-PCR. VH and CH, and VL and CL, are ligated together, respectively, using a conventional method, and the chimeric H chain DNA and chimeric L chain DNA obtained are inserted into respective appropriate expression vectors (for example, vectors comprising promoters that have transcription activity in CHO cells, COS cells, mouse myeloma cells and the like (e.g., CMV promoter, SV40 promoter and the like)). The DNAs that encode the two chains may be inserted into separate vectors, and may be inserted into a single vector in tandem. Host cells are transformed with the chimeric H chain and chimeric L chain expression vectors obtained. As the host cells, animal cells, for example, Chinese hamster ovary (CHO) cells, monkey-derived COS-7 cells, Vero cells, rat-derived GHS cells and the like, in addition to the above-described mouse myeloma cells, can be mentioned. For the transformation, any method applicable to animal cells can be used, with preference given to electroporation method and the like. It is possible to isolate a chimeric monoclonal antibody by culturing the host cells in a medium suitable thereto for a given period, and thereafter recovering the culture supernatant and purifying it in the same manner as the above. Alternatively, it is also possible to obtain a chimeric monoclonal antibody easily and in large amounts from milk or eggs of transgenic animals which are produced by a conventional method using germ line cells of an animal such as bovine, goat, or chicken as the host cells, for which a transgenic technique has been established and a know-how of mass propagation as a domestic animal (domestic fowl) has been compiled. Furthermore, it is also possible to obtain a large amount of the chimeric monoclonal antibody from seeds, leaves and the like obtained from a transgenic plant prepared by microinjection or electroporation for protoplast, the particle gun method, the Ti vector method and the like for intact cells, using as the host cell a cell of a plant for which transgenic technology has been established, and which is cultured in large amounts as a major crop, such as corn, rice, wheat, soy or tobacco.
When the chimeric monoclonal antibody obtained is digested with papain, Fab is obtained; when the same is digested with pepsin, F(ab')2 is obtained.
It is also possible to reformat into scFv by ligating DNAs that encode mouse VH and VL via a suitable linker, for example, a DNA that encodes a peptide consisting of 1 to 40 amino acids, preferably 3 to 30 amino acids, more preferably 5 to 20 amino acids (e.g., [Ser-(Gly)m]n or [(Gly)m-Ser]n (m is an integer from 0 to 10, n is an integer from 1 to 5) and the like); furthermore, it is possible to reformat into a minibody monomer by ligating a DNA that encodes CH3 via a suitable linker, or reformat into a scFv-Fc by ligating a DNA that encodes full-length CH via a suitable linker. The DNA encoding such an antibody molecule modified (coupled) by gene engineering can be expressed in a microorganism such as Escherichia coli or yeast under the control of a suitable promoter, to produce the antibody molecule in large amounts.
When DNAs encoding mouse VH and VL are inserted downstream of one promoter in tandem and introduced into Escherichia coli, a dimer called Fv is formed by monocistronic gene expression. When an appropriate amino acid in the framework regions (FRs) of VH and VL is substituted with Cys using molecule modeling, a dimer called dsFv is formed via the intermolecular disulfide bond between the two chains.
As used herein, “a humanized antibody” means an antibody wherein the sequences of all regions present in the variable region, other than the complementarity determining region (CDR), (i.e., FRs in the constant region and variable region) are derived from a human, and wherein only the sequence of CDR is derived from another mammalian species. The other mammalian species is preferably an animal species that permits easy preparation of a hybridoma, for example, mouse.
As examples of the method of preparing a humanized antibody, the methods described in U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761 and 5,693,762 or partially modified methods therefrom and the like can be mentioned. Specifically, DNAs that encode VH and VL derived from a non-human mammalian species (e.g., mouse) are isolated in the same manner as the above for a chimeric antibody, after which sequencing is performed by a conventional method using an automated DNA sequencer (e.g., manufactured by Applied Biosystems Co. and the like), and the base sequences obtained or amino acid sequences deduced therefrom are analyzed using a publicly known antibody sequence database [for example, Kabat database (see Kabat et al., “Sequences of Proteins of Immunological Interest”, edited by the US Department of Health and Human Services, Public Health Service, NIH, 5th edition, 1991) and the like] to determine the CDRs and FRs of the two chains. A base sequence wherein the CDR encoding region of base sequences that encode the L chain and H chain of a human antibody having FR sequences similar to the determined FR sequences [e.g., human κ type L chain subgroup I and human H chain subgroup II or III (see Kabat et al., 1991 (supra))] is substituted with a determined base sequence that encodes the CDR of another animal species, is designed, and the base sequence is divided into fragments of about 20 to 40 bases, and a sequence complementary to the base sequence is further divided into fragments of about 20 to 40 bases so that they alternatively overlap with the aforementioned fragments. It is possible to construct DNAs that encode VH and VL having a human-derived FR and a CDR derived from another mammalian species by synthesizing individual fragments using a DNA synthesizer, and hybridizing and ligating them in accordance with conventional methods. In order to transfer a CDR derived from another mammalian species to human-derived VH and VL more quickly and more efficiently, it is preferable to use PCR-based site directed mutagenesis. As examples of such a method, the sequential CDR grafting method described in JP-A-5-227970 and the like can be mentioned. Such methods include, for example, the sequential CDR transplantation described in JP-A-HEI-5-227970. By joining the VH- and VL-encoding DNAs thus obtained to human derived CH- and CL-encoding DNAs, respectively, in the same manner as the above for a chimeric antibody, and introducing them into an appropriate host cell, a cell or transgenic animal or plant that produces a humanized antibody can be obtained.
A humanized antibody, like a chimeric antibody, can be modified to scFv, scFv-Fc, minibody, dsFv, Fv and the like using gene engineering techniques, and can be produced in microorganisms such as Escherichia coli and yeast by using an appropriate promoter.
The technology for producing a humanized antibody can also be applied to, for example, the production of a monoclonal antibody that can preferably be administered to another animal species for which no hybridoma production technology has been established. For example, animals widely propagated as domestic animals (domestic fowls) such as bovine, swine, sheep, goat, and chicken, and pet animals such as dogs and cats, and the like can be mentioned as the subject animal species.
(iii) Preparation of Fully Human Antibody using Human Antibody-Producing Animal
Provided that a functional human Ig gene is introduced into a non-human warm-blooded animal having the endogenous immunoglobulin (Ig) gene knocked out (KO) therein, and that this animal is immunized with an antigen, a human antibody is produced in place of the antibody derived from the animal. Therefore, provided that an animal such as mice, for which a technique for producing a hybridoma has been established, is used, it is possible to acquire a fully human monoclonal antibody by the same method as the conventional method used to prepare a mouse monoclonal antibody. First, some of the human monoclonal antibodies generated by using a human antibody-producing mouse (see Immunol. Today, 17: 391-397, 1996) obtained by crossing a mouse transfected with minigenes of the human Ig H chain and L chain using an ordinary transgenic (Tg) technique with a mouse wherein the endogenous mouse Ig gene has been inactivated using an ordinary KO technique, are already in clinical stage, and to date production of anti-human Ig human antibody (HAHA) has not been reported.
Later, Abgenix Inc. [trade name: XenoMouse (see Nat. Genet., 15: 146-156, 1997; U.S. Pat. No. 5,939,598 and the like)] and Medarex Inc. [trade name: Hu-Mab Mouse (see Nat. 15 Biotechnol., 14: 845-851, 1996; US Patent No. 5,545,806 and the like)] established Tg mice transfected with even a larger human Ig gene using a yeast artificial chromosome (YAC) vector, thus enabling the production of human antibodies of richer repertoire. However, because the human Ig gene, for example, in the case of the H chain, exhibits its diversity as the VDJ exon, which is a variable combination of about 80 kinds of V fragments, about 30 kinds of D fragments and 6 kinds of J fragments, encodes the antigen binding site, the full length thereof is as large as about 1.5 Mb (14th chromosome) for the H chain, about 2 Mb (2nd chromosome) for the κL chain, and about 1 Mb (22nd chromosome) for the λL chain. To reproduce a diverse antibody repertoire like in humans in another animal species, it is desirable to introduce the full length of each Ig gene; however, a DNA that is insertable into a conventional transfection vector (plasmid, cosmid, BAC, YAC and the like) is normally several kb to several hundred kb in length, and it has been difficult to introduce the full length of Ig genes by the conventional technique for establishing a transgenic animal, which comprises inserting a cloned DNA into a fertilized egg.
Tomizuka et al. (Nat. Genet., 16: 133-143, 1997) prepared a mouse having the full-length human Ig gene by introducing a natural fragment of a human chromosome harboring the Ig gene (hCF) into a mouse [transchromosomic (TC) mouse]. That is, first, a human-mouse hybrid cell having human chromosomes in which the 14th chromosome comprising the H chain gene and the 2nd chromosome comprising the κL chain gene, both labeled with, for example, a drug-resistance marker and the like, is treated with a spindle formation inhibitor (e.g., colcemid) for about 48 hours to prepare a microcell wherein one to several chromosomes or fragments thereof are enveloped in nuclear membrane, and the chromosomes are introduced into a mouse ES cell by the micronuclear fusion method. A hybrid ES cell retaining the chromosomes having the human Ig gene or fragments thereof is selected using a medium containing a drug, and the cell is microinjected into a mouse embryo in the same manner as with the preparation of an ordinary KO mouse. A germ line chimera is selected among the chimeric mice obtained, with coat color as the index, and the like, to establish a TC mouse strain carrying the human 14th chromosome fragment (TC(hCF14)) and a TC mouse strain carrying the human 2nd chromosome fragment (TC(hCF2)). After preparing mice wherein the endogenous H chain gene and κL chain gene are knocked out (KO (IgH) and KO (Igκ)) by a conventional method, it is possible to establish a mouse strain having all the four kinds of gene modifications (double TC/KO) by repeating the crossing of these four strains.
Provided that the same method as that for producing an ordinary mouse monoclonal antibody is applied to a double TC/KO mouse prepared as described above, it is possible to prepare an antigen-specific human monoclonal antibody-producing hybridoma. However, there is the drawback of a lower efficiency of hybridoma acquirement than that with the ordinary mouse, because hCF2 containing the κL chain gene is unstable in the mouse cells.
Meanwhile, because the aforementioned Hu-Mab mouse has a structure wherein the variable region cluster is doubled although it has about 50% of the κL chain gene, it exhibits a κ chain diversity equivalent to that with full length (on the other hand, HuMab mouse exhibits a low H chain diversity and inadequate response to antigen because it carries only about 10% of the H chain gene), and the κ chain is stably retained in the mouse cells because it is inserted in mouse chromosome via a YAC vector (Igκ-YAC). Making use of this advantage, it is possible to obtain an efficiency for hybridoma acquirement and antigen affinity of antibody that are equivalent to those with the ordinary mouse, by crossing a TC(hCF14) mouse with a Hu-Mab mouse to prepare a mouse that stably retains both hCF14 and Igκ-YAC (trade name: KM mouse).
Furthermore, it is also possible to prepare a human antibody-producing animal in which the XL chain gene is further transfected to reconstruct the diverse human antibody repertoire more completely. Such an animal can also be obtained by producing a TC mouse in which the human 22nd chromosome or a fragment thereof harboring the λL chain gene is introduced in the same manner as the above (TC(hCF22)), and crossing the mouse with the above-described double TC/KO mouse or KM mouse, or can also be obtained by, for example, constructing a human artificial chromosome (HAC) comprising both the H chain locus and the λL chain locus, and introducing it into a mouse cell (Nat. Biotechnol., 18: 1086-1090, 2000).
When the antibody of the present invention is used as a pharmaceutical, the antibody of the present invention is desirably a monoclonal antibody, but it may be a polyclonal antibody. When the antibody of the present invention is a polyclonal antibody, it is not necessary to utilize a hybridoma; therefore, provided that a human antibody-producing animal is produced in the same manner as the above using an animal species for which no technique for preparing a hybridoma has been established but a transgenic technique has been established, preferably an ungulate such as bovine, it is also possible to produce a human antibody in larger amounts at low costs (see, for example, Nat. Biotechnol., 20: 889-894, 2002). The human polyclonal antibody obtained can be purified by collecting blood, ascites fluid, milk, egg and the like, preferably milk or egg, of the human antibody-producing animal, in combination with the same purification techniques as the above.
(iv) Preparation of Fully Human Antibody using Phage Display Human Antibody Library
Another approach to produce a fully human antibody is a method using phage display. This method sometimes encounters cases in which a mutation due to PCR is introduced into a site other than CDRs; for this reason, a few reports of cases of HAHA production in clinical stage are available; on the other hand, however, the method has advantages such as no risk of cross-species viral infection derived from the host animal and the indefinite specificity of the antibody (antibodies against forbidden clone, sugar chain and the like can also be easily prepared).
Examples of the method of preparing a phage display human antibody library include, but are not limited to, the methods described below.
Although a phage used is not subject to limitation, filamentous phage (Ff bacteriophage) is normally preferably used. As the method of presenting a foreign protein on the phage surface, a method comprising expressing and presenting the foreign protein as a fusion protein with any of the coat proteins g3p, and g6p to g9p on the coat protein can be mentioned; and a method comprising fusing the foreign protein to the N-terminal side of g3p or g8p is often used. As the phage display vector, besides 1) one in which the foreign gene is introduced in the form of fusion gene with the coat protein gene of the phage genome, to allow all the coat proteins presented on the phage surface to be presented as a fusion protein with the foreign protein, 2) one in which the gene encoding the fusion protein is inserted separately from the wild-type coat protein gene to allow the fusion protein and the wild-type coat protein to be expressed simultaneously, and 3) an Escherichia coli having a phagemid vector harboring the gene that encodes the fusion protein is infected with a helper phage having the wild-type coat protein gene to produce phage particles that express the fusion protein and the wild-type coat protein simultaneously, and the like can be mentioned; however, a phage display vector of the type 2) or 3) is used for the preparation of an antibody library, because in the case of 1), the capability of infection is lost when a large foreign protein is fused.
As specific vectors, those described by Holt et al. (Curr. Opin. Biotechnol., 11: 445-449, 2000) can be mentioned as examples. For example, pCES1 (see J. Biol. Chem., 274: 18218-18230, 1999) is an Fab-expressing phagemid vector wherein a DNA encoding the κL chain constant region allocated to downstream of the g3p signal peptide, and a DNA encoding CH3, His-tag, c-myc tag, and the amber stop codon (TAG) followed by the g3p coding sequence, allocated to downstream of the g3p signal peptide, are arranged under the control of one lactose promoter. When this is introduced into an Escherichia coli having an amber mutation, Fab is presented onto the g3p coat protein, but when it is expressed in the HB2151 strain and the like, which do not have an amber mutation, a soluble Fab antibody is produced. As the scFv-expressing phagemid vector, for example, pHEN1 (J. Mol. Biol., 222:581-597, 1991) and the like are used.
Meanwhile as examples of the helper phage, M13-KO7, VCSM13 and the like can be mentioned.
As another phage display vector, a vector designed to comprise sequences comprising a cysteine-encoding codon linked to each of the 3′ end of the antibody gene and the 5′ end of the coat protein gene to express the two genes simultaneously and separately (not in the form of a fusion protein), and to present the antibody onto the coat protein on the phage surface via S—S bonds between the introduced cysteine residues (CysDisplay™ technology of Morphosys Co.), and the like can be mentioned.
As the kind of human antibody library, a naive/non-immunized library, a synthetic library, an immunized library and the like can be mentioned.
The naive/non-immunized library is a library obtained by acquiring the VH and VL genes retained by a normal human by RT-PCR, and randomly cloning them into the above-described phage display vector. Normally, mRNA derived from lymphocytes of peripheral blood, bone marrow, tonsil and the like of a normal human, and the like are used as the template. A library prepared by selectively amplifying IgM-derived mRNA in which a class switch due to antigen sensitization has not occurred, to avoid V gene biases such as clinical history, is particularly called a naive library. Representatively, the library of CAT Co. (see J. Mol. Biol., 222: 581-597, 1991; Nat. Biotechnol., 14: 309-314, 1996), the library of MRC Co. (see Annu. Rev. Immunol., 12: 433-455, 1994), the library of Dyax Co. (see J. Biol. Chem., 1999 (supra); Proc. Natl. Acad. Sci. USA,14: 7969-7974, 2000) and the like can be mentioned.
The synthetic library is obtained by selecting a functional particular antibody gene in human B cells, and replacing of a portion of a V gene fragment, for example, an antigen-binding region such as CDR3, with a DNA encoding a random amino acid sequence of appropriate length, to construct a library. The synthetic library is recognized as being excellent in antibody expression efficiency and stability because the library can be constructed with the combination of the VH and VL genes, which produce functional scFv and Fab, since the beginning. Representatively, the HuCAL library of Morphosys Co. (see J. Mol. Biol., 296: 57-86, 2000), the library of BioInvent Co. (see Nat. Biotechnol., 18: 852, 2000), the library of Crucell Co. (see Proc. Natl. Acad. Sci. USA, 92: 3938, 1995; J. Immunol. Methods, 272: 219-233, 2003) and the like can be mentioned.
An immunized library is a library obtained by preparing an mRNA from lymphocytes collected from a human such as a patient with cancer, autoimmune disease, infectious disease and the like or a recipient of vaccination, having an elevated blood antibody titer against the target antigen, or from human lymphocytes and the like which are artificially immunized with the target antigen by the above-described in vitro immunization method, in the same manner as with the above-described naive/non-immunized library, and amplifying the VH and VL genes by RT-PCR, to construct a library. It is possible to obtain the desired antibody even from a library of relatively small size because the desired antibody gene is contained in the library already at the beginning.
The wider the diversity of the library is, the better; actually, however, an appropriate library size is about 108 to 1011 clones, taking into consideration of the number of phages handleable in the following panning operation (1011 to 1013 phages) and the number of phages necessary to isolate and amplify clones in ordinary panning (100 to 1,000 phages/clone); it is possible to screen for an antibody normally having a Kd value on the order of 10−9 with a library of about 108 clones.
The process for selecting an antibody against the target antigen by the phage display method is referred to as panning. Specifically, for example, a phage presenting an antigen-specific antibody is concentrated by repeating a series of operations of bringing an antigen-immobilized carrier and a phage library into contact with each other, washing out the unbound phage, thereafter eluting the bound phage from the carrier, and infecting the phage to Escherichia coli to proliferate it, about 3 to 5 times. As the carrier for immobilizing the antigen, various carriers used in ordinary antigen-antibody reactions or affinity chromatography, for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or microplates, tubes, membranes, columns, beads and the like comprising glass, metal and the like, and surface plasmon resonance (SPR) sensor chips, and the like can be mentioned. For the antigen immobilization, physical adsorption may be used, and a method using a chemical bond used to insolubilize and immobilize a protein or enzyme and the like is also acceptable. For example, a biotin-(strept)avidin system and the like are preferably used. When the endogenous ligand that is a target antigen is a small molecule such as a peptide, it is necessary to pay special attention to prevent masking of the portion used as the antigen determinant by conjugated with the carrier. For washing the unbound phage, a blocking solution such as BSA solution (once or twice), a PBS containing a surfactant such as Tween (3 to 5 times) and the like can be used. There is also a report mentioning that the use of citrate buffer (pH 5) and the like is preferable for the washing. For elution of the specific phage, an acid (e.g., 0.1 M hydrochloric acid and the like) is normally used; cleavage with a specific protease (for example, a gene sequence that encodes the trypsin cleavage site can be introduced into the linkage site between the antibody gene and the coat protein gene. In this case, E. coli infection and proliferation are possible even if all the coat protein is expressed in the form of a fusion protein because the wild-type coat protein is presented on the surface of the eluted phage), competitive elution with a soluble antigen, or elution by reduction of S—S bond (for example, in the aforementioned CysDisplay™, the antigen-specific phage can be recovered by dissociating the antibody and the coat protein by using a suitable reducing agent after performing panning) is also possible. When elution has been performed with an acid, the eluate is neutralized with Tris and the like, and the eluted phage is then infected to Escherichia coli, which is cultured; after which the phage is recovered by a conventional method.
After the phage presenting the antigen-specific antibody is concentrated by panning, the phage is infected to Escherichia coli and the cells are seeded onto a plate to perform cloning. The phage is again collected, and the antigen binding activity is confirmed by an antibody titer assay (e.g., ELISA, RIA, FIA and the like) or a measurement utilizing FACS or SPR.
Isolation and purification of the antibody from the selected phage clone that presents the antigen-specific antibody can be performed by, for example, when using a vector incorporating an amber stop codon at the linker site of the antibody gene and the coat protein gene as the phage display vector, infecting the phage to an Escherichia coli that does not have amber mutation (e.g., HB2151 strain) to produce and secrete soluble antibody molecules in periplasm or the medium, lysing the cell wall with lysozyme and the like, collecting the extracellular fraction, and purifying using the same purification technique as the above. Provided that the His-tag or c-myc tag has been introduced in advance, the antibody can easily be purified by using IMAC, an anti-c-myc antibody column and the like. When cleavage with a specific protease is utilized in panning, the antibody molecule is separated from the phage surface by an action with the protease, so that the desired antibody can be purified by performing the same purification operation as above mentioned.
The technology for producing a fully human antibody using a human antibody-producing animal and a phage display human antibody library can also be applied to the production of a monoclonal antibody of another animal species. For example, animals widely propagated as domestic animals (domestic fowls) such as bovine, swine, sheep, goat, and chicken, and pet animals such as dogs and cats, and the like can be mentioned as the subject animal species. In non-human animals, the utilization of an immunized library is more effective because there are fewer ethical problems concerning artificial immunization with the target antigen.
The polyclonal antibody to EDG2 or its partial peptide or a salt thereof can be manufactured by methods known per se. For example, an immunogen (an antigen such as EDG2s) or a complex of the immunogen and a carrier protein is prepared, and a warm-blooded animal is immunized with the antigen in a manner similar to the method described above for the manufacture of monoclonal antibodies. The product containing the anti-EDG2 antibody is collected from the immunized animal followed by separation and purification of the antibody.
In the complex of an immunogen and a carrier protein used to immunize a warm-blooded animal, the type of carrier protein and the mixing ratio of the carrier to hapten may be of any type in any ratio, as long as the antibody is efficiently produced to the hapten immunized by crosslinking to the carrier. For example, bovine serum albumin, bovine thyroglobulins, keyhole limpet hemocyanin, etc. is coupled to hapten with the weight ratio of approximately 0.1 to 20, preferably about 1 to about 5, per one hapten.
A variety of condensing agents (e.g., glutaraldehyde, carbodiimide, maleimide activated ester, activated ester reagents containing thiol group or dithiopyridyl group, etc.) can be used for the coupling of a carrier to hapten. The condensation product is administered to a warm-blooded animal either solely or together with carriers or diluents to the site in which the antibody may be prepared by the administration. In order to potentiate the antibody productivity upon the administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered.
The administration is usually made once approximately in approximately every 2 to 6 weeks and about 3 to about 10 times in total.
The polyclonal antibody can be collected from the blood, ascites, breast milk etc. of the blood of mammal immunized by the method described above, or from the blood, egg etc. when the immunized animal is a bird.
The polyclonal antibody titer in antiserum can be assayed by the same procedure as that for the determination of antiserum antibody titer described above. The separation and purification of the polyclonal antibody can be performed, following the method for the separation and purification of immunoglobulins performed as applied to the separation and purification of monoclonal antibodies described hereinabove.
When a partial peptide of EDG2 or a salt thereof is used as the antigen, its position on EDG2 is not subject to limitation; for example, a polypeptide or oligopeptide comprising a partial amino acid sequence of a region well conserved among various warm-blooded animals or a salt thereof can be used.
The (i) EDG2s, (ii) nucleic acid that encodes EDG2 or a partial peptide thereof (preferably, DNA), (iii) anti-EDG2 antibody, (iv) antisense EDG2, (v) EDG2 DNM or the inhibitory peptide of the present invention, and (vi) EDG2 antagonist, described above have the following applications, for example.
As shown in Examples below, the EDG2 gene is highly expressed in joint tissue, particularly in synovial membrane, and the expression thereof rises in OA synovial membrane. In OA synovial membrane, the expression of inflammatory cytokines rises transiently upon LPA stimulation, and subsequently the expression of cartilage substrate decomposing enzymes rises, but the rises in the expression thereof are suppressed by the addition of an siRNA against EDG2. These facts show that a substance capable of suppressing the expression or activity of EDG2 suppresses inflammatory cytokine expression in joints and/or joint destruction (e.g., articular cartilage destruction), and is effective in the prevention/treatment of bone/joint diseases, particularly diseases associated with cartilage substrate degeneration/production abnormalities or abnormalities of differentiation from cartilage progenitor cells to chondrocytes. Here, “a disease associated with” refers to a disease that is caused by the condition or that results in the condition.
Furthermore, the EDG2 gene is also highly expressed in the spinal cord, and is a dominant LPA receptor. The OA susceptibility polymorph i-EDG2-09 in the EDG2 gene described below exhibits an increased transcription activity of EDG2 promoter in a susceptible allele, and the polymorph correlates significantly with symptomatic OA with a complaint of knee pain. These facts show that a substance capable of suppressing the expression or activity of EDG2 not only suppresses joint destruction (e.g., articular cartilage destruction), but also acts to mitigate pain, which is the chief complaint of OA patients, and is effective in the prevention/treatment of bone/joint diseases with pain.
As stated above, EDG2 is thought to increase the expression of cartilage substrate decomposing enzyme and promote articular cartilage destruction by eliciting a rise in the expression of inflammatory cytokines in synovial cells, and to induce pain in bone/joint diseases by transmitting signals from LPA; therefore, if there is an abnormality in EDG2 or a nucleic acid (e.g., gene, mRNA and the like) that encodes the same in a living organism (emergence of highly active mutant), or if the amount expressed has been abnormally increased, or if an increased expression of an inflammatory cytokine in joints occurs due to a certain other factor, and cartilage substrate decomposition is abnormally accentuated, or if a painful symptom is manifested in a bone/joint disease, a) by (i) administering antisense EDG2 to the patient to introduce (and expressing) the same into a target cell, or (ii) introducing antisense EDG2 into an isolated target cell and expressing the same, and thereafter transplanting the cell to the patient, b) by administering an anti-EDG2 antibody to the patient to inactivate (neutralize) EDG2, c) by administering an EDG2 antagonist to the patient to block the action of EDG2, d) by administering EDG2 DNM or the inhibitory peptide of the present invention to the patient to block the action of EDG2, or e) by introducing/expressing a nucleic acid that encodes EDG2 DNM or the inhibitory peptide of the present invention into the patient in vivo or ex vivo in the same manner as with the above-described antisense EDG2, and the like, it is possible to reduce the amount and/or activity of EDG2 in the patient's body, suppress inflammatory cytokine expression and/or joint destruction (e.g., articular cartilage destruction), and mitigate pain, so as to prevent/treat diseases underlain by cartilage substrate abnormalities and the like, and bone/joint diseases with pain and the like.
Therefore, a) antisense EDG2, b) anti-EDG2 antibody, c) EDG2 antagonist, d) EDG2 DNM or the inhibitory peptide of the present invention, or e) a nucleic acid that encodes EDG2 DNM or the inhibitory peptide of the present invention can be used as a prophylactic/therapeutic agent for the above-described diseases, for example, diseases such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, and chondroma, preferably osteoarthritis [e.g., hip OA (HOA), knee OA (KOA)], more preferably KOA.
Here, the term EDG2 antagonist is used with a meaning that encompasses both substances that antagonistically bind to the LBD of EDG2, but have almost no or totally no influence on the equilibrium between the active form and inactive form of EDG2 (neutral antagonists) and substances that bind to an optionally chosen site of EDG2 to shift the equilibrium between the active form and inactive form of EDG2 toward the more inactive side (inverse agonists). As EDG2 antagonists, for example, publicly known antagonists such as the isoxazol derivatives disclosed in EP 1258484 Al (preferably, Ki16425 (see Mol. Pharmacol., 2003, 64(4): 994-1005) and the like), dioctylglycerol pyrophosphate, dioctylphosphatidic acid (Mol. Pharmacol., 2001, 60(4): 776-784), VPC32183, VPC32179, and VPC12249, which are commercially available from Avanti Polar Lipids, Inc., as well as substances selected by the screening method of the present invention described below, can also be included.
When an anti-EDG2 antibody, EDG2 antagonist, or EDG2 DNM or the inhibitory peptide of the present invention is used as the above-described prophylactic/therapeutic agent, the same can be prepared as a pharmaceutical formulation according to a routine means. Meanwhile, when a nucleic acid that encodes antisense EDG2, EDG2 DNM or the inhibitory peptide of the present invention is used as the above-described prophylactic/therapeutic agent, the nucleic acid, alone or after being inserted into an appropriate vector such as retrovirus vector, adenovirus vector, or adeno-associated virus vector, can be prepared as a pharmaceutical formulation according to a routine means. The nucleic acid can be administered as it is, or along with an auxiliary for promoting its ingestion, using a gene gun or a catheter such as a hydrogel catheter.
For example, antisense EDG2, anti-EDG2 antibody, EDG2 antagonist, EDG2 or the inhibitory peptide of the present invention or a nucleic acid that encodes EDG2 DNM or the inhibitory peptide of the present invention (hereinafter, sometimes generically referred to as “EDG2 suppressant”) can be used orally as tablets, capsules, elixirs, microcapsules and the like, coated with sugar as required, or can be used parenterally in the foam of an injection such as a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, by mixing an EDG2 suppressant with a publicly known physiologically acceptable carrier, a sweetener, a filler, a vehicle, an antiseptic, a stabilizer, a binder and the like, in a unit dosage form required for generally accepted preparation design, such a preparation can be produced. The active ingredient contents in these preparations are intended to ensure that an appropriate dose in the specified range is obtained.
Examples of additives that can be mixed in tablets, capsules and the like include binders such as gelatin, cornstarch, tragacanth and gum arabic, fillers such as crystalline cellulose, swelling agents such as cornstarch, gelatin, alginic acid and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, acamono oil or cherry, and the like can be used. When the formulation unit form is a capsule, a liquid carrier such as a grease (can be further contained in addition to the above-described type of material. A sterile composition for injection can be formulated according to an ordinary procedure for making a pharmaceutical preparation, such as dissolving or suspending an active substance in a vehicle like water for injection, or a naturally occurring vegetable oil such as sesame oil or coconut oil. The aqueous solution for injectable preparations is exemplified by saline, isotonic solutions containing glucose and another auxiliary (for example, D-sorbitol, D-mannitol, sodium chloride and the like) and the like, and may be used in combination with an appropriate solubilizer, for example, an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a non-ionic surfactant (e.g., Polysorbate 80™, HCO-50) and the like. The oily liquid is exemplified by sesame oil, soybean oil and the like, and may be used in combination with a solubilizer such as benzyl benzoate or benzyl alcohol.
Also, the above-described prophylactic or therapeutic agent may be combined with, for example, a buffer (for example, phosphate buffer solution, sodium acetate buffer solution), an analgesic (for example, benzalkonium chloride, procaine hydrochloride and the like), a stabilizer (for example, human serum albumin, polyethylene glycol and the like), a preservative (for example, benzyl alcohol, phenol and the like), an antioxidant and the like. The injectable preparation prepared is usually filled in an appropriate ampoule.
The preparations thus obtained are safe and less toxic, can be administered to human or other warm-blooded animals (e.g., rats, mice, hamsters, rabbits, sheep, goats, swine, bovine, horses, cats, dogs, monkey, chimpanzee, birds, etc.).
The dose of the EDG2 suppressant varies depending on the recipient, target organ, symptoms, method of administration and the like; in oral administration, the dose is normally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day for, for example, an OA patient (assumed to weigh 60 kg). In parenteral administration, a single dose varies depending on the subject to be administered, the subject organ, symptoms, route for administration, etc.; for example, in injection administration, the dose is normally about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day for an OA patient (as 60 kg body weight). In the case that the subject to be administered to is other than human, the corresponding dose as converted per 60 kg body weight can be administered.
As stated above, a substance capable of suppressing the activity of EDG2 suppresses inflammatory cytokine expression in joints and joint destruction (e.g., articular cartilage destruction) and mitigates pain, so that the substance is effective in the prevention/treatment of diseases underlain by cartilage substrate abnormalities and the like, and bone/joint diseases with pain. Accordingly, the present invention provides a screening method for a prophylactic/therapeutic substance for a bone/joint disease by measuring a change in the activity thereof using EDG2s. Specifically, by using EDG2s, or by constructing an expression system for recombinant EDG2, and using an affinity assay system using the expression system, a substance that reduces the bindability of EDG2 and ligand can be efficiently screened for.
Such compounds include (a) compounds that block or suppress cell-stimulating activities via EDG2 (for example, GTP-GDP exchange reaction promotion in conjugated Gα, intracellular cAMP production inhibition, inositol triphosphate (IP3) production promotion, intracellular Ca2+ uptake promotion, inflammatory cytokine expression induction, cartilage substrate decomposing enzyme expression induction, algesic action and the like) (antagonists), (b) compounds that reduce the binding force of EDG2 and ligand, and the like.
Accordingly, the present invention provides a screening method for a compound that alters the bindability of EDG2 and LPA or a salt thereof, comprising comparing (i) a case wherein EDG2 and LPA are brought into contact with each other and (ii) a case wherein EDG2, LPA and a test compound are brought into contact with each other.
In the above-described screening method, the amount of LPA bound to EDG2, cell-stimulating activities and the like in cases (i) and (ii) are measured and compared.
More specifically, the present invention provides:
In the methods (1) to (5) above, an EDG2 agonist (for example, VPC31143 and VPC31144 commercially available from Avanti Polar Lipids, Inc. and the like) can also be used in place of LPA.
The screening method of the present invention is hereinafter described specifically.
First, although the EDG2 used in the screening method of the present invention may be any one comprising the above-described EDG2, a cell membrane fraction of an organ of an EDG2-producing mammal is preferred. However, because human-derived organs, in particular, are difficult to obtain, the EDG2 used in the screening is suitably a human-derived EDG2s expressed in a large amount using a recombinant and the like.
An EDG2s is produced using the method described above, which is preferably performed by expressing a DNA that encodes EDG2 or an active peptide of the present invention in mammalian cells or insect cells. A cDNA is used as the DNA fragment encoding the desired portion of the protein, but is not construed as limiting. For example, a gene fragment or a synthetic DNA may also be used. For introducing a DNA fragment that encodes EDG2 or the active peptide of the present invention into host animal (insect) cells and efficiently expressing the same, it is preferable to insert the DNA fragment downstream of an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a human heat shock promoter, a cytomegalovirus promoter, an SRα promoter, the polyhedrin promoter of nuclear polyhedrosis virus (NPV), which is a baculovirus having insect hosts, or the like.
Therefore, the EDG2s used in the screening method of the present invention may be an EDG2s purified according to a method known per se, or may be used as cells producing the an EDG2s or a cell membrane fraction thereof.
In the above-described screening method, when using cells producing EDG2s, the cells may be fixed with glutaraldehyde, formalin and the like. This fixation can be performed according to a method known per se.
Cells producing EDG2s refer to host cells expressing EDG2s; as the host cells, yeasts, insect cells, animal cells and the like are used.
The aforementioned cell membrane fraction refers to a fraction rich in cell membrane obtained by a method known per se after cell disruption. As the method of cell disruption, cell squashing using a Potter-Elvehjem homogenizer, disruption using a Waring blender or Polytron (manufactured by Kinematica Inc.), disruption by ultrasonication, disruption by cell spraying through a thin nozzle under an increased pressure using a French press or the like, and the like can be mentioned.
For cell membrane fractionation, fractionation by centrifugal forces, such as fractional centrifugation or density gradient centrifugation, is mainly used. For example, a cell disruption fluid is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (normally about 1 to 10 minutes), the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm) normally for 30 minutes to 2 hours, and the precipitate obtained is used as the membrane fraction. The membrane fraction is rich in the EDG2s expressed and cell-derived membrane components such as phospholipids and membrane proteins.
The amount of EDG2s in the cells producing the EDG2s and the membrane fraction thereof is preferably 103 to 108 molecules per cell, more preferably 105 to 107 molecules. Note that as the amount expressed increases, LPA-binding activity per unit of membrane fraction (specific activity) increases so that it is possible to construct a highly sensitive screening system, and to assay a large number of samples in the same lot.
In performing (1) to (3) above for screening for a compound that alters the bindability of EDG2 and LPA, for example, an appropriate EDG2s-containing membrane fraction and labeled LPA are required.
As the EDG2s-containing membrane fraction, a membrane fraction containing natural type EDG2, or a recombinant EDG2s-containing membrane fraction possessing an equivalent activity and the like are desirable. Here, an equivalent activity refers to an equivalent LPA binding activity, signal transduction action or the like.
As the labeled LPA, one labeled with, for example, [3H], [32P], [125I], [14C] or the like, according to a conventional method is used.
Specifically, in performing screening for a compound that alters the bindability of EDG2 and LPA, cells producing EDG2s or a membrane fraction thereof is first suspended in a buffer suitable for the screening to prepare a standard preparation of EDG2s. The buffer may be any buffer that does not interfere with the binding of EDG2s and LPA, such as a phosphate buffer or Tris-hydrochloride buffer having a pH value of 4 to 10 (desirably a pH value of 6 to 8). To reduce non-specific binding, a surfactant such as CHAPS, Tween-80™ (Kao-Atlas Co.), digitonin or deoxycholate may be added to the buffer. Furthermore, to suppress the degradation of EDG2s by proteases, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratory Co.), or pepstatin may be added. A given amount (5,000 cpm to 500,000 cpm) of labeled LPA is added to 0.01 to 10 ml of a suspension of EDG2s, in the presence of a 10−4M to 10−10M test compound. To determine non-specific binding (NSB), a reaction tube containing the unlabeled ligand in large excess is also provided. The reaction is carried out at about 0 to about 50° C., desirably at about 4 to about 37° C., for about 20 minutes to about 24 hours, desirably for about 30 minutes to about 3 hours. After the reaction, the reaction mixture is filtered through glass fiber filter paper and the like, and washed with an appropriate amount of the same buffer; thereafter, the residual radioactivity on the glass fiber filter paper is counted using a liquid scintillation counter or a γ-counter. A test compound showing a specific binding (B-NSB) of, for example, not more than 50%, as the percent ratio to the count (B0-NSB) calculated by subtracting non-specific binding (NSB) from the count in the absence of antagonizing substance (B0), can be selected as a candidate substance capable of inhibiting the antagonism.
In performing the methods (4) to (5) above for screening for a compound that alters the bindability of EDG2 and LPA, for example, cell-stimulating activities via EDG2s (for example, GTP-GDP exchange reaction promotion in conjugated Gα, intracellular cAMP production inhibition, IP3 production promotion, intracellular Ca2+ uptake promotion, inflammatory cytokine expression induction, cartilage substrate decomposing enzyme expression induction, algesic action and the like) can be measured using a publicly known method or a commercially available measuring kit.
Specifically, first, EDG2s-producing cells are cultured on a multiwell plate and the like. In performing screening, the medium is previously replaced with a fresh medium or an appropriate buffer that does not exhibit toxicity to the cells, and the cells are incubated for a given length of time with the addition of a test compound and the like, after which the cells are extracted or the supernatant is recovered, and the resulting product is quantified according to each method. If it is difficult to test the production of the substance serving as an index for cell-stimulating activities because of a decomposing enzyme contained in the cell, the assay may be performed with the addition of an inhibitor of the decomposing enzyme. An activity such as cAMP production suppression can be detected as a production suppressing action on cells having the basic productivity increased with forscolin or the like.
In performing screening by measuring a cell-stimulating activity, an appropriate cell wherein EDG2s is expressed on the membrane is required. As the cell wherein EDG2s is expressed, a cell line that produces natural type EDG2, a cell line expressing the aforementioned recombinant EDG2s and the like are desirable.
As examples of test compounds, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like can be used; these compounds may be novel compounds or publicly known compounds.
The screening kit for a compound that alters the bindability of EDG2 and LPA or a salt thereof comprises EDG2s, a cell that produces EDG2s or a membrane fraction thereof and the like.
As examples of the screening kit of the present invention, the following can be mentioned.
Hanks' balanced salt solution (manufactured by Gibco Co.) supplemented with 0.05% bovine serum albumin (manufactured by Sigma Co.).
The solution is sterilized by filtration through a filter of 0.45 μm pore size and stored at 4° C., or may be prepared freshly just before use.
CHO cells allowed to express an EDG2, passaged with a 12-well plate at 5×105 cells/well, cultured at 37° C., 5% CO2, 95% air for 2 days.
LPAs (including analogues) labeled with a commercially available [3H], [32P], [125I], [14C] or the like
An aqueous solution of the LPA is stored at 4° C. or −20° C. and diluted to 1 μM with the assay buffer solution just before use.
LPA (including analogues) is dissolved in a PBS containing 0.1% bovine serum albumin (manufactured by Sigma Co.) to obtain a concentration of 1 mM, and preserved at −20° C.
PMB=[(B−NSB)/(B0−NSB)]×100 [Equation 1]
PMB: percent maximum binding
B: value obtained in the presence of test compound
NSB: non-specific binding
B0: maximum binding
In another preferred mode of embodiment, cells that express EDG2s on the cell membrane further express conjugated Gα. A family of Gαs that conjugates with EDG2 each can be identified by determining whether or not the signal transduction action of EDG2 is activated when LPA, which is a ligand, is allowed to act. As conjugated Gαs of EDG2, Gi/o, Gq/11 and the like are known. Preferably, as cells that express EDG2s and a conjugated Gα thereof, transformant cells co-transfected with EDG2 or a DNA that encodes the active peptide of the present invention and a DNA that encodes the conjugated Gα can be mentioned.
When LPA binds to EDG2s, the Gα activation domain (GAD) of the receptor and the receptor binding region of the conjugated Gα interact with each other to produce a conformational change in Gα, and GDP dissociates from the GTP/GDP-binding region and binds with GTP immediately. The Gα-GTP acts on an effector to promote or suppress the activity thereof. Meanwhile, when an antagonist is bound, a conformational change in the receptor does not occur and the Gα activation domain is in the inactivated state, so that the activated form Gα-GTP level decreases, and the action on the effector is inhibited. Here, provided that a GTP analogue that does not undergo hydrolysis by the GTPase activity of Gα, for example, 35S-labeled GTPγS, in place of GTP, is added to the system in advance, by measuring/comparing the radioactivities bound to the membrane in the presence and absence of a test compound, the effect of the test compound on the GDP-GTP exchange reaction in Gα can be evaluated, and a substance possessing EDG2 agonist/antagonist activity can be screened for. That is, if the radioactivity decreases in the presence of the test compound, the test compound is judged to have antagonist activity against EDG2.
In another preferred mode of embodiment, screening method is provided which comprises comparing the activity of an effector that interacts with Gα in the presence and absence of a test compound in a co-expression system for EDG2s and conjugated Gα. Therefore, cells that express EDG2s and a conjugated Gα thereof also further express an effector that accepts signals from the Gα. Gαs belonging to the Gi/o family have adenylate cyclase (AC) as the effector, and suppress the activity thereof. Gαs belonging to the Gq/11 family have phospholipase C (PLC) β as the effector, and promote the activity thereof.
In a screening system comprising AC as the effector, the action of Gα on the effector can be evaluated by directly measuring the AC activity. For the measurement of AC activity, any publicly known technique may be used; examples include, but are not limited to, a method wherein ATP is added to an AC-containing membrane fraction, and the resulting cAMP content is measured using an anti-cAMP antibody by a competitive immunoassay with cAMP labeled with an RI (for example, 125I), enzyme (for example, alkaline phosphatase, peroxidase and the like), fluorescent substance (for example, FITC, rhodamine and the like) and the like, a method wherein [α-32P] ATP is added to an AC-containing membrane fraction, and the resulting, and the resulting [32P] cAMP is separated using an alumina column and the like, and thereafter the radioactivity thereof is measured, and the like. When the conjugated Gα is Gi/o, if the AC activity increases in the presence of the test compound, the test compound is judged to have antagonist activity against EDG2, and conversely, if the AC activity decreases, the test compound is judged to have agonist activity against EDG2.
When an intact eukaryotic cell is used as the screening system, the action of Gα on AC can also be evaluated by measuring the cAMP content in the cell, or by labeling the cell with [3H] adenine, and measuring the radioactivity of the resulting [3H] cAMP. Although the cAMP content in the cell can be measured by incubating the cell in the presence and absence of a test compound for an appropriate length of time, and subjecting the extract obtained by cell disruption to the above-described competitive immunoassay, any other publicly known method can be used.
In another mode, a method is available wherein the cAMP content is evaluated by measuring the amount expressed of a reporter gene under the control of cAMP responding element (CRE). That is, a eukaryotic cell incorporating a vector containing an expression cassette with a DNA that encodes a reporter protein (for example, luciferase, GFP, peroxidase, alkaline phosphatase and the like) linked downstream of a CRE-containing promoter is cultured in the presence and absence of a test compound for an appropriate length of time, and the expression of the reporter gene in the extract obtained by cell disruption is measured/compared using a publicly known technique, whereby the cAMP content in the cell is evaluated.
Therefore, when the conjugated Gα is Gi/o, if the intracellular the cAMP content (or the amount expressed of reporter gene under the control of CRE) increases in the presence of the test compound, the test compound is judged to have antagonist activity against EDG2; conversely, if the cAMP content (or the amount of reporter gene expressed) decreases, the test compound is judged to have agonist activity against EDG2.
Meanwhile, in a screening system comprising PLCβ as the effector (i.e., if the conjugated Gα polypeptide is Gq/11), the action of Gα on the effector can be evaluated by directly measuring the PLCβ activity. PLCβ activity can be evaluated by, for example, adding [3H]-labeled phosphatidylinositol-4,5-diphosphate (PIP) to a PLCβ-containing membrane fraction, and measuring the amount of inositol phosphate (IP3) produced using a publicly known technique. PLCβ activity is measured and compared in the presence and absence of a test compound; if the PLCβ activity increases in the presence of the test compound, the test compound is judged to have agonist activity against EDG2, and conversely, if the PLCβ activity decreases, the test compound is judged to have antagonist activity against EDG2.
When an intact eukaryotic cell is used as the screening system, the action of Gα on PLCβ can also be evaluated by adding [3H]-labeled inositol to the cell, and measuring the radioactivity of the resulting [3H]-labeled IP3, or measuring the Ca2+ content in the cell. The Ca2+ content in the cell can be measured spectroscopically using a fluorescent probe (fura-2, indo-1, fluor-3, Calcium-Green I and the like), or can be measured using aequorin, a calcium-sensitive luminescent protein, and the like, after the cell is incubated in the presence and absence of a test compound for an appropriate length of time, but any other publicly known method can be used. As an apparatus suitable for spectroscopic measurements using a fluorescent probe, the FLIPR (Molecular Devices Co.) system can be mentioned.
As another mode, a method is available wherein the Ca2+ content is evaluated by measuring the amount expressed of a reporter gene under the control of TPA (12-O-tetradecanoylphorbor-13-acetate) responding element (TRE), upregulated by Ca2+. That is, a eukaryotic cell incorporating a vector containing an expression cassette with a DNA that encodes a reporter protein (for example, luciferase, GFP, peroxidase, alkaline phosphatase and the like) linked downstream of a TRE-containing promoter is cultured in the presence and absence of a test compound for an appropriate length of time, and the expression of the reporter gene in the extract obtained by cell disruption is measured/compared using a publicly known technique, whereby the Ca2+ content in the cell is evaluated.
Therefore, if the intracellular Ca2+ content (or the amount expressed of a reporter gene under the control of TRE) increases in the presence of the test compound, the test compound is judged to have agonist activity against EDG2, and conversely, if the intracellular Ca2+ content (or the amount of reporter gene expressed) decreases, the test compound is judged to have antagonist activity against EDG2.
By expressing a chimeric Gα wherein the region that interacts with the AC of Gi/o polypeptide (about 5 amino acids at the C-terminal) is substituted by the sequence of Gq/11, in a eukaryotic cell, irrespective of the conjugated Gα of EDG2, an EDG2 agonist/antagonist can be screened for using PLCβ as the effector.
In another preferred mode of embodiment, for example, as disclosed in WO 03/055507, EDG2 can be permanently activated by expressing a fusion protein wherein conjugated Gα is joined to the C-terminal side of EDG2 in a host eukaryotic cell such as a mammalian cell or an insect cell. In this case, without using a ligand such as LPA, agonist and inverse agonist can be screened for.
In another preferred mode of embodiment, yeast (for example, baker's yeast and the like) is used as the host cell. Yeast has STE2, which is a receptor of conjugation pheromone α factor, as the only GPCR. Upon receiving a ligand stimulation, STE2 activates GPA1, which is a conjugated Gα, and becomes dissociated from Gβ/Gγ. By the kinase cascade signal from the released Gβ/Gγ, the expression of FUS1 is induced, and conjugation occurs. Here, provided that STE2 is destroyed, and instead EDG2 is expressed in yeast, and conjugated Gα of EDG2 is co-expressed under the control of GPA1 promoter, upon LPA stimulation, EDG2, just like STE2, mediates intracellular signal transduction. Here, provided that EDG2 and conjugated Gα are co-expressed under the control of GPA1 promoter, and further fused with a gene that confers an auxotrophic mutation to FUS1, a reporter gene (for example, LacZ) and the like, signal activation can easily be detected. Details of this method are described in, for example, Yeast, 16: 11-22 (2000) and the like.
A compound obtained using the above-described screening method or screening kit or a salt thereof is a compound possessing an action to alter the bindability of EDG2 and LPA, specifically, (a) a compound possessing a cell-stimulating activity via LPA-EDG2 interaction (for example, GTP-GDP exchange reaction promotion in conjugated Gα, intracellular cAMP production inhibition, IP3 production promotion, intracellular Ca2+ uptake promotion, inflammatory cytokine expression induction, cartilage substrate decomposing enzyme expression induction, algesic action and the like) (agonist), (b) a compound not having the cell-stimulating activity (antagonist), (c) a compound that enhances the binding force of EDG2 and LPA, or (d) a compound that reduces the binding force of EDG2 and LPA.
Because an antagonist against EDG2 is capable of suppressing the bioactivity of LPA on EDG2, is useful as a safe pharmaceutical of low toxicity that suppresses LPA activity. A compound that reduces the binding force of EDG2 and LPA is useful as a safe pharmaceutical of low toxicity for reducing the bioactivity of LPA on EDG2. Therefore, these compounds can be used as suppressants of the expression of inflammatory cytokines in joints, joint destruction that can be elicited thereby (e.g., articular cartilage destruction), or pain in a bone/joint disease, and are useful as prophylactic/therapeutic drugs for a bone/joint disease resulting in an cartilage substrate abnormality, a symptomatic bone/joint disease with pain, for example, a disease such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, chondroma, or the like, preferably osteoarthritis [e.g., hip OA (HOA), knee OA (KOA)], more preferably KOA.
Meanwhile, an agonist against EDG2 possesses an action similar to the bioactivity of LPA on EDG2, and is therefore useful as a safe pharmaceutical of low toxicity that enhances LPA activity. A compound that enhances the binding force of EDG2 and LP is useful as a safe pharmaceutical of low toxicity for enhancing the bioactivity of LPA on EDG2. These compounds are useful as, for example, prophylactic/therapeutic drugs for schizophrenia (schizophrenia), obesity and the like.
A measurement of cell-stimulating activities in the above-described screening method, according to the purpose of screening, can also be performed by evaluating a more direct activity of EDG2.
For example, as stated above, EDG2, in response to LPA stimulation, induces in synovial cells, the expression of inflammatory cytokines (for example, TNFα, IL-1b, IL-6 and the like) and cartilage substrate decomposing enzyme (for example, collagenases such as MMP-1, MMP-3, and MMP-13, and the like). Therefore, by comparing the expression of any one of the above-described genes in a case wherein synovial cells with forcibly expressed EDG2 are cultured in the presence of LPA and a case wherein the same are cultured in the presence of both LPA and a test compound, a substance that suppresses the inflammatory cytokine expression induction and/or joint destruction (e.g., articular cartilage destruction) action of EDG2 can be screened for.
If the expression of the gene (genes) has decreased in the presence of the test compound, the test compound can be selected as a substance that suppresses the activity of EDG2.
As stated above, EDG2 is involved in pain in bone/joint diseases (for example, joint pain in OA, particularly knee pain in KOA). Therefore, for example, a pain test such as the von Frey test is performed on laboratory animals having EDG2 overexpressed therein (for example, EDG2 transgenic mouse, rat and the like), allocated to a test compound dosing group and a non-dosing group; if a significant analgesic effect is observed in the test compound dosing group, the compound can be selected as a substance that suppresses pain in bone/joint disease.
When a substance that suppresses EDG2 activity, obtained as described above (EDG2 activity suppressant) is used as the above-described prophylactic/therapeutic agent for bone/joint disease, the substance can be prepared as a pharmaceutical formulation in the same manner as the above for EDG2 antagonist and the like.
The preparations thus obtained are safe and less toxic, can be administered to human or other warm-blooded animals (e.g., rats, mice, hamsters, rabbits, sheep, goats, swine, bovine, horses, cats, dogs, monkey, chimpanzee, birds, etc.).
The dose of the EDG2 activity suppressant varies depending on the recipient, target organ, symptoms, method of administration and the like; in oral administration, the dose is normally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day for, for example, an OA patient (assumed to weigh 60 kg). In parenteral administration, a single dose varies depending on the subject to be administered, the subject organ, symptoms, route for administration, etc.; for example, in injection administration, the dose is normally about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day for an OA patient (as 60 kg body weight). In the case that the subject to be administered to is other than human, the corresponding dose as converted per human (60 kg body weight) can be administered.
As stated above, a substance that suppresses the expression of EDG2 also suppresses inflammatory cytokine expression in joints, joint destruction (e.g., articular cartilage destruction), and pain in bone/joint diseases, and is effective in the prevention/treatment of bone/joint diseases resulting in an cartilage substrate abnormality, or symptomatic bone/joint diseases with pain. Accordingly, the present invention provides a screening method for a substance that suppresses inflammatory cytokine expression in joints and/or joint destruction (e.g., articular cartilage destruction) and/or pain in a bone/joint disease, comprising comparing the expression of EDG2s in cells having the capability of producing EDG2s in the presence and absence of a test compound.
The amount of EDG2s expressed can also be measured at the transcription level by detecting the mRNA thereof using a nucleic acid capable of hybridizing to a nucleic acid that encodes EDG2 under high stringent conditions (that is, a nucleic acid comprising the aforementioned base sequence that encodes EDG2 or a portion thereof (hereinafter also referred to as “the sense EDG2”) or nucleic acid comprising a base sequence complementary to a base sequence that encodes EDG2 or a portion thereof (the antisense EDG2)). Alternatively, the amount expressed can also be measured at the translation level by detecting a protein (peptide) using the aforementioned anti-EDG2 antibody.
Accordingly, more specifically, the present invention provides:
In the screening methods (a) and (b) above, as cells having the capability of producing EDG2s, the same as used in the above-described screening method for an EDG2 activity suppressant are preferably used.
For example, a measurement of the mRNA level or protein (peptide) level of EDG2s can specifically be performed as described below.
As the test substance, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products and the like can be mentioned, and these substances may be novel substances or known substances.
As specific examples of the method of measuring the amount of EDG2s in the screening method (b) above,
In the quantitation method (ii) above, it is desirable that the two kinds of antibodies be ones that recognize different portions of EDG2s. For example, if one antibody is an antibody that recognizes the N-terminal portion of EDG2s, an antibody that reacts with the C-terminal portion of EDG2s can be used as the other antibody.
As labeling agents used for the assay methods using labeled substances, there are employed, for example, radioisotopes, enzymes, fluorescent substances, luminescent substances, etc. As the radioisotopes, there are employed, for example, [125I], [131I], [3H], [14C], etc. As the enzymes described above, stable enzymes with a high specific activity are preferred; for example, beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc. are used. Examples of the fluorescent substance used are fluorescamine, fluorescein isothiocyanate, etc. As the luminescent substances, there are employed, for example, luminol, luminol derivatives, luciferin, lucigenin, etc. Furthermore, the biotin-avidin system may also be used for binding of an antibody or antigen to the labeling agent.
As the sample liquid, a cell membrane-containing fraction obtained by a method known per se after cell disruption is used. As the method of cell disruption, cell squashing using a Potter-Elvehjem homogenizer, disruption using a Waring blender or Polytron (manufactured by Kinematica Inc.), disruption by ultrasonication, disruption by cell spraying through a thin nozzle under an increased pressure using a French press or the like, and the like can be mentioned. For cell membrane fractionation, fractionation by centrifugal forces, such as fractional centrifugation or density gradient centrifugation, is mainly used. For example, a cell disruption fluid is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (normally about 1 to 10 minutes), the supernatant is centrifuged at a higher speed (15000 rpm to 30000 rpm) normally for 30 minutes to 2 hours, and the precipitate obtained is used as the membrane fraction.
Also, as long as the labeling agent is detectable, intact cells may be used as the sample.
The methods for quantifying EDG2s using the anti-EDG2 antibody are not to be limited particularly. Any method can be used, so long as the amount of antibody, antigen, or antibody-antigen complex corresponding to the amount of antigen in a test fluid can be detected by chemical or physical means and can be calculated from a standard curve prepared from standard solutions containing known amounts of the antigen. For example, nephrometry, competitive method, immunometric method, and sandwich method are advantageously used, among which the sandwich method described below is particularly preferable in terms of sensitivity and specificity.
For immobilization of the antigen or antibody, physical adsorption may be used. Chemical binding methods conventionally used for insolubilization or immobilization of proteins, enzymes, etc. may be used as well. For the carriers, examples include insoluble polysaccharides such as agarose, dextran, cellulose, etc.; synthetic resin such as polystyrene, polyacrylamide, silicone, etc., or glass, etc.
In the sandwich method, the insolubilized anti-EDG2 antibody is reacted with a test fluid (primary reaction), then with a labeled form of another anti-EDG2 antibody (secondary reaction), and the activity of the labeling agent on the immobilizing carrier is assayed, whereby the amount of EDG2s in the test fluid can be quantified. The order of the primary and secondary reactions may be reversed, and the reactions may be performed simultaneously or with some time intervals. The labeling agent and the methods for insolubilization can be performed by modifications of those methods described above. In the immunoassay by the sandwich method, the antibody used for immobilized antibody or labeled antibody is not necessarily from one species, but a mixture of two or more species of antibodies may be used to increase the measurement sensitivity.
The anti-EDG2 antibody can be used for the assay systems other than the sandwich method, for example, the competitive method, immunometric method, nephrometry, etc.
In the competitive method, EDG2s in a test fluid and labeled EDG2s are competitively reacted with an antibody, and the unreacted labeled antigen (F) and the labeled antigen bound to the antibody (B) are separated (B/F separation). The amount of the labeled antigen in B or F is measured, and the amount of EDG2s in the test fluid is quantified. This reaction method includes a liquid phase method using a soluble antibody as an antibody, polyethylene glycol and a secondary antibody to the soluble antibody (primary antibody) for B/F separation, etc. and an immobilized method either using an immobilized antibody as the primary antibody (direct method), or using a soluble antibody as the primary antibody and an immobilized antibody as the secondary antibody (indirect method).
In the immunometric method, EDG2s in a test fluid and immobilized EDG2s are competitively reacted with a definite amount of labeled antibody, the solid phase is separated from the liquid phase, or EDG2s in a test fluid is reacted with an excess amount of labeled antibody, the immobilized EDG2s is then added to bind the unreacted labeled antibody to the solid phase, and the solid phase is separated from the liquid phase. Then, the amount of the labeled antibody in either phase is measured to quantify an amount of the antigen in the test fluid.
In the nephrometry, an amount of insoluble precipitates produced after the antigen-antibody reaction in gel or solution are measured. Even when the amount of EDG2s in a test fluid is small and only a small amount of precipitates is obtained, laser nephrometry utilizing scattering of laser can be advantageously employed.
For applying these individual immunological assay methods to the quantification methods of the present invention, any particular conditions, and setting of procedures and the like are not required. The assay systems for EDG2s may be constructed by adding ordinary technical consideration in the art to conventional conditions and procedures in the respective methods. For the details of these general technical means, reference can be made to the reviews and texts.
For example, Meth. Enzymol., Vol. 70: (Immunochemical Techniques (Part A)), ibidem Vol. 73 (Immunochemical Techniques (Part B)), ibidem Vol. 74 (Immunochemical Techniques (Part C)), ibidem Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibidem Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibidem Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by Academic Press) and the like can be referenced to.
As described above, by using an anti-EDG2 antibody, the amount of EDG2s produced in cells can be quantified with high sensitivity.
In the screening methods (a) and (b) above, a substance that has increased the amount of the EDG2s expressed (mRNA content or protein (peptide) content) can be selected as EDG2 expression promoting drug, and a substance that has reduced the amount expressed can be selected as an EDG2 expression suppressant. An EDG2 expression suppressant can be used as a suppressant for the expression of inflammatory cytokines in joints, joint destruction that can be elicited thereby (e.g., articular cartilage destruction), or pain in a bone/joint disease, and is useful as a prophylactic/therapeutic drug for a bone/joint disease resulting in an cartilage substrate abnormality, a symptomatic bone/joint disease with pain, for example, a disease such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, chondroma or the like, preferably osteoarthritis [e.g., hip OA (HOA), knee OA (KOA)], more preferably KOA.
Meanwhile, an EDG2 expression promoting drug is useful as prophylactic/therapeutic drug for, for example, schizophrenia (schizophrenia), obesity and the like.
When an EDG2 expression suppressant is used as the above-described prophylactic/therapeutic agent, the same can be prepared as a pharmaceutical formulation in the same manner as with the aforementioned EDG2 antagonist and the like.
The preparations thus obtained are safe and less toxic, can be administered to human or other warm-blooded animals (e.g., rats, mice, hamsters, rabbits, sheep, goats, swine, bovine, horses, cats, dogs, monkey, chimpanzee, birds, etc.).
The dose of the EDG2 expression suppressant varies depending on the recipient, target organ, symptoms, method of administration and the like; in oral administration, the dose is normally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day for, for example, an OA patient (assumed to weigh 60 kg). In parenteral administration, a single dose varies depending on the subject to be administered, the subject organ, symptoms, route for administration, etc.; for example, in injection administration, the dose is normally about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day for an OA patient (as 60 kg body weight). In the case that the subject to be administered to is other than human, the corresponding dose as converted per 60 kg body weight can be administered.
The above-described screening method for an EDG2 expression suppressant can also be used to evaluate the pharmacological effect of an existing suppressing agent for inflammatory cytokine expression in joints, a suppressing agent for joint destruction (e.g., articular cartilage destruction), a suppressing agent for pain in bone/joint disease and the like on the recipient. That is, from a subject receiving any one of the above-described drugs, a biological sample, for example, articular fluid, articular cartilage or the like is collected, and the EDG2 mRNA content or EDG2 protein content in the sample is measured using the above-described method. For example, if the amount of EDG2 expressed decreases significantly compared with the level before drug administration, the drug can be judged to have a prophylactic/therapeutic effect on the subject (drug recipient).
Because a nucleic acid comprising a base sequence that encodes EDG2 or a portion thereof (hereinafter also referred to as “the sense EDG2”) or a nucleic acid comprising a base sequence complementary to the base sequence or a portion thereof (the antisense EDG2) is capable of detecting abnormalities in the EDG2-encoding DNA or mRNA (gene abnormalities) in a human or another warm-blooded animal (for example, rat, mouse, hamster, rabbit, sheep, goat, swine, bovine, horse, cat, dog, monkey, chimpanzee, bird and the like) when used as a probe and the like, it is useful as, for example, a genetic diagnostic agent for damage or mutation of the DNA, a splicing abnormality or decreased expression of mRNA, amplification of the DNA, increased expression of mRNA, and the like. The nucleic acid comprising a portion of the base sequence that encodes EDG2 is not subject to limitation, as long as it has a necessary length for a probe (for example, about 15 bases or more), and needs not encode a partial peptide of EDG2.
The above-described genetic diagnosis using the sense or antisense EDG2 can be performed by, for example, Northern hybridization known per se, quantitative RT-PCR, the PCR-SSCP method, allele-specific PCR, the PCR-SSOP method, the DGGE method, the RNase protection method, the PCR-RFLP method and the like.
As stated above, EDG2 raises the expression of inflammatory cytokines, induces joint destruction (e.g., articular cartilage destruction), or provokes pain in bone/joint disease. For this reason, it is thought that if the subject is suffering a bone/joint disease resulting in an cartilage substrate abnormality or a symptomatic bone/joint disease with pain, or is at an increased risk for suffering the same in the future, the expression of the EDG2 gene is elevated compared with normal state. Therefore, for example, if an increase in the expression of the EDG2 gene is detected as a result of Northern hybridization or quantitative RT-PCR for an RNA fraction extracted from cells of a subject warm-blooded animal, the subject can be judged as being suffering, or being likely to suffer in the future, a bone/joint disease resulting in an cartilage substrate abnormality, a symptomatic bone/joint disease with pain, for example, a disease such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, chondroma or the like.
Because the aforementioned anti-EDG2 antibody is capable of measuring the amount of EDG2 or a salt thereof in humans or other warm-blooded animals (for example, rat, mouse, hamster, rabbit, sheep, goat, swine, bovine, horse, cat, dog, monkey, chimpanzee, bird and the like), it is useful as, for example, a genetic diagnostic agent for decreased expression or increased expression of the protein and the like.
The above-described genetic diagnosis using an anti-EDG2 antibody can be performed by performing immunoassay using as cells having the capability of producing EDG2s a biological sample (e.g., articular fluid, biopsy and the like) collected from a subject warm-blooded animal in a screening method for an EDG2 expression suppressant using the aforementioned anti-EDG2 antibody (screening method (b)).
If as a result of the immunoassay, an increase in the EDG2 or a salt thereof in the sample is detected, the subject can be judged as being suffering, or being likely to suffer in the future, a bone/joint disease resulting in an cartilage substrate abnormality, a symptomatic bone/joint disease with pain, for example, a disease such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, chondroma and the like.
The present invention also provides a method of judging (diagnosing) genetic susceptibility to bone/joint diseases, including OA (hereinafter, sometimes simply referred to as “the diagnostic method of the present invention”). The diagnostic method of the present invention comprises detecting polymorphs in the subject's EDG2 gene.
Herein, “a (gene) polymorph” refers to a change (substitution, deletion, insertion, translocation, inversion and the like) in 1 or a plurality of bases on genomic DNA, the change being present in a population at a frequency of 1% or more; for example, one wherein one base has been substituted by another base (SNP), one wherein 1 to several tens of bases have been deleted or inserted (DIP), one wherein the copy number differs at a site where a sequence of 2 to several tens of bases as 1 unit is repeatedly present (one wherein the copy number is 2 to 4 bases is referred to as a microsatellite polymorph, one wherein the copy number is several to several tens of bases is referred to as a variable number of tandem repeat (VNTR)) and the like can be mentioned. The polymorph that can be utilized in the diagnostic method of the present invention may be of any type, as far as the following requirements are met.
The polymorph that can be utilized in the diagnostic method of the present invention is not particularly limited, as far as it is a polymorph present in the EDG2 gene, and the frequency of either one allele is higher in an optionally chosen population of patients with bone/joint disease significantly than in an optionally chosen population of patients without bone/joint disease; preferably, the polymorph is an SNP in the base shown by base number 24007 in the base sequence shown by SEQ ID NO:3 [corresponds to the 2820th (−2820) base upstream of the transcription initiation point (“A” shown by base number 26827 in the base sequence shown by SEQ ID NO:3). Hereinafter, the position of each polymorph in the EDG2 gene is indicated by a distance from the transcription initiation point.] [−2820 G>A (hereinafter, any polymorph as mentioned herein is denoted as ““position of polymorph” “base of major allele”>“base of minor allele””. “−” means that no base exists, and “N/ins” means that the base N has been inserted.); being the 9th polymorph, from the 5′ upstream side, of the 27 polymorphs found in the region consisting of the 5′ flanking region, first exon and first intron of the EDG2 gene in Examples below, the polymorph is referred to as “i-EDG2-09”. Other polymorphs are named in the same way.] or a polymorph that is in a linkage disequilibrium state of a linkage disequilibrium coefficient D′ of 0.9 or more with respect to the SNP. Here, “linkage disequilibrium coefficient D′” can be obtained using the following equation, if each allele of the first SNP of the two SNPs is written as (A, a), each allele of the second SNP as (B, b), and the frequencies of four haplotypes (AB, Ab, aB, ab) are written as PAB, PAb, PaB, and Pab:
D′=(PABPab−PAbPaB)/Min [(PAB+PaB)(PaB+Pab), (PAB+PAb)(PAb+Pab)]
[wherein Min [(PAB+PaB)(PaB+Pab), (PAB+PAb)(PAb+Pab)] means that the smaller of the values (PAB+PaB)(PaB+Pab) and (PAB+PAb)(PAb+Pab) is assumed.]
i-EDG2-09 is an SNP in the 2820th base upstream of the transcription initiation point of the EDG2 gene, registered with the NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP/) as RefSNP ID: rs10980705, wherein the base of the major allele is “G”, and the base of the minor allele is “A”. Of them, A allele is an OA-susceptible allele that is found at a high frequency in the OA patient group.
As polymorphs, other than i-EDG2-09, present in the region consisting of the 5′ flanking region, first exon and first intron of the EDG2 gene, 17 publicly known polymorphs registered with the NCBI SNP database [RefSNP ID: rs3030215 (−23718 to −23719→ACAGGAG/ins; i-EDG2-02), rs4576485 (−14799 T>C; i-EDG2-05), rs10980706 (−3262 A>C; i-EDG2-08), rs9919025 (−2254 C>T; i-EDG2-10), rs4978978 (−2068 C>G; i-EDG2-11), rs10980704 (−1828 A>T; i-EDG2-12), rs10980702 (−1340 G>T; i-EDG2-13), rs10980701 (−1076 G>C; i-EDG2-14), rs12344515 (−866 A>G; i-EDG2-15), rs7858454 (−77 A>G; i-EDG2-17), rs12236414 (1039 G>T; i-EDG2-18), rs7041855 (1637 A>C; i-EDG2-19), rs1861157 (3389 C>T; i-EDG2-21), rs3739706 (7369 C>T; i-EDG2-23), rs3739707 (7660 T>G; i-EDG2-24), rs3739708 (7747 A>T; i-EDG2-25) and rs2287005 (9053 A>G; i-EDG2-27)], and 9 novel polymorphs found in the present invention [−26825 to −26826→A/ins (i-EDG2-01), −18363 to −18364→T/ins (i-EDG2-03), −14946 G>A (i-EDG2-04), −13850 A>T (i-EDG2-06), −3289 C>T (i-EDG2-07), −425 T>C (i-EDG2-17), 1728 A>G (i-EDG2-20), 3624 G>A (i-EDG2-22) and 8510 A>G (i-EDG2-26)] can be mentioned. Of them, all polymorphs but i-EDG2-24 are mutually in the relationship of D′>0.9.
Of the above-described polymorphs, one wherein the frequency of either one allele is significantly higher in an optionally chosen population of patients with bone/joint disease than in an optionally chosen population of patients without bone/joint disease can be utilized in the diagnostic method of the present invention. Herein, hereinafter, such a polymorph is sometimes referred to as a bone/joint disease marker polymorph.
Provided that each of the population of patients with bone/joint disease and population of patients without bone/joint disease is a population consisting of a sufficient number of patients to give statistically reliable results, there is no particular limitation on the size thereof (number of samples), background factors of each sample (for example, birth place, age, sex, disease and the like) and the like. As examples of the bone/joint disease, osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, osteochondroma, osteoma, chondroma, congenital bone system disease and the like can be mentioned, and the disease is preferably osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system diseases resulting in an cartilage substrate abnormality or the like, more preferably, osteoarthritis (OA), still more preferably hip OA (HOA) or knee OA (KOA), particularly KOA. Because there is an ethical need for informed consent of the sample donor, as the population of patients without bone/joint disease, usually, a patient group other than a group of patients with diseases other than bone/joint diseases at a certain medical institution, or a group of subjects diagnosed as not being suffering a bone/joint disease in a screening checkup in a certain area and the like are preferably used.
As polymorphs, out of the above-described 26 polymorphs other than i-EDG2-09, wherein the frequency of either one allele is significantly higher in a population of OA patients than in a population of non-OA patients in case/control correlation analysis in a Japanese general population, i.e., i-EDG2-03, i-EDG2-12 and i-EDG2-25, can be mentioned. For all these polymorphs, the minor alleles (T/ins allele for i-EDG2-03, T allele for i-EDG2-12, T allele for i-EDG2-25) are OA susceptible alleles. These polymorphs are mutually nearly completely linked.
Therefore, in a preferred mode of embodiment, the diagnostic method of the present invention comprises detecting one or more polymorphs out of i-EDG2-03, i-EDG2-09, i-EDG2-12 and i-EDG2-25. More preferably, the polymorph utilized in the diagnostic method of the present invention is i-EDG2-09 or i-EDG2-12, particularly preferably i-EDG2-09. The i-EDG2-09 polymorph influences the transcription activity of the EDG2 gene; in A allele (−2820A), the transcription activity rises under conditions that activate the transcriptional factor AP-1. Therefore, considering the above-described functions of EDG2 (i.e., induction of inflammatory cytokine expression in joints, joint destruction (e.g., articular cartilage destruction) elicitation, elicitation of pain in bone/joint diseases and the like), it is suggested that A allele may exhibit susceptibility not only to osteoarthritis, but also to a bone/joint disease resulting in an cartilage substrate abnormality and symptomatic bone/joint diseases with pain, for example, a disease such as osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, or chondroma.
Because the i-EDG2-03, i-EDG2-12 and i-EDG2-25 polymorphs, particularly the i-EDG2-12 polymorph, are mutually nearly completely linked with i-EDG2-09, by detecting any one of these polymorphs, it is possible to determine whether or not a haplotype susceptible to the above-described bone/joint disease is retained with high accuracy.
In the diagnostic method of the present invention, polymorph detection can be achieved using any publicly known method of SNP detection. Examples of classical methods of detection include a method comprising performing hybridization with accurate control of stringency in accordance with, for example, the method of Wallace et al. (Proc. Natl. Acad. Sci. USA, 80, 278-282 (1983)), with a genomic DNA extracted from cells or the like of a subject as the sample, using as the probe a nucleic acid containing a continuous base sequence of about 15 to about 500 bases, which is a partial base sequence of the EDG2 gene [for example, the base sequence shown by SEQ ID NO:3 can be referred to. The base sequence corresponds to the base sequence shown by base numbers 112795 to 76917 in the base sequence (complementary strand sequence) registered as GenBank accession No. AC007157 (VERSION: AC007157.6, GI: 4646258, updated on Apr. 27, 1999; herein sometimes simply abbreviated as “AC007157.6”).], and which contains the base of one of the above-described polymorph sites (preferably, i-EDG2-03, i-EDG2-09, i-EDG2-12 or i-EDG2-25, more preferably i-EDG2-09 or i-EDG2-12, particularly preferably i-EDG2-09), and detecting only a sequence that is completely complementary to the probe, a method comprising performing hybridization with gradual reductions in reaction temperature from denaturation temperature using a pair of mixed probes which are the above-described nucleic acid and a nucleic acid resulting from substitution of the base(s) at the polymorph site by other base(s) in the above-described nucleic acid, with one of the nucleic acids labeled and the other unlabeled, to allow a sequence completely complementary to one probe to be hybridized in advance, to thereby prevent cross-reactions with the probe containing the mismatch. Here, As the labeling agent, for example, radioisotopes, enzymes, fluorescent substances, luminescent substances and the like are used. As the radioisotopes, for example, [125I], [131I], [3H], [14C] and the like are used. As the enzymes described above, stable enzymes with a high specific activity are preferred; for example, beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substances, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
Preferably, polymorph detection can be performed by, for example, the various methods disclosed in WO 03/023063, for example, the RFLP method, the PCR-SSCP method, ASO hybridization, the direct sequencing method, the ARMS method, the denaturant density gradient gel electrophoresis method, the RNase A cleavage method, the chemical cleavage method, the DOL method, the TaqMan PCR method, the invader method, the MALDI-TOF/MS method, the TDI method, the molecular beacon method, the dynamic allele-specific hybridization method, the padlock probe method, the UCAN method, the nucleic acid hybridization method using a DNA chip or DNA microarray, the ECA method and the like (see WO 03/023063, 5th line on page 17 to 20th line on page 28). Hereinafter, as representative methods, the TaqMan PCR method and the invader method used in Examples below are described in more detail.
The TaqMan PCR method is a method utilizing a fluorescently labeled allele-specific oligonucleotide (TaqMan probe) and a PCR with Taq DNA polymerase. As a TaqMan probe, an oligonucleotide consisting of a continuous base sequence of about 15 to about 30 bases being a partial base sequence of the EDG2 gene (see, for example, the base sequence shown by SEQ ID NO:3), and containing the base(s) at any of the above-described polymorph sites (preferably, i-EDG2-03, i-EDG2-09, i-EDG2-12 or i-EDG2-25, more preferably i-EDG2-09 or i-EDG2-12, particularly preferably i-EDG2-09), is used. For example, i-EDG2-03 is a polymorph in the base sequence shown by base numbers 8463 to 8464 in the base sequence shown by SEQ ID NO:3, i-EDG2-09 is a polymorph in the base shown by base number 24007 in the base sequence shown by SEQ ID NO:3, i-EDG2-12 is a polymorph in the base shown by base number 24999 in the base sequence shown by SEQ ID NO:3, and i-EDG2-25 is a polymorph in the base shown by base number 34573 in the base sequence shown by SEQ ID NO:3; therefore, as the probe for detection of each polymorph, an oligonucleotide consisting of a continuous partial base sequence of about 15 to about 30 bases, containing one of the numbered bases (or base sequence), is used.
The probe is labeled with a fluorescent dye such as FAM or VIC at the 5′ end thereof, and with a quencher (quenching substance) such as TAMRA at the 3′ end thereof; when the probe is in the form as it is, the quencher absorbs the fluorescence energy, so that fluorescence will not be detected. It is preferable that the probe be prepared for both alleles, and labeled with fluorescent dyes having mutually different fluorescence wavelengths (for example, FAM for one allele, VIC for the other) for the sake of one-time detection. To prevent a PCR extension reaction from occurring from the TaqMan probe, the 3′ end has been phosphorylated. Provided that PCR is performed along with a primer designed to amplify a partial sequence of genomic DNA comprising the region that hybridizes with the TaqMan probe and Taq DNA polymerase, the TaqMan probe hybridizes with the template DNA, and simultaneously an extension reaction from the PCR primer occurs; however, as the extension reaction proceeds, the TaqMan probe hybridized is cleaved by the 5′ nuclease activity of Taq DNA polymerase, whereby the fluorescent dye is released and becomes no longer influenced by the quencher, and fluorescence is detected. With the amplification of the template, fluorescence intensity increases exponentially.
For example, in the detection of the i-EDG2-09 polymorph, when the allele-specific oligonucleotide comprising the bases of the polymorph site (about 15 to about 30 bases long; A allele and G allele were labeled with FAM and VIC, respectively, at the 5′ end, and both were labeled with TAMRA at the 3′ end) was used as TaqMan probe, if the subject's genotype is AA or GG, strong fluorescence intensity of FAM or VIA, respectively, is observed, with almost no fluorescence of the other observed. Meanwhile, if the subject's genotype is AG, fluorescences from both FAM and VIC are detected.
In the invader method, unlike in the TaqMan PCR method, the allele-specific oligonucleotide (allele probe) itself is not labeled, but a sequence lacking complementarity to the template DNA (flap) is present on the 5′ side of the base of the polymorph site, and a complementary sequence specific for the template is present on the 3′ side. In the invader method, further, an oligonucleotide having a complementary sequence specific for the 3′ side of the polymorph site of the template (invader probe; the polymorph site which is the 5′ end of the base corresponding to the probe is optionally chosen) and a FRET (fluorescence resonance energy transfer) probe wherein a sequence that can assume a hairpin structure on the 5′ side is present, and after formation of the hairpin structure, the sequence continuous from the base pairing with the base at the 5′ end toward the 3′ side is a sequence complementary to the flap of the allele probe, are used. The 5′ end of the FRET probe is labeled with a fluorescent substance (for example, FAM, VIC and the like), and a quencher (for example, TAMRA and the like) is bound to the vicinity thereof; no fluorescence will be detected in the state as it is (hairpin structure).
When the genomic DNA which is the template is reacted with an allele probe and an invader probe, the invader probe's 3′ end invades the polymorph site upon complementary binding of the three entities. When the single-stranded portion of the allele probe (i.e., flap portion on the 5′ side of the polymorph site base) is cut out using an enzyme that recognizes the structure of this polymorph site (cleavase), the flap binds complementarily with the FRET probe, and the polymorph site of the flap enters the hairpin structure of the FRET probe. This structure is recognized and cleaved by the cleavase, whereby the terminally labeled fluorescent dye in the FRET probe is released and becomes no longer influenced by the quencher, and fluorescence is detected. Although allele probes wherein the base of the polymorph site does not match the template are not cleaved by the cleavase, even allele probes that are not cleaved are capable of hybridizing to the FRET probe, so that fluorescence is detected likewise. However, because reaction efficiency differs, fluorescence intensity is remarkably stronger in allele probes wherein the base of the polymorph site matches the template, than in allele probes wherein the base does not matches the template.
Usually, it is preferable that before being reacted with the three kinds of probes and cleavase, the template DNA be previously amplified by PCR using a primer capable of amplifying a region comprising the portion to which the allele probes and invader probe hybridize.
As a result of an examination of polymorphs as described above, if the subject is judged to retain an allele susceptible to a bone/joint disease, particularly if the subject is judged to be a homozygous with respect to the allele, the subject can be judged to have a high genetic susceptibility to the bone/joint disease. For example,
When examining two or more polymorphs, if one or more results disagree with other results, the results for the i-EDG2-09 and/or the i-EDG2-12 polymorph prevail, and if the results disagree within these bases, the results for the i-EDG2-09 polymorph prevail.
As stated above, the i-EDG2-03 polymorphis a novel polymorph that was for the first time found in the present invention, and is also a marker polymorph that can be utilized for the diagnosis of genetic susceptibility to bone/joint diseases. In more detail, the publicly known base sequence at the position of i-EDG2-03 is “TA” as shown by base numbers 8463 to 8464 in the base sequence shown by SEQ ID NO:3, but the present inventors identified a novel polymorph wherein “thymine (T)” is inserted between the two bases (DIP). Because the frequency of T/ins allele is significantly higher in a population of OA patients than in a population of non-OA patients, the allele is an allele susceptible to a bone/joint disease resulting in an cartilage substrate abnormality and symptomatic bone/joint diseases with pain, for example, a disease such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, or chondroma, preferably OA (for example, HOA or KOA), more preferably KOA.
Accordingly, the present invention also provides a nucleic acid being a partial base sequence of the EDG2 gene, and comprising a continuous base sequence of about 15 to about 500 bases (preferably about 15 to about 200 bases, more preferably about 15 to about 50 bases) containing the bases shown by base numbers 8463 to 8464 in the base sequence shown by SEQ ID NO:3 (but “T” is inserted between the two bases). A novel nucleic acid comprising such a polymorph site can preferably used in the above-described diagnostic method of the present invention to detect the i-EDG2-03 polymorph. Such a nucleic acid can be synthesized on the basis of the base sequence information shown by SEQ ID NO:3 using an automated DNA/RNA synthesizer.
The present invention also provides a kit to be used for the above-described the diagnostic method of the present invention. That is, the diagnostic kit of the present invention comprises one pair or more of nucleic acid probe and/or primer capable of detecting one or more polymorphs selected from the group consisting of a polymorph present in the EDG2 gene wherein the frequency of either one allele is higher in an optionally chosen population of patients with bone/joint disease than in an optionally chosen population of patients without bone/joint disease, preferably i-EDG2-09 or each polymorph present in the EDG2 gene in a linkage disequilibrium state (preferably, completely linked) wherein the linkage disequilibrium coefficient D′ is 0.9 or more.
Specifically, the nucleic acid probe used in the diagnostic kit of the present invention is a nucleic acid that hybridizes with the genomic DNA in the above-described region containing the bases of the polymorph site to be detected in the diagnostic method of the present invention; as far as it is specific for the target site, and is capable of easily detecting polymorphism, the length thereof (the base length of the portion that hybridizes with genomic DNA) is not particularly limited; for example, the length is about 15 bases or more, preferably about 15 to about 500 bases, more preferably about 15 to about 200 bases, still more preferably about 15 to about 50 bases.
The probe may comprise an additional sequence suitable for polymorphism detection (a sequence not complementary to the genomic DNA). For example, the allele probe used in the above-described invader method has an additional sequence called a flap at the 5′ end of the base of the polymorph site.
The probe may be previously labeled with an appropriate labeling agent, for example, a radioisotope (e.g., 125I, 131I, 3H, 14C and the like), enzyme (e.g., β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like), fluorescent substance (e.g., fluorescamine, fluorescein isothiocyanate and the like), luminescent substance (e.g., luminol, luminol derivative, luciferin, lucigenin and the like) and the like. Alternatively, in the vicinity of a fluorescent substance (e.g., FAM, VIC and the like), a quencher (quenching substance) that absorbs the fluorescence energy produced by the fluorescent substance may further be bound. In this mode of embodiment, upon the detection reaction, the fluorescent substance and the quencher become separated from each other and fluorescence is detected.
Preferably, the nucleic acid probe used in the diagnostic kit of the present invention is a nucleic acid comprising a continuous base sequence of about 15 to about 500 bases, preferably about 15 to about 200 bases, more preferably about 15 to about 50 bases, comprising the bases at a polymorph site selected from the group consisting of the polymorph site (i-EDG2-03) shown by base numbers 8463 to 8464 in the base sequence shown by SEQ ID NO:3, the polymorph site shown by base number 24007 (i-EDG2-09), the polymorph site shown by base number 24999 (i-EDG2-12) and the polymorph site shown by base number 34573 (i-EDG2-25), more preferably the i-EDG2-09 and/or i-EDG2-12 polymorph site, particularly preferably the i-EDG2-09 polymorph site. Here,
The nucleic acid primer used in the diagnostic kit of the present invention may be any one, as far as it is designed to be capable of specifically amplifying a region of the genomic DNA comprising the base(s) of the polymorph site to be detected in the above-described diagnostic method of the present invention. For example, a pair of nucleic acids which is a combination of a nucleic acid comprising about 15 to about 50 bases, preferably about 15 to about 30 bases, which is a partial base sequence of the base sequence shown by SEQ ID NO:3, which nucleic acid hybridizes with a portion of a complementary strand sequence on the 5′ side from the base(s) of the polymorph site to be detected, and a nucleic acid comprising a base sequence of about 15 to about 50 bases, preferably about 15 to about 30 bases, which nucleic acid hybridizes with a portion of the sequence on the 3′ side from the base(s) of the polymorph site, wherein the length of the fragment of the nucleic acid amplified thereby is about 50 to about 1,000 bases, preferably about 50 to about 500 bases, more preferably about 50 to about 200 bases, can be mentioned.
The primer may comprise an additional sequence suitable for polymorphism detection (a sequence not complementary to the genomic DNA), for example, a linker sequence.
The primer may be previously labeled with an appropriate labeling agent, for example, a radioisotope (e.g., 125I, 131I, 3H, 14C and the like), enzyme (e.g., β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like), fluorescent substance (e.g., fluorescamine, fluorescein isothiocyanate and the like), luminescent substance (e.g., luminol, luminol derivative, luciferin, lucigenin and the like) and the like.
Preferably, the nucleic acid primers used in the diagnostic kit of the present invention are a pair of nucleic acids capable of amplifying a continuous base sequence of about 50 to about 1,000 bases, preferably about 50 to about 500 bases, more preferably about 50 to about 200 bases, comprising the bases at a polymorph site selected from the group consisting of the polymorph site shown by base numbers 8463 to 8464 in the base sequence shown by SEQ ID NO:3 (i-EDG2-03), the polymorph site shown by base number 24007 (i-EDG2-09), the polymorph site shown by base number 24999 (i-EDG2-12) and the polymorph site shown by base number 34573 (i-EDG2-25), more preferably the i-EDG2-09 and/or i-EDG2-12 polymorph site, particularly preferably the i-EDG2-09 polymorph site.
The nucleic acid probe or primer used in the diagnostic kit of the present invention may be a DNA or an RNA, and may be single-stranded or double-stranded. In the case of a double strand, the same may be a double-stranded DNA, a double-stranded RNA, or a DNA/RNA hybrid. Therefore, when a nucleic acid having a certain base sequence is described herein, it should be understood that the term nucleic acid is used with a meaning that encompasses all of single-stranded nucleic acids having the base sequence, single-stranded nucleic acids having a sequence complementary to the base sequence, and double-stranded nucleic acids which are hybrids thereof unless otherwise stated.
The above-described nucleic acid probe or primer can be synthesized, for example, on the basis of information on the base sequence shown by SEQ ID NO:3 using an automated DNA/RNA synthesizer according to a conventional method.
The above-described nucleic acid probe and/or primer can be separately (or in a mixed form if possible) dissolved in water or an appropriate buffer solution (e.g., TE buffer and the like) to obtain an appropriate concentration (e.g., 1 to 50 μM at 2× to 20× concentration and the like), and preserved at about −20° C.
The diagnostic kit of the present invention may further contain other ingredients necessary for the implementation of the method as constituents thereof according to the method of polymorph detection. For example, provided that the kit is for detection of polymorphs by the TaqMan PCR method, the kit can further contain a 10×PCR reaction buffer solution, 10×MgCl2 aqueous solution, 10×dNTPs aqueous solution, Taq DNA polymerase (5 U/μL) and the like.
As shown in Examples below, an intranuclear factor that binds more selectively to A allele (−2820A) is present in i-EDG2-09, suggesting that the factor may act as a transcriptional factor. Therefore, a nucleic acid comprising a base sequence consisting of the base shown by base number 24007 in the base sequence shown by SEQ ID NO:3 (but the base is “adenine (A)”) and a base (or base sequence) adjacent thereto, by functioning as a decoy nucleic acid, is capable of inhibiting the binding of the intranuclear factor to the transcription regulatory region of the EDG2 gene to reduce the transcription activity of the EDG2 gene, and can be used as a suppressing agent for inflammatory cytokine expression in joints, joint destruction (e.g., articular cartilage destruction), and pain in a bone/joint disease, and the like, and can therefore be used as a prophylactic/therapeutic agent for a bone/joint disease resulting in an cartilage substrate abnormality and a symptomatic bone/joint disease with pain, for example, a disease such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, or chondroma, preferably OA (for example, HOA or KOA), more preferably KOA.
Here, “neighboring bases” may exist on any of the 5′ upstream and 3′ downstream sides of −2820A, and may exist on both sides. The base sequence consisting of −2820A and neighboring bases thereof (or base sequence) is preferably a base sequence consisting of about 5 to about 30 bases, more specifically base sequence consisting of a full length of about 5 to about 30 bases, which consist of −2820A, 0 to 20 bases on the 5′ upstream thereof, and 0 to 20 bases on the 3′ downstream of 16T.
When the above-described decoy nucleic acid is used as the above-described prophylactic or therapeutic agent, it can be prepared as a pharmaceutical formulation in the same manner as the aforementioned case of anti-EDG2 antagonist and the like.
The preparations thus obtained are safe and less toxic, can be administered to human or other warm-blooded animals (e.g., rats, mice, hamsters, rabbits, sheep, goats, swine, bovine, horses, cats, dogs, monkey, chimpanzee, birds, etc.).
The dose of the decoy nucleic acid varies depending on the subject to be administered, the subject organ, symptoms, route for administration, etc.; for example, in oral administration, the dose is normally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day for an OA patient (as 60 kg body weight). In parenteral administration, a single dose varies depending on the subject to be administered, the subject organ, symptoms, route for administration, etc.; for example, in injection administration, the dose is normally about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day for an OA patient (as 60 kg body weight). In the case that subject to be administered is other than human, the corresponding dose as converted per 60 kg body weight can be administered.
As stated above, an intranuclear factor that binds more selectively to A allele (−2820A) is present in i-EDG2-09, and it is suggested that the factor may act as a transcriptional factor; therefore, a substance that inhibits the binding of the factor to −2820A and the region therearound is effective in the prevention/treatment of bone/joint diseases, bone/joint diseases resulting in an cartilage substrate abnormality, and symptomatic bone/joint diseases with pain, for example, diseases such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, or chondroma, preferably OA (for example, HOA or KOA), more preferably KOA.
Accordingly, the present invention also provides a screening method for a prophylactic/therapeutic substance for the above-described bone/joint disease, comprising using a nucleic acid comprising a base sequence consisting of the base shown by base number 24007 in the base sequence shown by SEQ ID NO:3 (but the base is “A”) and a base (or base sequence) adjacent thereto and a transcriptional factor that selectively binds to the base sequence. For example,
When the binding of the nucleic acid and the transcriptional factor is used as the index, the screening can be performed by, for example, incubating the nucleic acid, previously labeled (e.g., 32P, digoxigenin and the like), and the transcriptional factor (which can be provided in the form of, for example, a nuclear extract derived from human or other warm-blooded animal cells, or can also be used after affinity purification from the extract using the nucleic acid) in the presence of a test compound, subjecting the reaction mixture to non-denatured gel electrophoresis, and detecting an increase or decrease in the signal intensity of a band corresponding to the nucleic acid-transcriptional factor complex.
As examples of the test compound, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like can be mentioned.
When the expression of a gene under the control of a promoter comprising the nucleic acid is used as the index, any promoter capable of functioning in animal cells can be used; for example, the SRα promoter, the SV40 promoter, the LTR promoter, the CMV (cytomegalovirus) promoter, the HSV-tk promoter and the like are used. The nucleic acid can be inserted to an appropriate position in the promoter using a gene engineering technique known per se. Alternatively, a native promoter of the EDG2 gene comprising −2820A may be used.
Although the gene under the control of a promoter comprising the nucleic acid is not subject to limitation, as long as it permits an easy measurement of the expression level thereof, reporter genes such as luciferase, GFP, alkaline phosphatase, peroxidase, β-galactosidase and the like are preferably used. Also, the EDG2 gene comprising −2820A can also be used as is. In this case, a cell or tissue derived from an animal (preferably a human) that naturally carries the EDG2 gene can be used as “an animal cell containing a gene under the control of a promoter comprising the nucleic acid”. When a reporter gene is used as the gene under the control of a promoter comprising the nucleic acid, a reporter gene ligated downstream of a promoter comprising the above-described nucleic acid using a gene engineering technique known per se can be inserted to an appropriate transfer vector, for example, a vector such as an Escherichia coli-derived plasmid (e.g., pBR322, pBR325, pUC12, pUC13), a Bacillus subtilis-derived plasmid (e.g., pUB110, pTP5, pC194), a yeast-derived plasmid (e.g., pSH19, pSH15), or a bacteriophage such as λ phage, and transferred to a host animal cell. The transfer vector may comprise another enhancer, polyA addition signal, selection marker, SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like as required. As examples of the selection marker, the dihydrofolate reductase gene [methotrexate (MTX) resistance], the ampicillin resistance gene, the neomycin resistance gene (G418 resistance) and the like can be mentioned.
The animal cell is not subject to limitation, as long as it is a cell capable of expressing the transcriptional factor; examples of useful animal cells include, but are not limited to, Huh-7 cells and the like. The animal cell can be transformed according to, for example, a method described in Saibo Kogaku (Cell Engineering), extra issue 8, Shin Saibo Kogaku Jikken Protocol (New Cell Engineering Experimental Protocol), 263-267 (1995), published by Shujunsha, and Virology, 52:456 (1973).
As examples of the test compound, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like can be mentioned.
Cells are cultured for a given time in an appropriate medium (e.g., minimal essential medium, Dulbecco's modified Eagle medium, Ham medium, F12 medium, RPMI1640 medium, William's E medium and the like) in the presence and absence of the test compound, after which the expressions of a gene under the control of a promoter comprising the nucleic acid are compared between those under the two conditions. The expression of the EDG2 gene can be detected and quantified by an immunoassay method such as ELISA using the above-described anti-EDG2 antibody, and the RT-PCR method.
Alternatively, it is also possible to screen for a prophylactic or therapeutic substance for bone and joint diseases by using animal cells that can be used in the above-described method, and comparing the intracellular localizations of the transcriptional factor, for example, the degree of transfer of the factor from cytoplasm to nucleus in the animal cells, in the presence and absence of the test compound. More specifically, for example, by immunostaining the cells with a fluorescein labeled antibody against the transcriptional factor, the transfer of the factor from cytoplasm to nucleus can be monitored. Alternatively, by using a transformant capable of expressing the transcriptional factor in the form of a fusion protein with a fluorescent protein such as GFP, it is also possible to directly monitor the transfer of the factor from cytoplasm to nucleus (see, for example, Biochem. Biophys. Res. Commun., 278: 659-664 (2000)).
In the above-described screening method, a test compound that has inhibited the binding of the nucleic acid and the transcriptional factor can be used as a suppressing agent for inflammatory cytokine expression in joints, joint destruction (e.g., articular cartilage destruction), pain in a bone/joint disease and the like, and can therefore be used as a prophylactic/therapeutic agent for a bone/joint disease resulting in an cartilage substrate abnormality and a symptomatic bone/joint disease with pain, for example, a disease such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, or chondroma, preferably OA (for example, HOA or KOA), more preferably KOA.
When the above-described binding inhibitory compound is used as the above-described prophylactic/therapeutic agent, the same can be prepared as a pharmaceutical formulation in the same manner as the above for EDG2 antagonist and the like.
The preparations thus obtained are safe and less toxic, can be administered to human or other warm-blooded animals (e.g., rats, mice, hamsters, rabbits, sheep, goats, swine, bovine, horses, cats, dogs, monkey, chimpanzee, birds, etc.).
The dose of the above-described binding inhibitory compound varies depending on the recipient, target organ, symptoms, method of administration and the like; in oral administration, the dose is normally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day for, for example, an osteoarthritis patient (assumed to weigh 60 kg). In parenteral administration, a single dose varies depending on the subject to be administered, the subject organ, symptoms, route for administration, etc.; for example, in injection administration, the dose is normally about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day for an osteoarthritis patient (as 60 kg body weight). In the case that the subject to be administered to is other than human, the corresponding dose as converted per human (60 kg body weight) can be administered.
As stated above, in each of the i-EDG2-03, i-EDG2-09, i-EDG2-12 and i-EDG2-25 polymorphs, the minor alleles (T/ins allele for i-EDG2-03, A allele for i-EDG2-09, T allele for i-EDG2-12, T allele for i-EDG2-25) are alleles susceptible to bone/joint diseases, including OA. For i-EDG2-09, in A allele, compared with G allele, the transcription activity of the EDG2 gene rises under conditions that activate the transcriptional factor AP-1. Meanwhile, because EDG2 induces inflammatory cytokine expression in joints, it is strongly suggested that EDG2 may raise the expression of cartilage substrate decomposing enzymes, and elicit joint destruction (e.g., articular cartilage destruction), and may elicit pain in a bone/joint disease. Judging from the above, it can be thought that in animal individuals having the above-described susceptible allele, inflammatory cytokine expression in joints rises with the overexpression of EDG2, and the risk for causing joint destruction (e.g., articular cartilage destruction), or for manifesting a painful symptom in a bone/joint disease is higher than in individuals having a non-susceptible allele. Therefore, for individuals having the above-described susceptible allele, administration of a substance that suppresses the expression or activity of EDG2 (EDG2 suppressant) is effective in the prevention/treatment of a bone/joint disease, particularly a bone/joint disease resulting in an cartilage substrate abnormality and a symptomatic bone/joint disease with pain, for example, a disease such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, or chondroma, preferably OA (for example, HOA or KOA), more preferably KOA.
Accordingly, the present invention also provides a suppressing agent for inflammatory cytokine expression in joints, joint destruction (e.g., articular cartilage destruction), and pain in a bone/joint disease, and a prophylactic/therapeutic agent for a bone/joint disease resulting in an cartilage substrate abnormality and a symptomatic bone/joint disease with pain, for example, a disease such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, or chondroma, preferably OA (for example, HOA or KOA), more preferably KOA, comprising the aforementioned EDG2 suppressant, wherein the suppressing agent or prophylactic/therapeutic agent is administered to an animal individual (preferably, a human) having an allele susceptible to any one of the polymorphs i-EDG2-03, i-EDG2-09, i-EDG2-12 and i-EDG2-25, which is preferably homozygous with respect to the susceptible allele.
In this case as well, the EDG2 suppressant can be prepared as a pharmaceutical formulation by the same technique as the above, and the same dose/route for administration as the above can be used.
EDG2, in response to stimulation by LPA, which is a ligand thereof, induces the expression of inflammatory cytokines, to thereby induce the expression of cartilage substrate decomposing enzymes and elicit articular cartilage destruction, and elicit pain in a bone/joint disease. Therefore, also by blocking the simulation by LPA, an effect as with the aforementioned EDG2 suppressant can be obtained.
Accordingly, the present invention also provides a suppressing agent for inflammatory cytokine expression in joints, joint destruction (e.g., articular cartilage destruction), or pain in a bone/joint disease, and a prophylactic/therapeutic agent for joint destruction (e.g., articular cartilage destruction), a bone/joint disease resulting in an cartilage substrate abnormality and a symptomatic bone/joint disease with pain, for example, a disease such as osteoarthritis, osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, sport-related joint disorder, congenital bone system disease (e.g., achondroplasia, multiple apophysial dysplasia, vertebral apophysial dysplasia, metaphysial dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostosis, hemihypertrophy, Ollier's disease, Maffucci's syndrome and the like), osteochondroma, osteoma, or chondroma, preferably OA (for example, HOA or KOA), more preferably KOA, comprising a substance that suppresses the production or activity of LPA.
Examples of the substance that inhibits LPA production include, but are not limited to, substances that inhibit the expression or activity of LPA production enzyme and the like. LPA is produced from phosphatidylcholine by the action of phospholipase D (PLD) and secretory phospholipase A2 (sPLA2), or by the action of phospholipase A2 (PLA2), lecithin cholesterol acyltransferase (LCAT) and autotaxin/lisophospholipase D (ATX/lysoPLD), or from monoacyl glycerol (MAG) by the action of MAG kinase. Therefore, by inhibiting the expression or production of one or more of these enzymes, the production of LPA can be inhibited. As the substance that inhibits the expression of the enzymes, antisense nucleic acids, siRNAs and the like of each enzyme gene can be mentioned. These molecules can be acquired on the basis of nucleotide sequence information on each enzyme gene known per se by the same technique as the above for antisense EDG2. Meanwhile, substances that inhibit the activity of the enzymes, neutralizing antibodies against each enzyme protein, publicly known enzyme inhibitors and the like can be mentioned. As the neutralizing antibody, a commercially available one can be used as it is, and the antibody can also be acquired with each enzyme protein or a peptide fragment thereof as the antigen, by the same technique as the above for anti-EDG2 antibody. As examples of LPA production enzyme inhibitors, PLA2 inhibitors such as chloropromadine, quinacrine, arachidonyl trifluoromethyl ketone, palmityl trifluoromethyl ketone and anti-annexin antibody, PLD inhibitors such as ethanol, 1-butanol, and 2,3-diphosphoglycerate, LCAT inhibitors such as DTNB and PCMPS, ATX inhibitors such as cyclic phosphatidic acid analogues and the like can be mentioned.
The substance that inhibits LPA activity is exemplified by, but is not limited to, the above-described EDG2 antagonists, as well as an antibody against LPA and the like. As the anti-LPA neutralizing antibody, a commercially available one can be used as it is, and the antibody can also be acquired by conjugating LPA as the hapten with an appropriate carrier protein (for example, KLH, albumin and the like), and treating this conjugate as the antigen by the same technique as the above for anti-EDG2 antibody.
An antisense nucleic acid and siRNA of LPA production enzyme gene can be prepared as a pharmaceutical formulation in the same manner as with antisense EDG2. An anti-LPA production enzyme neutralizing antibody or an anti-LPA neutralizing antibody can be prepared as a pharmaceutical formulation in the same manner as with anti-EDG2 antibody. Furthermore, an LPA production enzyme inhibitor can be prepared as a pharmaceutical formulation in the same manner as with EDG2 antagonist.
The dose of the above-described substance that inhibits the production or activity of LPA varies depending on the recipient, target organ, symptoms, method of administration and the like; in oral administration, the dose is normally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day for, for example, an OA patient (assumed to weigh 60 kg). In parenteral administration, a single dose varies depending on the subject to be administered, the subject organ, symptoms, route for administration, etc.; for example, in injection administration, the dose is normally about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day for an OA patient (as 60 kg body weight). In the case that the subject to be administered to is other than human, the corresponding dose as converted per 60 kg body weight of human can be administered.
In the description and drawings, the codes of bases and amino acids are denoted in accordance with the IUPAC-IUB Commission on Biochemical Nomenclature or by the common codes in the art, examples of which are shown below. For amino acids that may have the optical isomer, L form is presented unless otherwise indicated.
Furthermore, substituent group, protecting group and reagents which are often used in the present specification are denoted as follows.
The sequence identification numbers 1 to 3 in the sequence listing herein show the following sequences.
The present invention is hereinafter described more specifically by means of the following Examples, which, however, are for illustrative purposes only and do not limit the invention in any way.
As a technique for identifying an osteoarthritis (OA) susceptibility gene using a correlation analysis, a candidate gene approach to G protein coupled receptors (GPCRs) expressed in articular cartilage was adopted. By targeting GPCRs as candidate genes for OA susceptibility genes, it is possible to identify a direct drug discovery target with a low molecular compound. It is also possible to identify an OA susceptibility signal transduction pathway, and a molecule involved in the signal can also be investigated as a drug discovery target. However, it is unrealistic to deal with all GPCRs as candidate genes. Hence, it was decided to perform correlation analyses for GPCRs expressed in articular cartilage, which is the major lesion site in OA.
GPCR expression analyses were performed using microarray sets configured with oligonucleotide probes for about 60,000 kinds of target sequences (GeneChip U95 and U133, Affymetrix). A series of operations for biotinylated cRNA preparation, array hybridization and the like were performed as directed in the Affymetrix GeneChip expression analysis manual. RNAs of OA articular cartilage and normal articular cartilage were purchased from Direct Clinical Access Co. with the approval of the in-house ethical committee of Takeda Pharmaceutical Co., Ltd. First, with 5-10 μg of total RNA as the template, using the T7-poly T primer and Superscript II (Invitrogen), a first strand cDNA was synthesized. Next, using E. coli DNA polymerase I (Invitrogen) and a ligase (Invitrogen), a second strand cDNA was synthesized. Using the double-stranded cDNA obtained, in vitro transcription (Ambion) was performed in the presence of biotinylated UTP and CTP (Enzo Diagnostics). Biotinylated cRNA was fragmented by being incubated in a buffer containing Tris (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate at 94° C. for 30 minutes, and array hybridization was performed. Washing and staining were performed using dedicated Fluidics Station (Affymetrix). Signals were detected using dedicated Confocal Scanner (Molecular Dynamics). By the above-described method, a microarray analysis was performed using biotinylated cRNA probes prepared from total RNAs extracted from knee OA patient-derived articular cartilages (n=5), hip OA patient-derived cartilages (n=5), normal knee articular cartilages (n=3) and normal hip articular cartilage (n=3); GPCRs receiving a judgment “presence” in any person were identified as “articular cartilage expression GPCRs”. As a result, 64 genes were selected as cartilage expression GPCRs (Table 1).
For performing a correlation analysis, it is necessary to determine the genotypes of single nucleotide polymorphism (SNP) present on each gene in each individual in a diseased group (cases) and a control group (controls). Hence, it was decided to determine genotypes (genotyping) using invader probes stocked by the RIKEN SNP Research Center (RIKEN SRC). For the 64 cartilage expression GPCR genes selected in Example 1, database search was performed for the presence or absence of invader probes stocked by the RIKEN SRC. As a result, 47 genes were found to have invader probes registered.
For 8 genes wherein the number of probes per gene is 1, it was decided to perform genotyping using the probe without limitation on conditions. For the remaining 39 genes wherein the number of probes per gene is 2 or more, it was decided to select haplotype tagging SNPs (htSNPs) using the genotype data from the RIKEN SRC, and use them as genotyping probes. The procedures for selection of the htSNPs are described below. First, all invader probes present in the 39 genes were checked against the RIKEN SRC's genotyping data, and 544 probes fulfilling Hardy-Weinberg's equilibrium (HWE) were selected (P≧0.01). These 544 probes included probes wherein the minor allele frequency (MAF) was 10% or more; from genotype data thereon, haplotype blocks were estimated using Gabriel's method. For each haplotype block estimated, SNPs that express all the haplotypes occurring at a frequency of 1% or more in the block were selected as htSNPs. Of the 39 genes for which selection of htSNPs was performed, 37 genes were found to have 1 to 11 htSNPs per gene. The total number of htSNPs for these genes was 162. For the remaining 2 genes (GRM7 and GRM8), the number of htSNPs was 45 and 47, respectively. These 2 genes, because of necessity for typing a larger number of htSNPs than other genes, were excluded from this screening. SNPs on X chromosome were also excluded from the subjects of analysis. After the processes shown above, 167 SNPs for 44 genes were finally identified as subject SNPs for genotyping (Table 2).
For the correlation analysis, the 2-stage stepwise screening technique was used. The case population used in the screening was a group of patients who visited a number of medical institutions in Japan, in whom an OA change in the knee joint was observed in X-ray diagnostic imaging (radiographic OA patient population). For primary screening, genotyping was performed on genomic DNAs extracted from a population of 368 Japanese knee OA patients and a population of 323 controls by the invader method according to the method of Ohnishi et al. [J Hum Genet. 2001; 46(8):471-7.]. Of the 167 SNPs subjected to the genotyping, 4 SNPs exhibited no polymorphism. Also, 5 SNPs did not fulfill the HWE (P≧0.01) in the control population; furthermore, 6 SNPs did not permit genotyping because probes did not work. For 155 SNPs, excluding these SNPs, a correlation analysis was performed by X2 test or Fisher's exact test; 13 SNPs exhibited a correlation of P<0.05. For these 13 SNPs, genotyping was performed in a population of 276 Japanese knee OA patients that did not overlap the primary screening, and a correlation analysis was performed using genotype data on a population of 654 controls registered with the RIKEN SRC database; 2 SNPs (rs3739216 and rs3739708) exhibited a correlation of P<0.05. However, in rs3739216, the tendencies of MAF in primary screening and secondary screening reversed between the knee OA population and the control population; an analysis on both screening data in combination yielded P>0.05, so that the data was rejected. Eventually, rs3739708 passed the two stages of screening (Table 3). This screening suggested that knee OA susceptibility polymorphs may be present in a linkage disequilibrium (LD) region where rs3739708 is present.
a11, 12 and 22 indicate a minor allele homozygote and heterozygote and a major allele homozygote, respectively.
bFrequency of genotype 11
By the 2-stage screening shown in Example 3, rs3739708, which exhibited a high correlation with knee OA, was selected. Judging from the common disease-common variant hypothesis, true knee OA susceptibility polymorphs are thought to be present in LD regions as in rs3739708. We performed genotyping of the knee OA patient group used in the primary screening for polymorphs present in an about 800 kb region, including rs3739708, using invader probes or TaqMan probes already designed by Applied biosystems Co. (probes denoted as Invader or TaqMan in the “Probe type” column in Table 4). Of them, polymorphs wherein the MAF is 10% or more were used to calculate the linkage disequilibrium constant D′ for rs3739708. As a result, polymorphs having a relation of D′>0.9 to rs3739708 were rs7041855 and rs3739707 (
aMinor allele frequency in the diseased population in primary screening
To identify true knee OA susceptibility polymorphs, polymorph discovery was performed on the rs3739708 LD region according to the method of Iida et al. [J Hum Genet. 2005; 50(1):42-5.]. The method is briefly described below. Genomic DNAs were extracted from 48 persons, and 16 kinds of mixed genomes of 3 individuals each were used. On the basis of a genome sequence reported in the Genbank database (AC007157.6), primers that amplify the region between rs11794726 and rs12353088 were prepared. Using these primers, PCR was performed on 20 ng of each mixed genomic DNA. The PCR was performed using the GeneAmp PCR system (Applied biosystems), under the conditions shown below. Specifically, after initial denaturation was performed at 94° C. for 2 minutes, a reaction involving three temperatures, at 94° C. for 30 seconds (thermal denaturation), at 60° C. for 30 seconds (annealing), and at 72° C. for 2 minutes (extension reaction), as one cycle, was carried out in 35 cycles, and finally, a final extension reaction was carried out at 72° C. for 7 minutes. Sequencing was performed by a cycle sequence method using a fluorescent dye terminator, with the PCR reaction product as the template. All gene polymorphs were determined using the PolyPhred computer program. As a result, it was found that in the region for which polymorph discovery was performed, there were 24 SNPs and 3 insertion/deletion polymorphs (
aBased on calculations in disease population in primary screening.
For the polymorphs whose existence was demonstrated in Example 5, a TaqMan probe for genotyping was prepared by Assays-by-Design service (Applied biosystems). Using this TaqMan probe, with genomic DNAs (368 persons) collected from individuals in the knee OA patient group used in the primary screening as the samples, a genotyping reaction was carried out on a 384-multiwell plate. Detection was performed using ABI Prism 7900 (Applied biosystems), and the data was analyzed using Sequence Detection Software (Applied biosystems). For the polymorphs for which no TaqMan probe could be prepared, a PCR primer that amplifies the polymorph region was prepared, PCR was performed with genomic DNA as the template, and genotyping was performed according to the method described in Example 5. Using genotype data on selected polymorphs wherein the MAF is 10% or more, and polymorphs, including rs3739708, present in the 800 kb region used in Example 4, D′ was calculated pairwise, and an LD map was generated (
For all polymorphs present in the rs3739708 linkage disequilibrium region, genotyping was performed on 644 knee OA patients (the population used in the primary and secondary screening) and a population of 640 controls, according to the methods described in Examples 4 and 6, and a correlation analysis was performed. As a result, three polymorphs exhibiting a stronger correlation than with rs3739708 (i-EDG2-25) were identified (Table 6). All these polymorphs exhibited the strongest correlation in a minor allele recessive model. These polymorphs were mutually in a nearly complete linkage (Δ>0.85).
a11, 12 and 22 indicate a minor allele homozygote and hetezygote and a major allle homozygote, respectively.
Using the genotyping data obtained in Example 7, a haplotype analysis in the rs3739708 linkage disequilibrium region was performed. The SNPs used in the haplotype analysis were 13 SNPs wherein the minor allele frequency exceeded 10%. As a result, it was found that in this region, eight haplotypes accounted for 95% of the entire study population (population of knee OA patients and controls) (Table 7). When these haplotypes were subjected to a correlation analysis, no haplotype exhibiting a higher correlation than the three SNPs identified in Example 7 was observed (Table 7). This finding strongly suggests that in the rs3739708 linkage disequilibrium region, true disease-susceptible polymorphs may be present in the three SNPs that exhibited a high correlation in Example 7.
For four polymorphs highly correlated with knee OA present in the rs3739708 LD region (i-EDG2-03, 09, 12 and 25), a correlation analysis in a Mie Prefecture cohort (Miyagawa Village and Nansei Town) was performed. The correlation analysis was performed on a population of subjects judged to have OA on X-ray images (238 subjects, OA population) for a case group, and a population of subjects who had no OA findings on X-ray images and had no pain (279 subjects, non-OA population) for a control group. As a result, i-EDG2-09, 12 and 25 exhibited a correlation of P<0.05 in a minor allele recessive model (Table 8). This result, combined with Example 7, means that the correlations of i-EDG2-09, 12 and 25 with knee OA were reproduced in the two independent case/control populations. The final correlation value for i-EDG2-09 is 0.0000015 as obtained by multiplying the P-values for two independent populations (general Japanese population and cohort population). This remains a significant value even after Bonferroni's correction is performed with the number of SNPs used in the primary screening (155 SNPs).
a11, 12 and 22 indicate a monor allele homozygote and heterozygote and a major allele homozygote, respectively.
bP values and odds ratios were calculated using a recessive model (11 versus 12 + 22).
ci-EDG2-5 is an SNP identical to rs3739708.
Results of a correlation analysis suggested that the EDG2 gene might be a knee OA susceptibility gene. EDG2 is a GPCR having lysophosphatidic acid (LPA) as a ligand thereof. For LPA, four receptors, including EDG2, have been reported (EDG2, EDG4, EDG7 and GPR23). To analyze the tissue distributions of these four LPA receptors, an expression quantitation analysis by quantitative real-time PCR in various human tissues was performed. On 500 ng of total RNA of each of various human tissues (Human Total RNA Master Panel, Clontech), reverse transcription was performed using Multiscribe reverse transcriptase (Applied biosystems) and a random hexamer, whereby a cDNA was obtained. Quantitation of the LPA receptor genes was performed using QuantiTect STBR Green PCR (Qiagen) on the ABI PRISM 7700 sequence detection system (Applied biosystems) as directed in the documentation attached. The number of copies of each gene was calculated using a working curve. The amount of each gene was standardized by being divided by the number of copies of the G3PDH gene. The primers used in the gene quantitation are shown in Table 9. A Toyobo product was used as the primer for the G3PDH gene. As a result, the EDG2 gene was found to be strongly expressed in the whole brain and spinal cord. In these organs, the amount of the EDG2 gene expressed was about 20 to 100 times as high as other LPA receptor genes; this gene is a dominant LPA receptor (
humanEDG2
humanEDG4
humanEDG7
humanGPR23
humanTNFA
humanIL1B
humanIL6
rabbitEdg2
rabbitTnfa
rabbitIl1b
rabbitIl6
rabbitMmp1
rabbitMmp3
rabbitMmp13
rabbitG3pdh
To analyze the expression of the four LPA receptors examined in Example 10 in joint tissue, an expression quantitation analysis by quantitative real-time PCR was performed on articular cartilages and synovial tissues of knee OA patients. The method used was in accordance with Example 10. Total RNA was extracted from each tissue (articular cartilage n=4, synovial membrane n=3) using Isogen (Nippon Gene), and reverse transcription was performed on 500 ng of total RNA using Multiscribe reverse transcriptase (Applied biosystems) and a random hexamer, whereby a cDNA was obtained. Quantitation of the LPA receptor genes was performed using QuantiTect STBR Green PCR (Qiagen) on the ABI PRISM 7700 sequence detection system (Applied biosystems) as directed in the documentation attached. The number of copies of each gene was calculated using a working curve. The amount of each gene was standardized by being divided by the number of copies of the G3PDH gene. The primers used in the gene quantitation are shown in Table 9. A Toyobo product was used as the primer for the G3PDH gene. As a result, the EDG2 gene was found to be expressed in knee OA articular cartilage and OA synovial membrane. The amount of the EDG2 gene expressed was higher in synovial membrane at a level about 10 times as high as in articular cartilage. In synovial tissue, the amount of the EDG2 gene expressed was about 25 times as high as other LPA receptor genes, showing that the EDG2 gene is a dominant LPA receptor in synovial membrane (
Because the EDG2 gene is an LPA receptor that is dominant in OA synovial membrane, it was decided to perform a functional analysis of OA susceptibility polymorphs using synovial cells. E11 cells are a cell line established by immortalizing fibroblast-like cells from a rheumatoid arthritis patient synovial membrane with simian SV40 large T antigen (Abe et al, J Rheumatol. 1997 March; 24(3):420-9.). In the 5′-flanking region of EDG2, where i-EDG2-09 and 12 are present, an AP-1-binding motif is present. Hence, to determine whether or not a transcription activation mechanism by AP-1 is present in E11 cells, E11 cells were stimulated with phorbol 12-myristate 13-acetate (PMA), and the luciferase activity of a construct wherein the binding motif of AP-1 is inserted in front of the luciferase gene (PathDirectAP-1cis-Reporting system, Stratagene) was measured. The method used is described below. The E11 cells were cultured using Dulbecco's Modified Eagle's Medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS), antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin) and L-glutamine (200 mM). On the day before the transfection, the E11 cells were seeded to a 24-multiwell plate to obtain a cell density of 5×104 cells/well. The following day, using Fugene-6 (Roche diagnostics), 200 ng of the reporter construct and 4 ng of pRL-TK (Promega) as the internal standard for correction of transfection efficiency, per well were transfected. Twenty-four after the transfection, the cells were cultured on a medium containing 0.1, 1 and 10 ng/ml PMA. Twenty-four hours after the stimulation, the cells were solubilized, and luciferase activity was measured using PicaGene Dual SeaPansy System (Toyo Ink). The number of cases was n=2. As a result, it was found that the luciferase activity rose PMA dose-dependently (
The results in Example 12 revealed that in E11 cells, a mechanism for AP-1 activation by PMA stimulation exists. Hence, an investigation was made to determine whether PMA alters the expression of the EDG2 gene in E11 cells over time. The method used is described below. E11 cells were seeded to a 24-multiwell plate to obtain a cell density of 1×105 cells/well. The cells were cultured until they became confluent, after which they were stimulated with PMA (10 ng/ml); 0, 2, 4, 9, 12 30 and 24 hours later, total RNAs were recovered using ISOGEN. In these total RNAs, the amount expressed of the EDG2 gene relative to G3PDH was measured in accordance with the method described in Example 11. The number of cases was n=4. As a result, from 2 hours to 12 hours after the addition of PMA, the amount expressed of the EDG2 gene rose over time (
An AP-1-binding motif described in a report by Duncliffe et al. [Immunity. 1997 February; 6(2):175-85.] is present 1 to 7 bp upstream of i-EDG2-09 out of the 2 polymorphs that exhibited a high correlation with knee OA, and an NFAT-binding motif described in a report by Guo et al. [J Biol Chem. 2001 Dec. 28; 276(52):48871-8.] is present 11 to 16 by upstream. The existence of a transcriptional factor-binding motif where AP-1 and NFAT are located in tandem is known in a plurality of genes [Macian F, Lopez-Rodriguez C and Rao A., Oncogene. 2001 Apr. 30; 20(19):2476-89. Review.]. Meanwhile, in the surrounding sequence, including i-EDG2-12, no distinct transcriptional factor binding sequence was found. Hence, the influence of i-EDG2-09 on the EDG2 gene transcription activity was analyzed by luciferase reporter assay. Oligonucleotides (sense strand and antisense strand) wherein 4 bp at the restriction endonuclease NheI adhesion end is added on the 5′ side of a region centering at i-EDG2-09, and comprising the AP-1 and NFAT-binding motifs (a region at −2809 to −2839 from the transcription initiation point of EDG2), and 4 bp at the XbaI adhesion end is added on the 3′ side, were prepared for both alleles of i-EDG2-09 (G or A). After these oligonucleotides were converted to double-stranded DNAs for each allele, reporter constructs wherein each allele was integrated in tandem 5 times in the NheI site of the pGL3-promoter vector (Promega), which is a luciferase reporter vector, while retaining the orientation were prepared for G allele and A allele. These constructs were transfected to E11 cells cultured in Dulbecco's Modified Eagle's Medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin). The transfection was performed by the procedures shown below. On the day before the transfection, the E11 cells were seeded to a 24-multiwell plate to obtain a cell density of 5×104 cells/well. The following day, using Fugene-6 (Roche Diagnostics), 200 ng of the reporter construct and 4 ng of pRL-TK (Promega) as the internal standard for correcting the transfection efficiency per well were transfected. Twenty-four hours after the transfection, the cells were stimulated with a medium containing 10 ng/ml PMA. Five hours after the stimulation, the cells were solubilized, and luciferase activity was measured using the PicaGene Dual SeaPansy System (Toyo Ink). As a result, for both constructs, with the addition of PMA, the luciferase activity rose significantly. Without addition of PMA, there was no difference in transcription activity between G allele and A allele; however, with addition of PMA, a 2.4 times higher transcription activity was confirmed in A allele than in G allele (
In Example 14, i-EDG2-09 was found to exhibit a difference in transcription activity across alleles. Hence, to determine whether or not this difference in transcription activity depends on a difference in the binding affinity of intranuclear factor for each allele, a gel shift assay was performed using a nuclear extract from PMA-stimulated E11 cells. The method used is described below. After E11 cells were made to be confluent in a 100 mm dish, they were cultured on a medium containing PMA (10 ng/ml) for 0, 1, 2, 4 and 8 hours. After elapse of each time, nuclear extract protein of E11 cells was prepared according to the method of Andrews et al. [Nucleic Acids Res. 1991 Nov. 25; 19(22):6263-8.]. Oligonucleotide annealing and DIG labeling were performed using a DIG gel shift kit (Roche Diagnostics). The sequences of the oligonucleotides used are shown in Table 10. The DIG-labeled probe was mixed with a nuclear extract from PMA-stimulated E11 cells, and incubated at room temperature for 20 minutes, to form a DNA-protein complex. The DNA-protein complex formed was separated in 0.5% Tris-borate-EDTA buffer using 6% polyacrylamide gel. In the competitive experiments of the DIG-labeled probe and a non-labeled probe, the non-labeled probe in an amount 125 times the amount of the labeled probe was added prior to mixing the labeled probe and the nuclear extract. After electrophoresis, the DNA-protein complex was transferred to nitrocellulose membrane and crosslinked by baking (120° C., 30 minutes), after which signals from the labeled probe were detected using a chemiluminescence detection system (Roche Diagnostics Co.).
First, a gel shift assay was performed using the oligonucleotide of A allele and a nuclear extract from E11 cells stimulated with PMA over time. As a result, specific bands were confirmed on a lane wherein the nuclear extract of E11 cells and labeled oligo were mixed (
The inflammatory cytokine expression induction reaction of LPA, which is a ligand of EDG2, in human articular cartilage cells was analyzed. The method used is described below. Normal human knee articular cartilage cells (NHAC-kn, Takara) were embedded in alginate beads, and cultured in the attached differentiation medium (CDM) for 3 weeks. After incubation in sodium citrate solution (55 mM in 0.15M NaCl solution), the alginate beads were dissolved, and the cells were recovered via centrifugal operation. The cells were twice washed with 155 mM NaCl solution, after which they were seeded to a 24-well plate at a ratio of 1×105 cells/well. Twelve hours before performing LPA (1-Oleoyl-sn-Glycero-3-Phosphate, Sigma) stimulation, the medium was replaced with a DMEM/F-12 medium containing 0.1% fatty acid free bovine serum albumin (Sigma). Twelve hours after the medium replacement, the cells were stimulated with 10 μM LPA; 0, 2, 4, 8, 12 and 24 hours later, total RNA was extracted using ISOGEN (Nippon Gene), and the amounts expressed of TNFA and IL1B, which are inflammatory cytokine genes, were quantified by a method in accordance with Example 10. The number of cases was n=4. As a result, the amount expressed of the TNFA gene rose about 2 folds transiently 2 hours after addition of LPA (
The inflammatory cytokine expression induction reaction of LPA, which is a ligand of EDG2, in OA patient-derived fibroblast-like synovial cells was analyzed. The method used is described below. OA patient-derived fibroblast-like synovial cells (HFLS-OA) were purchased from Cell application Co. The HFLS-OA was cultured using the attached Synoviocyte growth medium. The number of passages for the cells used was 4 or 5. The HFLS-OA was seeded to a 24-well plate at a ratio of 1×105 cells/well, and cultured using the attached Synoviocyte growth medium until they became confluent. Twelve hours before performing LPA stimulation, the medium was replaced with Ham's F-12 (Invitrogen) medium containing 0.1% fatty acid free bovine serum albumin (Sigma). In the time-related change analysis, 12 hours after the medium replacement, the cells were stimulated with 10 μM LPA; 0, 1, 2, 4, 8, 12 and 24 hours later, total RNA was extracted using ISOGEN (Nippon Gene), and the amounts expressed of the TNFA, IL1B, and IL6 genes, which are inflammatory cytokine genes, and of the MMP1, MMP3, and MMP13 genes, which are Matrix metalloproteinase (MMP) genes, were quantified by a method in accordance with Example 10 (n=4). In the dose dependency test, 12 hours after the medium replacement, the cells were stimulated with 0, 0.1, 0.3, 1, 3 and 10 μM LPA; 2 hours later, total RNA was extracted using ISOGEN (Nippon Gene), and the amounts expressed of the TNFA, IL1B, and IL6 genes, which are inflammatory cytokine genes, were quantified in the same manner as the above for the time-related change test (n=3). The sequences of the primers used are shown in Table 9. As a result of the time-related change test of inflammatory cytokine genes with LPA stimulation, the amounts of TNFA, IL1B and IL6 expressed rose transiently, with peaks reached 2, 4 and 1 hour later, respectively (
In Example 14, the A allele of i-EDG2-09, which is a knee OA susceptibility polymorph, raised the transcription activity of the EDG2 gene; therefore, AA homozygous individuals possibly have highly expressed EDG2 in their synovial cells and an increased inflammatory cytokine gene expression raising reaction by LPA stimulation. The induced inflammatory cytokines may induce MMPs in joints to cause cartilage destruction and raise the susceptibility to radiographic OA. At the same time, inflammatory cytokines may raise the susceptibility to symptomatic OA by lowering the threshold value of the primary afferent nerve present in the primary synovial membrane.
The inflammatory cytokine expression induction reaction of LPA in a rabbit synovial cell line was analyzed. The method used is described below. A rabbit synovial cell line (HIG-82) was purchased from ATCC. The HIG-82 was cultured using Ham's F-12 medium (Invitrogen) containing 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin). The HIG-82 was seeded to a 24-well plate at a ratio of 1×105 cells/well, and cultured until they became confluent. Twelve hours before performing LPA stimulation, the medium was replaced with Ham's F-12 medium containing 0.1% fatty acid free bovine serum albumin (Sigma). In the time-related change analysis, 12 hours after the medium replacement, the cells were stimulated with 10 μM LPA; 0, 2, 4, 8, 12 and 24 hours later, total RNA was extracted using ISOGEN (Nippon Gene), and the amounts expressed of the TNFA and IL1B genes, which are inflammatory cytokine genes, and of the MMP1, MMP3 and MMP13 genes, which are MMP genes, were quantified by a method in accordance with Example 10 (n=4). In the dose dependency test, 12 hours after the medium replacement, the cells were stimulated with 0, 0.1, 1 and 10 μM LPA; 2 hours later, total RNA was extracted using ISOGEN (Nippon Gene), and the amounts expressed of the TNFA and IL1B genes, which are inflammatory cytokine genes, were quantified by the same method as with the time-related change test (n=4). As a result of the time-related change test of inflammatory cytokine genes with LPA stimulation, the amounts expressed of the TNFA and IL1B genes rose transiently, with peaks reached 2 hours later (
For i-EDG2-09, the relationship between the severity of knee OA and the frequency of disease-susceptible genotype (A homozygote) was examined in a general population of Japanese patients with knee OA.
As a result, in the population, as the severity of disease increased, the frequency of the disease-susceptible genotype tended to be higher (
To analyze the involvement of EDG2 in the LPA-induced inflammatory cytokine raising reaction in human OA synovial cells, obtained in Example 17, an investigation using an siRNA was performed. The method used is described below. An siRNA against EDG2 was designed by utilizing an siRNA design support system available from the Takara Bio Web site (http://bio.takara.co.jp). Synthesis of the siRNA was outsourced to Takara Bio. The sequence is shown in Table 11. The control siRNA used was such that the sequence of an siRNA against asporin was scrambled. OA patient-derived fibroblast-like synovial cells (HFLS-OA) were purchased from Cell application Co. The HFLS-OA was cultured using the attached Synoviocyte growth medium. The number of passages for the cells used was 4 or 5. The HFLS-OA was seeded to a 24-well plate at a ratio of 1×105 cells/well, and cultured using the attached Synoviocyte growth medium. The following day, the siRNA against EDG2 was introduced into the HFLS-OA to obtain a final concentration of 11 nM, using the Trans-IT-TKO transfection regent (Mirus Co.), according to the attached protocol. On the day after the transfection, the medium was replaced with Ham's F-12 (Invitrogen) medium containing 0.1% fatty acid free bovine serum albumin (Sigma). Twelve hours after the medium replacement, the cells were stimulated with 10 μM LPA; 2 hours later, total RNA was extracted using ISOGEN (Nippon Gene), and the amount of the IL6 gene expressed was quantified by a method in accordance with Example 10 (n=4). In the cells incorporating the siRNA against EDG2, the amount of EDG2 expressed was significantly suppressed (
Furthermore, the involvement of EDG2 in the LPA-induced inflammatory cytokine raising reaction in human OA synovial cells was analyzed using an antagonist against EDG2. The method used is described below. As the EDG2 antagonist, Ki16425 (Sigma) was used. OA patient-derived fibroblast-like synovial cells (HFLS-OA) were purchased from Cell application Co. The HFLS-OA was cultured using the attached Synoviocyte growth medium. The number of passages for the cells used was 4 or 5. The HFLS-OA was seeded to a 24-well plate at a ratio of 1×105 cells/well, and cultured using the attached Synoviocyte growth medium until they became confluent. Twelve hours before performing LPA stimulation, the medium was replaced with Ham's F-12 (Invitrogen) medium containing 0.1% fatty acid free bovine serum albumin (Sigma). Twelve hours after the medium replacement, the cells were treated with Ki16425 (0, 0.3, 1, 3, 10 and 33 μM) for 30 minutes. Thereafter, the cells were stimulated with 10 μM LPA; 2 hours later, total RNA was extracted using ISOGEN (Nippon Gene), and the amount expressed of the IL6 gene was quantified by a method in accordance with Example 10 (n=4). As a result, Ki16425 suppressed the rise in the expression of the IL6 gene by LPA dose-dependently (
A forcible expression vector comprising an ORF sequence of human EDG2 [hEDG2-pcDNA3.1(+)] was purchased from UMR cDNA Resource Center. This plasmid was introduced into McA-RH7777 (Dainippon Pharmaceutical Co., Ltd.) using Lipofectamine LTX (Invitrogen), and the cells were cultured in a medium containing Geneticin for 14 to 20 days to acquire cells that stably express EDG2.
Next, an evaluation was made using FLIPR tetra (Molecular Devices) to confirm that stimulation with LPA raised intracellular Ca concentrations via the EDG2 expressed. That is, these cells were seeded to a 384-well plate, 0.03 to 10 μM LPA (Sigma) was added to each well, and it was confirmed that rises in intracellular Ca2+ concentration occurred. LPA dose-dependently.
Furthermore, this elevation of intracellular Ca2+ concentration by LPA stimulation (0.3 μM) was suppressed dose-dependently by previously adding Ki16425 (Sigma), which has antagonizing action on EDG2 (10−8 to 10−5 M).
According to the present invention, genetic susceptibility to bone and joint diseases can easily be determined. Anti-EDG2 antibody, antisense EDG2, EDG2 antagonists, DNM5 of EDG2, and substances capable of regulating the expression or activity thereof can be used as prophylactic/therapeutic agents for bone/joint diseases.
This application is based on a patent application No. 2007-155792 filed in Japan, the contents of which are incorporated in full herein by this reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-155792 | Jun 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP08/60700 | 6/11/2008 | WO | 00 | 4/7/2010 |
