JP6714263B2 - Immunomodulator and screening method - Google Patents

Description

The present invention relates to immunomodulators and screening methods.

Fibroblast growth factor 23 (FGF23) is a humoral factor that is considered to be produced by bone tissue, particularly bone cells. FGF23 is deeply involved in the maintenance of phosphorous homeostasis, and reduces blood phosphorus levels by suppressing renal tubular phosphorus reabsorption and suppressing intestinal phosphorus absorption through reduction of blood levels of active vitamin D3. Let The specific action of FGF23 in the kidney is considered to be due to the action of FGF23 on the complex of FGF receptor (FGFR) and Klotho protein.

Moreover, the FGF23-Klotho signal is said to be involved in anti-aging, and FGF23 knockout mice show various aging-like symptoms with a short life (for example, see Non-Patent Document 1).

On the other hand, FGF23 is known to be expressed in spleen other than bone tissue, and it is considered that the control mechanism may be different between the expression of FGF23 in bone tissue and that in spleen (for example, Non-Patent Document 2).

FASEB J, 20, 720-722 (2006). Nephron Physiol, 111, 59-66 (2009).

However, the physiological significance of FGF23 expression in the spleen has not been fully elucidated.
An object of the present invention is to provide a screening method for a substance useful for regulation of immune function and an immunomodulator based on the physiological significance of FGF23 expression in the spleen.

The present invention includes the following aspects.
[1] An immunomodulator containing a FGF23-Klotho modulator as an active ingredient.
[2] The immunomodulator according to [1], wherein the FGF23-Klotho modulator is an FGF23-Klotho inhibitor.
[3] The immunomodulator according to [1], wherein the FGF23-Klotho modulator is an FGF23-Klotho activator.
[4] The immunomodulator according to any one of [1] to [3], which comprises one or more substances selected from the group consisting of vitamin D derivatives, nucleic acid oligos, anti-FGF23 antibodies and anti-Klotho antibodies.
[5] The immunomodulator according to [4], wherein the nucleic acid oligo is composed of natural or non-natural RNA or DNA.
[6] The immunomodulator according to [1] or [3], which is an antibody production enhancer.
[7] A screening method for a candidate substance useful for the regulation of immune function, which comprises (a) detecting the effect of a test substance on the FGF23-Klotho system, and (b) detecting the effect without using the test substance. In this case, selecting a test substance as a candidate substance that has a different effect on the FGF23-Klotho system in this case.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a screening method for a substance useful for regulation of immune function and an immunomodulator based on the physiological significance of FGF23 expression in the spleen.

It is a figure which shows the influence of the FGF23-Klotho signal in B cell. It is a figure which shows the mRNA expression level of the FGF23 in an osteoblast cell line 7 days after differentiation induction. It is a figure which shows the mRNA expression level of the FGF23 in the osteoblast cell line 14 days after differentiation induction.

“FGF23” is Fibroblast growth factor 23. The FGF23 precursor encoded by the human FGF23 gene is a protein consisting of 251 amino acids, and includes, for example, NCBI reference sequence number: NP_065689.1, accession number: NM_020638.2.

The FGF23-Klotho modulator is a substance capable of modulating FGF23-Klotho signal transduction, and includes an FGF23-Klotho inhibitor and an FGF23-Klotho activator.

The FGF23-Klotho inhibitor is at least one selected from the group consisting of a substance that inhibits the expression of FGF23 or Klotho and a substance that inhibits the function of FGF23 or Klotho. The FGF23-Klotho inhibitor may be a substance that directly inhibits the expression of FGF23 or Klotho, a substance that inhibits the binding between FGF23 and the complex of Klotho and FGFR, or an inhibitor of the interaction between Klotho and FGFR. It may be a substance or a substance that inhibits the action of active FGFR.
FGF23-Klotho inhibitors include vitamin D derivatives, nucleic acid oligos, anti-FGF23 antibodies and anti-Klotho antibodies.

The vitamin D derivative in the FGF23-Klotho inhibitor, for example, inhibits an increase in FGF23 expression due to active vitamin D. The vitamin D derivative may be a substance that suppresses the expression of active vitamin D or a substance that competitively inhibits the FGF23 expression action of active vitamin D.

Inhibition of FGF23 or Klotho expression can be achieved by utilizing, for example, an RNA interference effect on the expression of the FGF23 gene or Klotho gene. RNA interference is a method reported as a method of suppressing gene expression using RNA (Genes and Development, 16, 948-958 (2002)), and is a double gene having the same or similar sequence as the target gene. This is a phenomenon in which when the strand RNA is introduced into cells, the expression of both the introduced foreign gene and the target endogenous gene is suppressed. Specifically, it is possible to inhibit the expression of the FGF23 gene or the Klotho gene by using siRNA or an antisense nucleic acid that exhibits an RNA interference effect on the expression of the FGF23 gene or the Klotho gene.

“Nucleic acid oligo” means an oligomer of nucleic acid that controls the function of FGF23 gene or Klotho gene or FGF23 or Klotho protein, and includes siRNA, shRNA, antisense nucleic acid, decoy nucleic acid, and nucleic acid aptamer.

Preferred nucleic acid oligos include siRNA or shRNA. siRNA means a double-stranded RNA consisting of a short chain within a range that does not show toxicity in cells, and is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs. Can be Moreover, shRNA is RNA in which a single-stranded RNA constitutes a double strand via a hairpin structure.

siRNAs and shRNAs need not be completely identical to the target gene, but have at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence homology.

The portion of the double-stranded RNA in which siRNA and shRNA are paired with each other in RNA is not limited to those that are perfectly paired, but mismatches (corresponding bases are not complementary), bulges (bases corresponding to one strand) , Etc.) may be included in the unpaired portion. In the present invention, both the bulge and the mismatch may be contained in the double-stranded RNA region of the dsRNA where RNAs pair with each other.

The "antisense nucleic acid" is an antisense nucleic acid complementary to the transcription product of DNA encoding FGF23 or Klotho. There are several factors as described below as the action of the antisense nucleic acid to suppress the expression of the target gene. That is, degradation by RNase activity due to RNA double-strand recognition, inhibition of transcription initiation by triplex formation, transcriptional repression by hybridization with a site where an open loop structure is locally formed by RNA polymerase, and RNA under synthesis Inhibition of transcription by hybridizing with, suppression of splicing by hybridization at intron/exon junction, suppression of splicing by hybridization with spliceosomal site, suppression of translocation from nucleus to cytoplasm by hybridization with mRNA, translation initiation Translational inhibition by hybridization with factor binding site, inhibition of peptide chain extension by hybridization with mRNA translation region and polysome binding site, and gene expression inhibition by hybridization with nucleic acid-protein interaction site. .. Among these, it inhibits the process of transcription, splicing, or translation to suppress the expression of the target gene.

The antisense sequence may suppress the expression of the target gene by any of the above actions. In one embodiment, it is considered effective to inhibit the translation of a gene by designing an antisense sequence complementary to the untranslated region near the 5'end of FGF23 or Klotho mRNA. However, sequences complementary to the coding region or the 3'untranslated region may also be used. Thus, a sequence complementary to the untranslated region as well as the translated region of the gene can be used. Thus, not only the translation region of a gene, but also the nucleic acid containing the antisense sequence of the sequence of the untranslated region is included in the antisense nucleic acid used in the present invention. The antisense nucleic acid has a complementarity of preferably 90% or more, and most preferably 95% or more with the transcription product of the target gene. The length of the antisense RNA that effectively inhibits the expression of the target gene using the antisense sequence is not particularly limited.

The "anti-FGF23 antibody" or "anti-Klotho antibody" is an antibody that specifically binds to FGF23 or Klotho, and examples thereof include an antibody that inhibits FGF23-Klotho signal transduction in the case of an FGF23-Klotho inhibitor. ..
The "anti-FGF23 antibody" or "anti-Klotho antibody" can be obtained as a polyclonal antibody or a monoclonal antibody using known means. The origin of the antibody is not particularly limited, but is preferably of mammalian origin, more preferably of human origin. Examples of mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by a host transformed with an expression vector containing the antibody gene by a genetic engineering technique. These antibodies inhibit the functional expression of the FGF23-Klotho signaling system by binding to FGF23 or Klotho.

An antibody-producing hybridoma can be basically prepared using a known technique as follows. That is, using FGF23 or Klotho as a sensitizing antigen, this is immunized according to a usual immunization method, and the resulting immune cells are fused with a known parent cell by a usual cell fusion method, and a usual screening method is used. It can be prepared by screening monoclonal antibody-producing cells. For example, human FGF23 or Klotho used as an antigen for antibody acquisition can be obtained by using the gene sequence or amino acid sequence of FGF23 or Klotho disclosed in a known document.

The antibody may be a conjugated antibody bound to various molecules such as polyethylene glycol (PEG), radioactive substance, and toxin. Such a conjugated antibody can be obtained by chemically modifying the obtained antibody. The method for modifying the antibody has already been established in this field. The “antibody” in the present specification also includes these conjugated antibodies.

Antibodies include not only divalent antibodies represented by IgG, but also monovalent antibodies or polyvalent antibodies represented by IgM, as long as they bind to FGF23 or Klotho. The polyvalent antibody of the present invention includes a polyvalent antibody having all the same antigen binding sites, or a polyvalent antibody having some or all different antigen binding sites.

Further, the antibody may be a bispecific antibody as long as it binds to FGF23 or Klotho. Bispecific antibody refers to an antibody having a variable region that recognizes a different epitope in the same antibody molecule, and the epitope may be present in different molecules or may be present in the same molecule. May be That is, the bispecific antibody can also have an antigen binding site that recognizes a different epitope of FGF23 or Klotho. It is also possible to use a bispecific antibody in which one recognition site recognizes FGF23 or Klotho and the other recognition site recognizes an antigen other than FGF23 or Klotho.

Methods for producing bispecific antibodies are known. For example, a bispecific antibody can be prepared by binding two kinds of antibodies having different recognition antigens. The antibody to be bound may be 1/2 molecule of an antibody having an H chain and an L chain, respectively, or 1/4 molecule of an antibody consisting of an H chain only. Alternatively, bispecific antibody-producing fused cells can be prepared by fusing hybridomas that produce different monoclonal antibodies. Furthermore, bispecific antibodies can be prepared by genetic engineering techniques.

The antibody may be a minibodies as long as it binds to FGF23-Klotho. The minibodies include antibody fragments in which a part of the full-length antibody (whole antibody, such as whole IgG) is deleted. Partial deletion of the antibody molecule is acceptable as long as it binds to Arid5A. The antibody fragment of the present invention preferably contains either or both of a heavy chain variable region (VH) and a light chain variable region (VL). The amino acid sequence of VH or VL can include additions, deletions and/or substitutions. Further, as long as it binds to Arid5A, one or both of VH and VL can be deleted. The antibody fragment may be chimerized or humanized. Specific examples of the antibody fragment include Fab, Fab′, F(ab′)2, and Fv. Further, specific examples of the low molecular weight antibody include, for example, Fab, Fab′, F(ab′)2, Fv, scFv (single-chain Fv), diabody (Diabody), sc(Fv)2 (single-). chain (Fv) 2) and the like. Multimers of these antibodies (for example, dimers, trimers, tetramers, polymers) are also included in the antibodies of the present invention.
Antibody fragments can be produced by enzymatically digesting antibodies.

The FGF23-Klotho activator is at least one selected from the group consisting of a substance that promotes the expression of FGF23 or Klotho and a substance that enhances the function of FGF23 or Klotho. The FGF23-Klotho activator may be a substance that directly promotes the expression itself of FGF23 or Klotho, a substance that promotes the binding of FGF23 to the complex of Klotho and FGFR, or an interaction between Klotho and FGFR. It may be a substance that promotes or a substance that enhances the action of active FGFR.
FGF23-Klotho activators include phosphoric acid, vitamin D derivatives, nucleic acid oligos, anti-FGF23 antibodies and anti-Klotho antibodies.

The vitamin D derivative in the FGF23-Klotho activator promotes, for example, FGF23 expression in target cells. The vitamin D derivative may be a substance that becomes an agonist of the vitamin D receptor or a substance that cooperatively enhances the action of active vitamin D.
Vitamin D derivatives include active vitamin D, maxacalcitol, paricalcitol, falecalcitol triol and the like.

The nucleic acid oligo in the FGF23-Klotho activator is not particularly limited as long as it promotes the expression of at least one of FGF23 and Klotho.
The anti-FGF23 antibody and the anti-Klotho antibody are not particularly limited as long as they increase the activity of at least one of FGF23 and Klotho. Anti-Klotho antibodies include, for example, anti-Klotho and anti-FGFR bispecific antibodies.

FGF23-Klotho activation results in enhanced antibody production against Klotho-expressing B cells in, for example, the spleen. That is, the immunomodulator is preferably an antibody production enhancer.

Pharmaceutically acceptable materials such as preservatives and stabilizers may be added to the immunomodulator containing the FGF23-Klotho modulator as an active ingredient. Pharmaceutically acceptable may be a material having the above-mentioned FGF23-Klotho-regulating action per se, or a material having no such a regulating action, which can be administered together with the above-mentioned immunomodulator. It means a pharmaceutically acceptable material. Further, it may be a material having no regulatory action and having a synergistic effect or an additive stabilizing effect when used in combination with the FGF23-Klotho regulator.

Examples of pharmaceutically acceptable materials include sterilized water, physiological saline, stabilizers, excipients, buffers, preservatives, surfactants, chelating agents (EDTA, etc.), binders and the like.

Examples of the surfactant include nonionic surfactants, for example, sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate, and other sorbitan fatty acid esters; glycerin monocaprylate, glycerin monomyriate, glycerin monostearate. Typical examples include those having HLB6 to 18 such as glycerin fatty acid ester such as rate.

The surfactant may also include anionic surfactants such as sodium acetylsulfate, sodium laurylsulfate and sodium oleylsulfate, and alkylsulfates having an alkyl group having 10 to 18 carbon atoms; polyoxyethylene. Polyoxyethylene alkyl ether sulfate having an average number of added moles of ethylene oxide of 2 to 4 and an alkyl group having 10 to 18 carbon atoms such as sodium lauryl sulfate; a carbon atom of an alkyl group such as sodium lauryl sulfosuccinate Typical examples are alkyl sulfosuccinic acid ester salts having 8 to 18 carbon atoms; natural surfactants such as lecithin, glycerophospholipids; sphingolinolipids such as sphingomyelin; sucrose fatty acid esters of fatty acids having 12 to 18 carbon atoms. As an example:

One or two or more of these surfactants can be added to the immunomodulator in combination. A preferred surfactant for use in the immunomodulator is a polyoxyethylene sorbitan fatty acid ester such as polysorbate 20, 40, 60 or 80, with polysorbate 20 and 80 being especially preferred. Further, polyoxyethylene polyoxypropylene glycol represented by poloxamer (Pluronic F-68 (registered trademark)) and the like are also preferable.

As the buffer, phosphoric acid, citric acid buffer, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, caprylic acid, deoxycholic acid, salicylic acid, triethanolamine, fumaric acid, etc. Examples thereof include organic acids, carbonate buffers, Tris buffers, histidine buffers, imidazole buffers, and the like.

Alternatively, the solution preparation may be prepared by dissolving it in an aqueous buffer solution known in the field of solution preparation. The concentration of the buffer solution is generally 1 to 500 mM, preferably 5 to 100 mM, more preferably 10 to 20 mM.

The immunomodulator may also contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulin, amino acids, saccharides such as polysaccharides and monosaccharides, carbohydrates and sugar alcohols.

Examples of sugars and carbohydrates such as polysaccharides and monosaccharides include dextran, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, raffinose and the like.

Examples of sugar alcohols include mannitol, sorbitol, inositol and the like.

In the case of an aqueous solution for injection, for example, physiological saline, an isotonic solution containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and the like can be mentioned. You may use together with adjuvants, such as alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG etc.), a nonionic surfactant (polysorbate 80, HCO-50) etc.
If desired, it may further contain a diluent, a solubilizing agent, a pH adjusting agent, a soothing agent, a sulfur-containing reducing agent, an antioxidant and the like.

In addition, if necessary, it can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly[methylmethacrylic acid]), colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.). ) (See "Remington's Pharmaceutical Science 16th edition", Oslo Ed., 1980, etc.). Furthermore, a method for making a drug a sustained-release drug is also known and can be applied to the present invention (Langer et al., J. Biomed. Mater. Res. 1981, 15: 167-277; Langer, Chem. Tech. 1982, 12: 98-105; U.S. Pat. No. 3,773,919; European Patent Application Publication (EP) 58,481; Sidman et al., Biopolymers 1983, 22: 547-556; EP 133,988).
The pharmaceutically acceptable carrier to be used is appropriately selected or a combination of the above depending on the dosage form, but is not limited thereto.

When the immunomodulator is used as a medicine for humans and other animals, it is possible to administer these substances themselves not only directly to patients but also to formulate and administer them by known pharmaceutical methods. When formulating, the above-mentioned pharmaceutically acceptable materials may be added.

The immunomodulator can be administered in the form of a drug, and can be administered orally or parenterally, systemically or locally. For example, intravenous injection such as drip, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric solution, etc. can be selected, and the administration method should be selected appropriately depending on the age and symptoms of the patient. You can The effective dose is selected within the range of 0.001 mg to 100 mg per 1 kg of body weight. Alternatively, a dose of 0.1 to 1000 mg, preferably 0.1 to 50 mg per patient can be selected. In the case of anti-Arid5A antibody, for example, a preferable dose and administration method are effective doses such that free antibody is present in the blood. Weekly) 0.1 mg to 40 mg, preferably 1 mg to 20 mg, divided into 1 to several times, for example, twice/week, once/week, once/2 weeks, once/4 weeks, etc. Intravenous injection such as drip or subcutaneous injection, etc. The dosing schedule is 2 times/week or 1 time/week to 1 time/2 weeks, 1 time/3 weeks, 1 time/4 weeks, while observing the condition after administration and observing trends in blood test values. It is also possible to make adjustments such as extending the administration interval.

The present invention relates to a B cell differentiation regulator containing a FGF23-Klotho regulator as an active ingredient. FGF23 acts on B cells expressing Klotho to regulate B cell differentiation, for example, in the spleen. Specifically, the regulation of B cell differentiation includes enhancement of antibody production and terminal differentiation into plasma cells.

The present invention relates to a method for screening a candidate substance useful for regulating immune function. The screening method includes detecting an effect of a test substance on the FGF23-Klotho system, and selecting a test substance whose detected effect is different from the effect on the FGF23-Klotho system when the test substance is not used as a candidate substance. Including.

The screening method includes detecting the influence of a test substance on the FGF23-Klotho system. As a function of the FGF23-Klotho signal transduction system, in cells coexpressing Klotho and FGFR, activation of intracellular signal due to FGF23 is brought about. Specifically, suppression of phosphorus reabsorption in renal tubular cells, suppression of expression of active vitamin D producing enzyme 1α-hydroxylase, conversion of active vitamin D into a metabolite having low activity 24-hydroxylation In addition to promoting enzyme expression, it is thought to function as an antibody production enhancement in B cells and a differentiation signal in B cells. Therefore, examples of the effect on the FGF23-Klotho system include suppressing or promoting the expression of FGF23 or Klotho, inhibiting or enhancing the function of FGF23 or KlothoA by binding to FGF23 or Klotho, and competitively with FGF23 with FGFR. Examples thereof include, but are not limited to, inhibiting or enhancing the function of FGF23 by binding.

The screening method further includes selecting, as a candidate substance, a test substance whose detected effect is different from the effect on the FGF23-Klotho system when the test substance is not used. The difference in the detected influence includes the case where the detected influence is large and the case where the detected influence is small.

The first aspect of the screening method is to contact a test substance with FGF23, contact FGF23 contacted with the test substance with Klotho and FGFR, and evaluate the biological action or biochemical action of FGFR. Including doing.

The amino acid sequence of FGF23 used is as described above. The FGF23 used in the screening method includes a protein functionally equivalent to the above-mentioned known FGF23. Examples of such proteins include, but are not limited to, mutants of FGF23, alleles, variants, homologs, partial peptides of FGF23, and fusion proteins with other proteins.

A mutant of FGF23 is a naturally-occurring protein having an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and/or added in the amino acid sequence described above, and has the amino acid sequence described above. A protein functionally equivalent to the protein can be mentioned. Further, a protein encoded by a naturally-occurring DNA that hybridizes with a DNA having the above-described nucleotide sequence under stringent conditions, and a protein functionally equivalent to the protein having the above-described amino acid sequence, It can be mentioned as a mutant of FGF23.

Although the number of amino acids to be mutated is not particularly limited, it is generally considered to be within 30 amino acids, preferably within 15 amino acids, and more preferably within 5 amino acids (for example, within 3 amino acids). The mutated amino acid residue is preferably mutated to another amino acid whose amino acid side chain property is conserved. For example, the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G). , H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, T, Y), a sulfur atom-containing side Chain-containing amino acids (C, M), carboxylic acid- and amide-containing side chains (D, N, E, Q), base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) can be mentioned (all in parentheses represent one letter of the amino acid). It is already known that a polypeptide having an amino acid sequence modified by deleting, adding and/or substituting one or more amino acid residues for an amino acid sequence retains its biological activity. ing.

"Functionally equivalent" means that the target protein has a biological function or biochemical function equivalent to that of FGF23. FGF23 interacts with a complex containing Klotho and FGFR to express a biological function or a biochemical function. Examples of biological and biochemical functions of FGF23 include homeostasis of phosphorus and calcium and homeostasis of vitamin D metabolism.

Methods well known to those skilled in the art for preparing a DNA that encodes a “functionally equivalent protein” to a target protein include methods utilizing hybridization technology and polymerase chain reaction (PCR) technology. To be That is, for those skilled in the art, it is common practice to isolate a DNA having a high homology with FGF23 by using the nucleotide sequence of FGF23 or a part thereof as a probe and an oligonucleotide that specifically hybridizes to FGF23 as a primer. Is to do. Thus, the DNA encoding a protein having the same function as FGF23, which can be isolated by the hybridization technique or the PCR technique, is also included.

To isolate such DNA, the hybridization reaction is preferably performed under stringent conditions. In the present invention, stringent hybridization conditions refer to 6M urea, 0.4% SDS, 0.5xSSC conditions or stringency hybridization equivalent thereto. By using conditions with higher stringency, for example, 6 M urea, 0.4% SDS, 0.1xSSC, it is possible to expect isolation of DNA with higher homology. The DNA thus isolated is considered to have high homology with the amino acid sequence of the target protein at the amino acid level. High homology means at least 50% or more, more preferably 70% or more, further preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) of the entire amino acid sequence. Refers to the identity of. The identities of amino acid sequences and nucleotide sequences are determined using the algorithm BLAST (Proc. Natl. Acad. Sci. ISA 87:2264-2268, 1990, Proc Natl Acad Sci USA 90:5873, 1993) by Carlin and Artur. it can. Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215:403, 1990). When the base sequence is analyzed using BLASTN, the parameters are, for example, score=100 and wordlength=12. When analyzing an amino acid sequence using BLASTX, the parameters are, for example, score=50 and wordlength=3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

The species of FGF23-derived organism used in the screening method is not limited to a particular species. For example, human, monkey, mouse, rat, guinea pig, pig, cow, yeast, insect and the like can be mentioned.

The state of FGF23 used in the first aspect is not particularly limited, and may be, for example, a purified state, a state expressed in cells, a state expressed in a cell extract or the like.

The purification of FGF23 can be performed by a known method. Further, examples of cells expressing FGF23 include cells expressing endogenous FGF23 and cells expressing exogenous FGF23. Examples of the cells expressing endogenous FGF23 include, but are not limited to, cells derived from animal tissues and cultured cells. The cultured cells are not particularly limited and, for example, commercially available cells can be used. There are no particular limitations on the biological species from which the cells expressing endogenous FGF23 are derived, and examples thereof include humans, monkeys, mice, rats, guinea pigs, pigs, cows, yeasts and insects. The cell expressing the exogenous FGF23 can be prepared by, for example, introducing a vector containing a DNA encoding FGF23 into the cell. The vector can be introduced into cells by a general method, for example, calcium phosphate precipitation method, electric pulse perforation method, lipofectamine method, microinjection method and the like. The cell having the exogenous FGF23 can be prepared, for example, by inserting a DNA encoding FGF23 into a chromosome by a gene transfer method utilizing homologous recombination. The biological species from which the cells into which such exogenous FGF23 is introduced are not limited to mammals, and may be any biological species for which a technique for intracellularly expressing a foreign protein has been established.

Examples of the cell extract expressing FGF23 include those obtained by adding a vector containing a DNA encoding FGF23 to the cell extract contained in the in vitro transcription/translation system. The in vitro transcription/translation system is not particularly limited, and a commercially available in vitro transcription/translation kit or the like can be used.

The “test substance” is not particularly limited and includes, for example, a single substance such as a natural compound, an organic compound, an inorganic compound, a nucleic acid, a protein, a peptide, a compound library, a nucleic acid library, a peptide library, and a gene library. Examples thereof include rally expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts. The test substance can be appropriately labeled and used as necessary. Examples of the label include a radiolabel and a fluorescent label. In addition to the above-mentioned test substances, a mixture obtained by mixing a plurality of these test substances is also included.

Moreover, in the present invention, the “contact” is performed according to the state of the FGF23. For example, when FGF23 is in a purified state, it can be performed by adding a test substance to the purified preparation. In addition, if it is expressed in cells or expressed in cell extract, by adding a test substance to the cell culture solution or the cell extract, or by directly administering to a test animal, respectively. ,It can be carried out. When the test substance is a protein, for example, a vector containing a DNA encoding the protein is introduced into cells expressing FGF23, or the vector is added to a cell extract expressing FGF23. It is also possible to do so. Further, for example, it is also possible to utilize the two-hybrid method using yeast or animal cells.

In the first embodiment, next, FGF23 contacted with the test substance is contacted with Klotho and FGFR. The Klotho and FGFR may be cells expressing Klotho and FGFR, or may be a cell extract expressing Klotho and FGFR. The contact method can be carried out depending on the states of Klotho and FGFR.

In the first aspect, the biological or biochemical action of FGFR for evaluating the biological or biochemical action of FGFR includes, for example, angiogenesis, wound healing, embryogenesis and the like. ..

The second aspect of the screening method includes contacting the test substance with FGF23, detecting binding between the test substance and FGF23, and selecting a test substance that binds with FGF23 as a candidate substance.

The method for detecting the binding between the test substance and FGF23 is not particularly limited. The binding between the test substance and FGF23 can be detected, for example, by a label (for example, a label that can be quantitatively measured, such as a radiolabel or a fluorescent label) attached to the test substance bound to FGF23. Also, a labeling agent can be bound to FGF23. The binding can also be detected by immobilizing the test substance or FGF23 on a resin or a chip. The functional change of FGF23 caused by binding of the test substance to FGF23 can also be detected as an index.
In this embodiment, next, a test substance that binds to FGF23 is selected as a candidate substance. Selected substances include substances that modulate the FGF23-Klotho signaling system.

The third aspect of the screening method is to bring a test substance into contact with a cell expressing FGF23, to measure the expression level of FGF23, and to compare the expression level of FGF23 with that when the test substance is not contacted. Selecting a test substance with a change in a candidate substance. Examples of cells expressing FGF23 include osteoblasts, T cells, dendritic cells and the like.

The expression level of FGF23 can be measured by a method known to those skilled in the art. For example, the transcription level of the gene can be measured by extracting the mRNA of the gene according to a standard method and carrying out the Northern hybridization method or the RT-PCR method using this mRNA as a template. Furthermore, it is also possible to measure the expression level of the gene using DNA array technology.

The translation level of the gene can also be measured by collecting a fraction containing FGF23 encoded by the gene according to a standard method and detecting the expression of the FGF23 by an electrophoresis method such as SDS-PAGE. Moreover, it is also possible to measure the translation level of a gene by carrying out a Western blotting method using an antibody against FGF23 and detecting the expression of FGF23. Furthermore, the translation level of the gene can be measured by detecting the expression of FGF23 by ELISA using an antibody against FGF23. The antibody described above can be used as the antibody against FGF23.

In the third aspect, a test substance having an altered expression level of the FGF23 is then selected as a candidate substance as compared with the case where the test substance is not contacted. The selected candidate substances include substances that promote or suppress the expression of FGF23, and can regulate the FGF23-Klotho signal transduction system.

A fourth aspect of the screening method is to prepare a cell or a cell extract having a DNA functionally linked to a reporter gene downstream of a promoter region of a DNA encoding FGF23, and to prepare the prepared cell or cell extract. Expression of the reporter gene in contact with the test substance, measurement of the expression level of the reporter gene in the cell or cell extract in contact with the test substance, and comparison with the case where the test substance is not contacted Selecting a test substance with a decreased or increased level as a candidate substance.

In the fourth aspect, “operably linked” means that the expression of the reporter gene is induced by binding of the transcription factor to the promoter region of the FGF23 gene, and the promoter region of the FGF23 gene and the reporter gene Are bound together. Therefore, even when the reporter gene is bound to another gene to form a fusion protein with another gene product, the expression of the fusion protein is expressed by binding the transcription factor to the promoter region of the FGF23 gene. Is induced, it is included in the meaning of "functionally linked".

The reporter gene is not particularly limited as long as its expression can be detected, and examples thereof include CAT gene, lacZ gene, luciferase gene, β-glucuronidase gene (GUS) and GFP, which are commonly used by those skilled in the art. A gene etc. can be mentioned. In addition, the reporter gene also includes a DNA encoding Arid5A.
A cell or a cell extract having a DNA in which a reporter gene is operably linked to the downstream of the promoter region of the DNA encoding FGF23 can be prepared by the method described above.

The expression level of the reporter gene can be measured by a method known to those skilled in the art depending on the type of the reporter gene used. For example, when the reporter gene is the CAT gene, the expression level of the reporter gene can be measured by detecting the acetylation of chloramphenicol by the gene product. When the reporter gene is the lacZ gene, the color development of the dye compound due to the catalytic action of the gene expression product is detected, and when the reporter gene is the luciferase gene, the fluorescence of the fluorescent compound due to the catalytic action of the gene product is detected. Of the β-glucuronidase gene (GUS) by detecting G. glucuronase (ICN) or 5-bromo-4-chloro-3-indolyl-β by the catalytic action of the gene expression product. -The expression level of the reporter gene can be measured by detecting the color development of glucuronide (X-Gluc).

When the FGF23 gene is used as a reporter, the expression level of the gene can be measured by the method described above.

In the fourth embodiment, a test substance that reduces or increases the expression level of the reporter gene is then selected as compared with the case where the test substance is not contacted. The selected substance includes a substance that promotes or suppresses the expression of FGF23, and can regulate the FGF23-Klotho signal transduction system.

The fifth aspect of the screening method includes contacting the test substance with Klotho, detecting binding of the test substance with Klotho, and selecting a test substance that binds with Klotho as a candidate substance.

The method for detecting the binding between the test substance and Klotho is not particularly limited. The binding between the test substance and Klotho can be detected by, for example, a label (for example, a label capable of quantitative measurement such as a radiolabel or a fluorescent label) attached to the test substance bound to Klotho. Also, a labeling agent can be bound to Klotho. The binding can also be detected by immobilizing the test substance or Klotho on a resin, a chip or the like. The functional change of Klotho caused by the binding of the test substance to Klotho can also be detected as an index.
In this embodiment, next, a test substance that binds to Klotho is selected as a candidate substance. Selected substances include substances that modulate the FGF23-Klotho signaling system.

The sixth aspect of the screening method comprises contacting a test substance with cells expressing Klotho, measuring the expression level of Klotho, and comparing the expression level of Klotho as compared with the case where the test substance is not contacted. Selecting a test substance with a change in a candidate substance. Examples of cells that express Klotho include B cells, renal tubular cells, parathyroid cells, and the like.

The expression level of Klotho can be measured by a method known to those skilled in the art. For example, the transcription level of the gene can be measured by extracting the mRNA of the gene according to a standard method and carrying out the Northern hybridization method or the RT-PCR method using this mRNA as a template. Furthermore, it is also possible to measure the expression level of the gene using DNA array technology.

The translation level of the gene can also be measured by collecting a fraction containing Klotho encoded by the gene according to a standard method and detecting the expression of the Klotho by an electrophoresis method such as SDS-PAGE. Moreover, it is also possible to measure the translation level of a gene by carrying out a Western blotting method using an antibody against Klotho and detecting the expression of Klotho. As the antibody against Klotho, those described above can be used.

In the sixth aspect, a test substance having a changed expression level of Klotho is then selected as a candidate substance as compared with the case where the test substance is not contacted. The selected candidate substances include substances that promote or suppress Klotho expression, and can regulate the FGF23-Klotho signal transduction system.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

(Example 1)
The spleen was excised from C57BL/6 wild-type mice (8 to 11 weeks old, male), fixed with PFA (paraform aldehyde), embedded in OCT compound, and then a thin-section sample was prepared at a thickness of 10 μm. .. The sliced sample was treated with 10% BSA (bovine serum albumin) in PBS (phosphate buffer) for 30 minutes, and then FITC-labeled anti-human Klotho antibody and Alexa Fluor 647-labeled anti-FGF23 antibody were added to the sliced sample. Let react for minutes. After washing three times with PBS, a mounting agent for fluorescent staining (Vector Shield Hardening Medium) of Vector Laboratories containing DAPI (for nuclear staining) was added and mounted, and a Laser Scanning Microscopy by Carl Zeiss Microscopy was performed. , Image recorded.
From the observed image, it was found that Alexa Fluor 647-labeled FGF23-expressing cells were present near the FITC-labeled Klotho-expressing cells. That is, it was suggested that FGF23 may act only on B cells in the marginal zone of the spleen or that there may be an intercellular interaction between FGF23-expressing cells and Klotho-expressing cells.

(Example 2)
Mouse cell line A20 expressing both Klotho and FGFR was stimulated with FGF23 at a concentration of 10 ng/mL at 37° C. for 4 hours to extract RNA. CDNA was synthesized from the extracted RNA, and quantitative PCR was performed using primers specific to Bcl-2 and Bcl-XL as genes for proliferation system and IgG for antibody production (differentiation system gene).
The results are shown in Figure 1. From FIG. 1, no significant increase in the expression of the proliferation system genes BcL-2 and Bcl-XL was observed. On the other hand, IgG gene was significantly increased by FGF23 stimulation, and an increase in antibody production was observed. The increase in antibody production is considered to be due to the differentiation of B cells into antibody-producing cells. Therefore, it was suggested that the FGF23-Klotho signal is not a proliferation signal but a differentiation signal in B cells.

(Example 3)
Osteoblast cell line MC3T3-E cells were cultured in a medium containing 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate to induce differentiation, and matured osteoblasts were cultured on the 7th day and calcified on the 14th day. Used as cells (bone cells).
After removing the differentiation-inducing reagent from the culture medium, various active vitamin D derivatives (VDRA) were added, respectively, and cultured for 24 hours. After the culture was completed, RNA was extracted using TRizol and the expression level of FGF-23 mRNA was analyzed by real-time PCR. The results are shown in Figures 2 and 3.
The VDRA used was calcitriol, maxacalcitol, paricalcitol, and falecalcitol. Since each VDRA has a different affinity for the vitamin D receptor (VDR), the concentration added was determined by the ratio of the affinity for VDR.

From FIGS. 2 and 3, it can be seen that the expression level of the FGF23 gene is increased by contacting the osteoblast cell line with the activated vitamin D derivative. It is also found that the expression level of the FGF23 gene differs depending on the type of active vitamin D derivative and the stage of cell differentiation.

INDUSTRIAL APPLICABILITY The immunomodulator of the present invention provides a pharmaceutical composition having an immune function regulating action by a mechanism different from conventional ones. In addition, the present invention can provide a method for screening a useful substance having a regulatory action on immune function.

Claims (2)

An antibody production enhancer containing FGF23 . A method for screening useful candidate substance in the regulation of immune function,
(A) Detecting the effect of the test substance on the FGF23-Klotho system when the test substance is contacted with B cells, and (b) the detected effect is on the FGF23-Klotho system when the test substance is not used. Selecting a test substance with a different effect as a candidate substance,
Including the method.

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