JP2006240990A - Klotho protein and anti-klotho protein antibody and use of them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicinal composition containing a klotho protein lowering phosphorus in blood and/or an activated vitamin D in the blood and an anti-klotho protein antibody elevating the phosphorus in the blood and activated vitamin D in the blood, and capable of being utilized for the treatment or prevention of diseases associated with phosphorus and activated vitamin D. <P>SOLUTION: This medicinal composition contains the klotho protein as an active ingredient for lowering the phosphorus in the blood and/or activated vitamin D in the blood. The medicinal composition contains the anti-klotho protein antibody as an active ingredient for elevating the phosphorus in the blood and/or activated vitamin D in the blood. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

【０１１４】
上記ハイブリドーマクローンのうち、3つのハイブリドーマクローン2C3B、3C1E、1D6Aを、平成13年12月26日付で、独立行政法人 産業技術総合研究所 特許生物寄託センター（茨城県つくば市東１丁目１番地１中央第６）にブダペスト条約に基づき国際寄託した。また、ハイブリドーマクローン2C5Lを、2003年1月6日付で、独立行政法人 産業技術総合研究所 特許生物寄託センター（茨城県つくば市東１丁目１番地１中央第６）にブダペスト条約に基づき国際寄託した。受託番号は以下の通りである。
2C3B：FERM BP-7838
3C1E：FERM BP-7839
1D6A：FERM BP-7840
2C5L：FERM BP-8268
【０１１５】
〔実施例１７〕 モノクローナル抗体の調製
17-(1) 抗FGF-23抗体を含む培養上清の調製
抗FGF-23抗体産生ハイブリドーマを10μg/mlのウシインシュリン（米国、Sigma社）、5.5μg/mlのヒトトランスフェリン（米国、Sigma社）、0.01mMのエタノールアミン（米国、Sigma社）、5ng/mlの亜セレン酸ナトリウム（米国、Sigma社）を含有するeRDF培地（日本、極東製薬社）に馴化した。抗体を調製するためのハイブリドーマの培養はスピナ−フラスコで実施した。培養液を孔径が0.2μmのフィルター（米国、Pall Gelman Laboratory社）を通すことでハイブリドーマ等の雑排物を除去し、抗体を含む培養上清を回収した。
【０１１６】
17-(2) プロテインGを用いたモノクローナル抗体の精製
抗FGF-23抗体を含む培養上清をプロテインG Sepharose４FFカラム（米国、Amersham Pharmacia Biotech社）に通して抗体をカラムに吸着させ、0.1M グリシン緩衝液（pH2.8）を用いて溶出させた。溶出画分は1 M Tris-HCl を添加してpH7.2に調整した。このように調製した抗体溶液を分画分子量10000カットの透析膜（米国、Spectrum Laboratories社）を用いてPBS(-)で透析置換し、孔径0.22μmのメンブランフィルターMILLEX-GV（米国、Millipore社）でろ過滅菌し、精製抗FGF-23抗体を得た。精製抗体の濃度は280nmの吸光度を測定し、1mg/mlを1.35ODとして算出した。
【０１１７】
17-(3) プロテインAを用いたモノクローナル抗体の精製
プロテインA担体カラム（日本、免疫生物研究所社）を用い、吸着緩衝液としてグリシン緩衝液(pH8.9)、溶出緩衝液としてクエン酸緩衝液（pH 4.0）を用いて抗FGF-23抗体を含む培養上清から抗体をアフィニティー精製した。この抗体を含む溶出画分に1M Tris-HClを添加してpH7.2付近に調整した。次に透析膜を用いてこの抗体を含む溶液をPBS(-)に置換し、さらに孔径0.22μmのメンブランフィルターでろ過滅菌し、精製抗FGF-23抗体を得た。精製抗体の濃度は280nmの吸光度を測定し、1mg/mlを1.35ODとして算出した。
【０１１８】
このようにして得られた各精製モノクローナル抗体を、産生するハイブリドーマの名前を用いて示すことにする。例えばハイブリドーマ1D6Aが産生する抗体に関しては1D6A抗体と記載することにする。
【０１１９】
〔実施例１８〕 EGR１プロモータ・ルシフェラーゼを用いたklothoタンパク質発現PEAK細胞でのアッセイ系での抗FGF-23抗体の影響
抗FGF-23抗体の中和活性能が実施例９のアッセイ系で測定できるかどうかを調べる為に、実施例１５で作製した抗FGF-23抗体（2C3B）を用いて検討した。方法は実施例９に従って行ない、FGF-23添加直前に2C3B抗体を最終濃度２．５μｇ/mlになるように添加した。その結果、2C3B抗体はFGF-23によるルシフェラーゼの上昇を有意に抑制した（図１１）。このことから、EGR１プロモータ・ルシフェラーゼを用いたklothoタンパク質発現細胞でのアッセイ系はFGF-23とklothoタンパク質のシグナル伝達を抑制するような物質の探索に有効であることが示された。
【０１２０】
〔実施例１９〕 FGF-23と可溶化型klothoタンパク質によるCHOras clone1細胞のERKリン酸化亢進のアッセイ系への抗FGF-23抗体の影響
抗FGF-23抗体の中和活性能が実施例8のアッセイ系で測定できるかどうかを調べる為に、実施例１５で作製した抗FGF-23抗体（2C3B）を用いて検討した。
【０１２１】
方法は実施例８にしたがって行ない、FGF-23と２C３B抗体をあらかじめ混合した状態で細胞へ添加した。その結果、図１２に示すように2C3B抗体濃度依存的にFGF-23と可溶化型klothoタンパク質によるERKリン酸化亢進を抑制した。このことから、FGF-23とklothoタンパク質を用いたERKリン酸化亢進検出のアッセイ系はFGF-23とklothoタンパク質シグナル伝達を抑制するような物質の探索に有効であることが示された。
【０１２２】
〔実施例２０〕 抗FGF-23抗体"2C3B抗体"のマウス投与における効果
抗ヒトFGF-23モノクローナル抗体の正常マウスへの影響を検討するため、以下の実験を実施した。正常マウス（BALB/c、雄、12週齢）を4匹ずつからなる５つの群に無作為に分別し、図１３に示すように、第1群には媒体としてPBSを、第2には0.67mg/mlの抗ヒトFGF-23モノクローナル抗体（2C3B）を、また第３群には対照として0.67mg/mlの抗TPOモノクローナル抗体を、それぞれ0.15mlずつ尾静脈内に単回投与した。投与から24時間後に、エーテル麻酔下、心採血を実施し、マイクロテイナ(米国 ベクトンディッキンソン社)を用いて血清を分離した。得られた血清中のリン酸濃度をリン-テストワコー(日本国 和光純薬社)、血清1,25D濃度を1,25(OH)2D RIAキットTFB(米国 テイエフビー社)をそれぞれ用いて添付の文書に従い測定した。投与から採血までの間、各群毎にプラスチックケージで飼育し、リンおよびカルシウムを1%ずつ含む固形食CE-2(日本国 日本クレア社)および水道水を自由摂取させた。
【０１２３】
得られた結果を図１３に示す。測定値は各群における平均値+/-標準偏差で表されている。＊を付与した群は、student-tにより有意差検定を実施した結果、媒体（PBS）投与群および抗TPO抗体投与群の双方に対し、p<0.01を示した群を表す。
【０１２４】
【発明の効果】
実施例に示すように、klothoタンパク質の投与により血中の1,25ジヒドロキシビタミンD濃度および血清リン濃度が低下する。また、抗kolothoタンパク質抗体の投与により血中の1,25ジヒドロキシビタミンD濃度および血清リン濃度が上昇する。これらの実施例は、klothoタンパク質および抗kolothoタンパク質抗体が血中の1,25ジヒドロキシビタミンD濃度および血清リン濃度を変動させ、リンおよび／またはビタミンＤが関与する疾患の治療および／または予防に使用し得ることを示している。
【０１２５】
【配列表】

【０１２６】
【配列表フリーテキスト】
配列番号４〜２２、２８〜３０、３２〜３５：合成DNA
配列番号２４〜２７、３１：合成ペプチド
【図面の簡単な説明】
【図１】 ヒトFGF23組換え体を単回投与してから１時間後、８時間後でのマウス腎臓におけるEGR1遺伝子の発現変化をRT-PCR法で解析した結果を示す図である。
【図２】 ヒトFGF23組換え体を単回投与してから５分後、１０分後でのマウス腎臓におけるERKタンパク質のリン酸化変化をウェスタンブロッティング法で解析した結果を示す図である。
【図３】 全長klothoタンパク質発現PEAK細胞にヒトFGF23組換え体を添加して１０分後のERKタンパク質のリン酸化変化をウェスタンブロッティング法で解析した結果を示す図である。
【図４】 EGR1プロモータの下流にルシフェラーゼを結合したレポーター遺伝子をklotho発現プラスミド共にトランスフェクションしたPEAK細胞でのヒトFGF23刺激時のルシフェラーゼ活性変化を調べた図である。
【図５】 PEAK細胞に可溶化マウスklothoタンパク質を安定発現するCHOras clone1細胞の培養上清とヒトFGF23を添加したときのPEAK細胞でのERKのリン酸化亢進を調べた図である。
【図６】 可溶化マウスklothoタンパク質一過性発現PEAK rapid細胞上清サンプルおよびその対照としてPEAK rapid細胞の培養上清サンプルをmklotho-97抗体、mklotho-118抗体、mklotho-299抗体およびmklotho-941抗体を用いてウエスタンブロット法により解析した結果である。
【図７】 可溶化マウスklothoタンパク質一過性発現PEAK rapid細胞上清サンプル、その対照としてPEAK rapid細胞の培養上清サンプル、およびそれらをFGF-23固定化レジンに供した時のFGF-23結合物サンプルを、mklotho-118抗体またはmklotho-941抗体を用いてウエスタンブロット法により解析した結果である。一方、図YY2Bは同様なサンプルを、銀染色法により染色した結果である。
【図８Ａ】 細胞移植後14日目における可溶化型klothoタンパク質発現細胞移植群（klotho, n=5）および対照CHO細胞移植群（control, n=5）の1,25ジヒドロキシビタミンD(1,25D)濃度を示したグラフである。結果は平均値±標準誤差として表されており、対照群との有意差検定をStudent's tにより実施した。
【図８Ｂ】 細胞移植後27日目における可溶化型klothoタンパク質発現細胞移植群（klotho, n=6）および対照CHO細胞移植群（control, n = 6）の血清リン濃度ならびに血清カルシウム濃度を示したグラフである。結果は平均値±標準誤差として表されており、対照群との有意差検定をStudent's tにより実施した。
【図９】 抗klothoタンパク質抗体（p118）群および媒体投与群の投与２４時間後の血清リン濃度、9時間後の血清活性型ビタミンD濃度を示した図である。結果は平均値±標準誤差として表されており、対照群との有意差検定をStudent's tにより実施した。
【図１０Ａ】 CHO-FGF23H細胞培養上清をSDS-ポリアクリルアミドゲル電気泳動で分離し、抗FGF-23ポリクローナル抗体でウエスタンブロッティングにより組換え体FGF-23タンパク質およびその代謝物を検出した図である。培養上清中には全長のFGF-23Hタンパク質と配列番号３５のアミノ酸配列の配列番号179番目と180番目の間で切断されたて生じたN末側断片とC末側断片が存在する。hFGF23-48抗体および hFGF23-148抗体で、全長のFGF-23Hタンパク質とN末側断片ペプチドが認識された。Ｎ末側断片は小断片化されたペプチドの存在が認められる。抗His6-tag抗体では、全長タンパク質とC末側断片が検出された。
【図１０Ｂ】 CHO-FGF23細胞培養上清から精製したFGF-23全長タンパク質とN末側断片およびC末側断片をSDS-ポリアクリルアミドゲル電気泳動にて分離し、CBB染色して検出した図である。
【図１１】 EGR1プロモータの下流にルシフェラーゼを結合したレポーター遺伝子をklotho発現プラスミド共にトランスフェクションしたPEAK細胞でのヒトFGF-23刺激時に、抗FGF-23抗体（２C3B）を添加した時のルシフェラーゼ活性変化を調べた図である。FGF-23刺激により上昇するルシフェラーゼ活性が２C3B同時添加により抑制されることが示された。
【図１２】 CHOras clone1細胞に可溶化klothoタンパク質発現CHOras clone1細胞の培養上清とFGF23を添加したときに引き起こされるERKのリン酸化亢進を、抗FGF23抗体（2C3B）が抑制するかどうかを調べた図である。FGF23と可溶化klothoタンパク質発現CHOras clone1細胞培養上清を同時添加したときのERKリン酸化亢進は、2C3B抗体濃度依存的に抑制されることが示された。
【図１３】 2C3B抗体、抗TPO抗体、および媒体を投与後、24時間目における血清中リン酸、血清中1,25-ジヒドロキシビタミンD濃度を示した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a Klotho protein that changes phosphate metabolism and vitamin D metabolism, an antibody that recognizes the Klotho protein, a complex of the Klotho protein and an FGF23 polypeptide, and uses of these substances.
[0002]
[Prior art]
Phosphorus in the living body exists in bones, cells, and extracellular fluids, and plays an important role essential for the origin of mechanical strength of bones, energy metabolism of the living body, maintenance of cell functions, and the like. Because of this importance, the serum phosphorous concentration is maintained at a substantially constant level, suggesting the existence of a control mechanism for maintaining the homeostasis of blood phosphate concentration. Phosphorus balance in the body is regulated by excretion in the kidney and absorption in the intestinal tract, but the kidney is considered to be important as a phosphorous balance organ. Specifically, regulation of phosphate transporter protein in the proximal tubule of the kidney affects the regulation of blood phosphorus concentration. The involvement of parathyroid hormone, 1α, 25-dihydroxyvitamin D3, FGF23, etc. as substances that affect the change and regulation of serum phosphate levels is currently considered, but parathyroid hormone and 1α, 25 -Dihydroxyvitamin D3 plays an important role in regulating calcium metabolism rather than phosphorus metabolism.
[0003]
FGF23 is a substance discovered as a factor that strongly reduces serum phosphorus and serum 1α, 25-dihydroxyvitamin D (WO 02/14504, and Shimada T, et al: Proc Natl Acad Sci USA, 2001 May 22; 98 (11): p6500-p6505). A substance discovered as a result of a search for a tumor-specific high-expressing gene of neoplastic osteomalacia with a decrease in serum phosphorus. By reducing the phosphate transporter of the renal proximal tubule, serum phosphate Cause a decline. It also suppresses the expression of 1α hydroxylase, a synthase of 1α, 25-dihydroxyvitamin D3 (hereinafter referred to as active vitamin D3) in the kidney, and conversely, the activity of 24 hydroxylase, a degrading enzyme of active vitamin D3. By increasing the expression, the concentration of active vitamin D3 in the blood is decreased. As described above, FGF23 has been shown to have the effect of lowering both serum phosphorus and serum 1α, 25-dihydroxyvitamin D, but it has not been clarified regarding substances and pathways that transmit the signal.
[0004]
On the other hand, klotho mice with mutant phenotypes resembling human aging symptoms have been shown to exhibit various phenotypes due to insertion mutations of other genes into the klotho locus, which are short-lived, Growth retardation, senile skin atrophy, emphysema, hearing loss, cardiac dysfunction, Menkeberg-type arteriosclerosis, hyporotational bone density decrease, ectopic calcification, infertility, glycemic control abnormality, thymic atrophy, B cell differentiation It is a disorder etc. (WO98 / 29544, Kuro-o M, et al: Nature 1997 Vol 390 p45-51). In addition, it has been shown that these phenotypes are restored by introducing a full-length klotho gene into a klotho mouse using a viral vector (WO98 / 29544, Kuro-o M, et al: Nature 1997 Vol 390 p45-51). ). In addition, the extracellular region of klotho is composed of two regions, KL1 and KL2. By administering a protein fused with KL1 region and immunoglobulin to klotho mice, thymus, spleen, testis atrophy and dystrophy. It has been shown that calcification is suppressed (WO00 / 27885). In addition to the various symptoms of klotho mice listed above, it has been reported that klotho mutant mice have increased serum phosphate levels and increased active vitamin D (Yoshida T, et al : Endocrinology. 2002 Feb; 143 (2): 683-9.) In addition, the expression of klotho protein in renal failure in which phosphorus and vitamin D homeostasis are abnormal is compared to that in normal kidney. (Koh H, et al: Biochem Biophys Res Commun. 2001 Feb 2; 280 (4): 1015-20.). Increased expression of 1α-hydroxylase is known in klotho mutant mice, and abnormal vitamin D metabolism due to abnormal klotho gene is considered, but the function of klotho protein itself is unclear. However, the molecular mechanisms involved in phosphate metabolism and vitamin D metabolism in the klotho gene abnormality have not been clarified.
[0005]
[Patent Document 1]
WO02 / 14504
[Patent Document 2]
WO98 / 29544
[Patent Document 3]
WO00 / 27885
[Non-Patent Document 1]
Shimada T, et al: Proc Natl Acad Sci USA, 2001 May 22; 98 (11): p6500-p6505
[Non-Patent Document 2]
Kuro-o M, et al: Nature 1997 Vol 390 p45-51
[Non-Patent Document 3]
Endocrinology. 2002 Feb; 143 (2): 683-9
[Non-Patent Document 4]
Koh H, et al: Biochem Biophys Res Commun. 2001 Feb 2; 280 (4): 1015-20
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a pharmaceutical composition comprising a klotho protein that lowers blood phosphorus and / or blood active vitamin D and an anti-klotho protein antibody that increases blood phosphorus and / or blood active vitamin D And neoplastic osteomalacia involving phosphorus and vitamin D, ADHR, XLH, renal osteodystrophy, dialysis osteopathy, osteoporosis, hypophosphatemia, Kuru disease, osteomalacia, tubular dysfunction, bone Reduction, hypocalcemia, hyperphosphatemia, hyperparathyroidism, ectopic calcification, pruritus, osteosclerosis, Paget's disease, hypercalcemia, hypoparathyroidism, bone pain, muscle weakness An object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of diseases such as skeletal deformity, growth disorder and hypo-1,25Demia.
[0007]
[Means for Solving the Problems]
This invention is made | formed in view of such a condition, The objective is to provide the substance which can control the blood phosphorus level and the blood active vitamin D density | concentration, and its use.
[0008]
Regardless of whether the full-length klotho protein or solubilized klotho protein is present, the present inventors have the same signal transduction (increased ERK phosphorylation and increased egr1 expression) by FGF23 in the presence of the klotho protein as in the kidney in animals. Found to be caused by. It was also revealed that FGF23 and klotho protein bind to each other. We also found that the interaction between FGF23 and klotho protein is important for the regulation of phosphorus and vitamin D metabolism. In addition, administration of antibodies recognizing klotho protein to mice successfully increased the concentration of phosphorus and active vitamin D in the blood, and transplantation of solubilized klotho protein-expressing cells into nude mice We succeeded in lowering the phosphorus concentration and active vitamin D concentration. From these results, the present invention has been completed, and by the present invention, blood phosphorus, blood vitamin D can be controlled by controlling blood phosphorus and blood vitamin D, or the related diseases can be diagnosed. I think that it is possible. Accordingly, the present invention is as follows.
[1] A pharmaceutical composition for reducing blood phosphorus concentration and / or blood active vitamin D concentration, comprising klotho protein as an active ingredient,
[2] The pharmaceutical composition of [1], wherein the klotho protein is a solubilized klotho protein,
[3] A pharmaceutical composition for increasing blood phosphorus level and / or blood active vitamin D level, comprising an anti-klotho protein antibody as an active ingredient,
[4] Neoplastic osteomalacia, ADHR, XLH, renal osteodystrophy, dialysis osteopathy, osteoporosis, hypophosphatemia, kulu disease, osteomalacia, tubular dysfunction, osteopenia, hypocalcemia , Hyperphosphatemia, hyperparathyroidism, ectopic calcification, pruritus, osteosclerosis, Paget's disease, hypercalcemia, hypoparathyroidism, bone pain, muscle weakness, skeletal deformity, growth disorder And a pharmaceutical composition according to any one of [1] to [3] for use in the treatment and / or prevention of a disease involving phosphorus and / or vitamin D selected from the group consisting of low 1,25D blood, and
[5] A method for screening a substance that changes blood phosphorus concentration and / or blood active vitamin D concentration, wherein FGF23 and klotho protein are brought into contact with each other, and an intracellular signal due to interaction between FGF23 and klotho protein is obtained. A method of screening by detecting.
Hereinafter, the present invention will be described in detail.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a klotho protein that changes blood phosphorus concentration and active vitamin D concentration, an antibody that recognizes klotho protein, a complex of FGF23 and klotho protein, and uses of these substances.
[0010]
By using the present invention, a pharmaceutical composition comprising at least one of klotho protein, an antibody recognizing klotho protein, and a complex of FGF23 and klotho protein is used for pathological conditions involving phosphorus, vitamin D, for example, neoplastic bone Softening disease, ADHR, XLH, renal osteodystrophy, dialysis osteopathy, osteoporosis, hypophosphatemia, kulu disease, osteomalacia, tubular dysfunction, osteopenia, hypocalcemia, hyperphosphatemia , Hyperparathyroidism, ectopic calcification, pruritus, osteosclerosis, Paget's disease, hypercalcemia, hypoparathyroidism, bone pain, muscle weakness, skeletal deformity, growth disorder and low 1,25D blood It can be used for the treatment or prevention of diseases such as infectious diseases.
[0011]
Solubilized klotho
The solubilized klotho cDNA can be prepared by a known method based on the human-derived klotho cDNA sequence described in SEQ ID NO: 1 (Genebank accession No. AB005142). For example, it can be prepared by preparing a cDNA library from cells or organs expressing klotho protein and performing hybridization using a part of the klotho cDNA sequence (SEQ ID NO: 1) as a probe. In addition, RNA is prepared from cells and organs expressing klotho protein, cDNA is prepared by reverse transcriptase, oligo DNA is synthesized based on the klotho cDNA sequence (SEQ ID NO: 1), and this is used as a primer. It is also possible to carry out PCR reaction and amplify and prepare klotho cDNA.
[0012]
The base sequence of the klotho cDNA obtained as described above is determined. The nucleotide sequence can be determined by a known method such as a chemical modification method of Maxam-Gilbert or a dideoxynucleotide chain termination method using M13 phage. Usually, an automatic nucleotide sequencer (eg, 373A DNA manufactured by PERKIN-ELMER) is used. Sequencing is performed using a sequencer or the like.
[0013]
SEQ ID NO: 1 illustrates the base sequence of human klotho cDNA, SEQ ID NO: 2 illustrates the amino acid sequence of human full-length klotho protein, and SEQ ID NO: 3 illustrates the amino acid sequence of solubilized human klotho protein. Alternatively, as long as it has a low activity vitamin D blood-inducing activity, one or several amino acids in the amino acid sequence may be mutated such as deletion, substitution, or addition. The homology at the amino acid sequence level of human Klotho protein and mouse Klotho protein is 85% (SEQ ID NO: 2 shows the amino acid sequence of human klotho protein, SEQ ID NO: 23 shows the amino acid sequence of mouse klotho protein), mouse klotho protein is predicted to have the same function as human klotho protein. Therefore, in the present invention, the klotho protein includes not only a human-derived klotho protein but also a mouse-derived klotho protein or a klotho protein having a homology of amino acids with human klotho protein of 80% or more, preferably 85% or more. It is. A protein comprising the amino acid sequence of klotho protein derived from these non-human animal species, as long as it has hypophosphatemia-inducing activity and / or low-activity vitamin D-semia-inducing activity, Several amino acids may have mutations such as deletion, substitution and addition. Whether the protein has hypophosphatemia-inducing activity and / or low-activity-type vitamin Demia-inducing activity can be determined by transplanting cells expressing the protein into mice with reference to the method described in Example 13 below. Alternatively, the protein may be administered to a mouse or the like, and serum phosphorus concentration or 1,25 dihydroxyvitamin D concentration may be measured.
[0014]
For example, one or several, preferably 1 to 10, more preferably 1 to 5 amino acids may be deleted from the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 23. One or several amino acids, preferably 1 to 10, more preferably 1 to 5 amino acids may be added to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 23. Alternatively, SEQ ID NO: 2, One or several, preferably 1 to 10, more preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 3 or 23 may be substituted with other amino acids.
[0015]
In addition, as a substitution method, conservative substitution within a family that retains some characteristics of amino acids may be performed. The families generally classified from the side chain characteristics of amino acid side chains include the following.
(1) Acidic amino acid family: Aspartic acid, glutamic acid
(2) Basic amino acid family: lysine, arginine, histidine
(3) Nonpolar amino acid family: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
(4) Uncharged polar amino acid family: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine
(5) Aliphatic hydroxy amino acid family: serine, threonine
(6) Amide-containing amino acid family: asparagine, glutamine
(7) Aliphatic amino acids: alanine, valine, leucine, isoleucine
(8) Aromatic amino acid family: phenylalanine, tryptophan, tyrosine
(9) Hydrophobic amino acid family: leucine, isoleucine, valine
(10) Small amino acid family: alanine, serine, threonine, methionine, glycine
[0016]
In the present invention, in order to introduce a mutation into at least a part of the amino acid sequence of the solubilized klotho protein of the present invention, a technique of introducing a mutation into the base sequence of DNA encoding the amino acid is employed.
[0017]
Mutation can be introduced into DNA by a known method such as Kunkel method or Gapped duplex method, or a method analogous thereto. For example, a mutation is introduced based on a site-directed mutagenesis method using a mutant oligonucleotide as a primer, but a mutation introduction kit (for example, Mutan-K (TAKARA), Mutan-G (TAKARA), Mutations can also be introduced using TAKARA's LA PCR in vitro Mutagenesis series kit).
[0018]
Furthermore, it hybridizes with the probe prepared from the above DNA of the present invention (SEQ ID NO: 1) under stringent conditions and has hypophosphatemia-inducing activity and / or low-activity vitamin D-semia-inducing activity. A DNA encoding a protein is also included in the DNA of the present invention. Such a DNA sequence has a homology of 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more, with the sequence of the DNA represented by SEQ ID NO: 1. As described above, these DNA sequences include klotho proteins derived from animal species other than human, such as mouse klotho protein. The probe means one having a complementary sequence with respect to the full length of the sequence shown in SEQ ID NO: 1, or one having a complementary sequence to a continuous sequence (partial sequence) having a length of 17 bases or more.
[0019]
Here, the stringent condition means that the sodium concentration is 750 mM or higher, preferably 900 mM or higher, and the temperature is 40 ° C. or higher, preferably 42 ° C. Specifically, the conditions are 6 × SSC, 5 × Denhardt, 0.5% SDS, 50% Formamide, 42 ° C. 6 × SSC means 900 mM NaCl and 90 mM sodium citrate. Denhardt's solution (Denhardt) is a solution containing BSA (bovine serum albumin), polyvinylpyrrolidone and Ficoll400, and 50 × Denhardt is composed of 1% BSA, 1% polyvinylpyrrolidone and 1% Ficoll 400 (5 × Denhardt Means 50 times Denhardt one-tenth the concentration).
[0020]
Whether or not the protein encoded by the DNA of the present invention has hypophosphatemia-inducing activity and / or low-activity vitamin Demia-inducing activity can be determined by referring to the method described in Example 13 below. The expressed cells may be transplanted into mice, or the same protein may be administered to mice and the like, and the serum phosphorus concentration or 1,25 dihydroxyvitamin D concentration may be measured.
[0021]
Once the base sequence of the DNA of the present invention is determined, then the DNA of the present invention can be obtained by chemical synthesis or by PCR using primers synthesized from the determined base sequence.
[0022]
Production of recombinant vector and transformant containing DNA of the present invention
(1) Production of recombinant vector
The recombinant vector of the present invention can be obtained by ligating (inserting) the DNA of the present invention into an appropriate vector. The vector for inserting the DNA of the present invention is not particularly limited as long as it can replicate in the host, and examples thereof include plasmid DNA, phage DNA, and the like.
[0023]
Examples of plasmid DNA include plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), and plasmids derived from yeast (eg, YEp13, YEp24, YCp50, etc.). Examples of the phage DNA include λ phage. In addition, animal viruses such as retroviruses, adenoviruses or vaccinia viruses, or insect virus vectors such as baculoviruses can also be used. Furthermore, a fusion plasmid in which GST, His-tag, etc. are linked can also be used.
[0024]
In order to insert the DNA of the present invention into a vector, first, the purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and linked to the vector. Adopted.
[0025]
The DNA of the present invention needs to be incorporated into a vector so that the function of the DNA is exhibited. Therefore, in addition to a promoter and the DNA of the present invention, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence) and the like can be linked to the vector of the present invention. it can. Examples of the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.
[0026]
(2) Production of transformants
The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, the host is not particularly limited as long as it can express the DNA of the present invention. For example, Escherichia coli ( Escherichia coli ) Escherichia, such as Bacillus subtilis ( Bacillus subtilis Bacillus genus, Pseudomonas petitida ( Pseudomonas putida Bacteria belonging to the genus Pseudomonas, or Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) And the like. Animal cells such as COS cells, CHO cells, HEK293 cells, and insect cells such as Sf9 and Sf21 can also be used.
[0027]
When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention can autonomously replicate in the bacterium, and at the same time, is composed of a promoter, a ribosome binding sequence, the DNA of the present invention, and a transcription termination sequence. Is preferred. Moreover, the gene which controls a promoter may be contained. Examples of Escherichia coli include Escherichia coli ( Escherichia coli ) JM109, HB101, etc., and Bacillus subtilis, for example, Bacillus subtilis ( Bacillus subtilis ) And the like. Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example, T7 promoter derived from Escherichia coli or phage, such as trp promoter, lac promoter, PL promoter, PR promoter, etc. is used. An artificially designed and modified promoter such as a tac promoter may be used. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.
[0028]
When yeast is used as a host, for example, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Schizosaccharomyces pombe ( Schizosaccharomyces pombe ), Pichia Pastoris ( Pichia pastoris ) Etc. are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast. For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter, etc. Is mentioned. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.
[0029]
When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3 cells, human FL, HEK293, HeLa or Jurkat cells are used. As the promoter, SRα promoter, SV40 promoter, LTR promoter, β-actin promoter and the like may be used, and human cytomegalovirus early gene promoter and the like may be used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.
[0030]
When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used. As a method for introducing a recombinant vector into insect cells, for example, calcium phosphate method, lipofection method, electroporation method and the like are used.
[0031]
Klotho protein of the present invention
The klotho protein of the present invention is a protein having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 23, or has a homology of 80% or more, preferably 85% or more with these proteins. It is a klotho protein derived from animal species other than humans and mice, and has the above-mentioned hypophosphatemia-inducing activity and / or low-activity vitamin D-inducing activity. Furthermore, in the klotho protein of the present invention, one or several amino acids in the aforementioned amino acid sequence are substituted or missing as long as it has hypophosphatemia-inducing activity and / or low-activity vitamin Demia-inducing activity. Also included are proteins having lost and / or modified amino acid sequences. Further, such substitutions, deletions, modifications and additions may be in combinations.
[0032]
The klotho protein having hypophosphatemia-inducing activity and / or low-activity vitamin D-semia-inducing activity of the present invention has the nucleotide sequence shown in SEQ ID NO: 1 or the nucleotide numbers 1 to 2934 of the nucleotide sequence shown in SEQ ID NO: 1. Suitable host cell in a form capable of expressing a sequence containing a number, or a sequence of DNA encoding a klotho protein of another animal species or a sequence corresponding to base numbers 1 to 2934 of the base sequence of human klotho DNA It can be produced by preparing a transformed cell by introducing into the cell and expressing the DNA introduced into the transformed cell. In addition, the protein produced in this way may be subject to alteration of the polypeptide chain such as cleavage and glycosylation by the protein modification mechanism possessed by the host.
[0033]
The klotho protein of the present invention can be obtained by culturing the transformant and collecting it from the culture. “Culture” means any of cultured cells, cultured cells, or disrupted cells or cells in addition to the culture supernatant.
The method for culturing the transformant of the present invention is carried out according to a usual method used for culturing a host.
[0034]
A medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms, and efficiently cultures the transformants. As long as the medium can be used, either a natural medium or a synthetic medium may be used. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like. Examples of the inorganic substance include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
[0035]
The culture is usually performed at 37 ° C. for 4 to 48 hours under aerobic conditions such as shaking culture or aeration and agitation culture. During the culture period, the pH is maintained at 6.0 to 8.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.
[0036]
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector having a T7 promoter inducible with isopropyl-β-D-thiogalactopyranoside (IPTG), IPTG or the like can be added to the medium. Further, when culturing a microorganism transformed with an expression vector using a trp promoter inducible with indoleacrylic acid (IAA), IAA or the like can be added to the medium.
[0037]
Examples of a medium for culturing a transformant obtained using animal cells as a host include generally used RPMI1640 medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to these mediums. Culture is usually 5% CO ₂ Perform in the presence at 37 ° C. for 1-10 days. During culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary. When the klotho protein of the present invention is produced in cells or cells after culturing, the target polypeptide is obtained by crushing the cells or cells by sonication, repeated freeze-thawing, homogenizer treatment, etc. Collect. When the klotho protein of the present invention is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, From the above, the polypeptide of the present invention can be isolated and purified.
[0038]
The in vivo activity of the klotho protein of the present invention was evaluated by experiments in which the above-described recombinant cells expressing the klotho protein of the present invention were transplanted subcutaneously into nude mice.
[0039]
The cells transplanted in this study grow and form tumors under the skin of nude mice. Accordingly, the polypeptide of the present invention produced and secreted by cells is characterized by being released into the body fluid of mice. In this experiment, as shown in FIG. 8 (Example 13), mice transplanted with cells expressing the klotho protein of the present invention were transplanted with control CHO cells into which the DNA of the present invention had not been introduced, and tumors were transplanted. Obvious hypophosphatemia was exhibited as compared to individuals that formed or that did not form tumors. This revealed that the klotho protein of the present invention has hypophosphatemia-inducing activity.
[0040]
In the present invention, the protein can also be modified. For example, polyethylene glycol, dextran, poly (N-vinyl-pyrrolidone), polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, polyvinyl alcohol and the like are appropriately selected and used. As the modification method, any known method can be adopted, and for example, it is disclosed in detail in JP-T-10-510980.
[0041]
Anti-klotho protein antibody
The anti-klotho protein antibody in the present invention is an antibody or a part of an antibody having reactivity to the klotho protein or a part thereof as defined above. The antibody of the present invention includes a heavy chain and / or a light chain having an amino acid sequence in which one or several amino acids are deleted, substituted or added in each amino acid sequence of the heavy chain and / or light chain constituting the antibody. Monoclonal antibodies consisting of chains and capable of binding to the klotho protein are also included. In the amino acid sequence of the anti-klotho protein antibody of the present invention, the partial modification (deletion, substitution, insertion, addition) of the amino acid as described above is performed by partially modifying the base sequence encoding the amino acid sequence. Can be introduced. These modification techniques are well known to those skilled in the art, and commercially available mutagenesis kits and the like can be used.
[0042]
(1) Antibodies that specifically recognize and bind to the partial structure of klotho protein
In order to obtain antibodies useful for detecting klotho protein and regulating biological activity, it is effective to obtain antibodies that recognize structural features of klotho protein, antibodies that have high affinity, antibodies that can neutralize biological activity, etc. is there. Since the structure and antigenicity of the Klotho protein are unknown, a plurality of peptides corresponding to the partial sequence of the klotho protein were synthesized, and antibodies against each peptide were obtained. Using these, the klotho protein secreted by the expression cells was detected by Western blotting, and it was revealed that the mature klotho protein was expressed in the expression cells and in the culture supernatant. In addition, the antibodies obtained showed various binding specificities for mature klotho protein and peptides generated by its cleavage.
[0043]
(2) Antibody production
The antibody of the present invention can be produced, for example, by the following production method. That is, for example, a klotho protein or a part thereof as defined above, or a conjugate with an appropriate substance (for example, bovine serum albumin) for enhancing the antigenicity of an antigen, if necessary, an immunostimulator (Freund's Adjuvant etc.) and immunize non-human mammals including human antibody-producing transgenic mice. Alternatively, immunization can be performed by administering an expression vector incorporating a DNA encoding klotho protein. Polyclonal antibodies can be obtained from serum obtained from immunized animals. Monoclonal antibodies are prepared from antibody-producing cells obtained from immunized animals and myeloma cells that do not have autoantibody-producing ability (myeloma cells). The hybridomas are cloned and specific for the antigen used for immunization. It is produced by selecting clones that produce monoclonal antibodies that exhibit specific affinity.
[0044]
More specifically, it can be produced as follows. A hybridoma secreting a monoclonal antibody can be prepared according to the method of Kohler and Milstein et al. (Nature, 1975 Vol. 256: 495-497) and the like. That is, antibody-producing cells contained in the spleen, lymph node, bone marrow, tonsil, etc., preferably lymph node or spleen obtained from the immunized animal as described above, preferably mouse, rat, guinea pig, hamster, rabbit Alternatively, it is prepared by cell fusion with a myeloma cell having no autoantibody production ability derived from a mammal such as a human. Hybridoma clones that produce monoclonal antibodies are screened by culturing the hybridomas in, for example, a microtiter plate, and the reactivity of the culture supernatant in the wells in which proliferation has been observed with the immunoantigen, for example, enzyme immunoassay methods such as ELISA. This can be done by measuring.
[0045]
A monoclonal antibody can be produced from a hybridoma by culturing the hybridoma in vitro and isolating it from the culture supernatant. It can also be cultured in vivo in ascites of mice, rats, guinea pigs, hamsters or rabbits and isolated from ascites.
[0046]
In addition, a gene encoding a human monoclonal antibody is cloned from an antibody-producing cell such as a hybridoma and incorporated into an appropriate vector, which is then used as a host (eg, mammalian cell line, E. coli, yeast cell, insect cell, plant cell, etc.). Recombinant antibodies introduced and produced using genetic recombination techniques can be prepared (PJDelves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY, P. Shepherd and C. Dean., Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS, JWGoding., Monoclonal Antibodies: principles and practice., 1993 ACADEMIC PRESS). Furthermore, a transgenic bovine, goat, sheep or pig in which the gene of the target antibody is incorporated into an endogenous gene is produced using a transgenic animal production technique, and the antibody gene is derived from the milk of the transgenic animal. It is also possible to obtain a large amount of monoclonal antibodies. When hybridomas are cultured in vitro, the hybridomas are grown, maintained and stored according to various characteristics such as the characteristics of the cell type to be cultured, the purpose of the test study and the culture method, and monoclonal antibodies are produced in the culture supernatant. It is possible to carry out using any nutrient medium that is derived from a known nutrient medium or a known basal medium, such as that used for the preparation.
[0047]
As shown in Example 10, the polyclonal antibody of the present invention was prepared by binding a chemically synthesized partial peptide of klotho protein and bovine thyroglobulin, which is a carrier protein, and immunizing a rabbit. The antibody against each peptide induced by immunization was purified by an affinity column on which the peptide used for immunization was immobilized. The characteristics of the antibody thus obtained were examined by Western blotting and the reactivity with the klotho protein was clarified.
[0048]
Complex of FGF23 and klotho protein
The complex of FGF23 and klotho protein referred to in the present invention can be produced by mixing an FGF23 molecule (described in WO02 / 14504) and a klotho protein as defined above. The FGF23 molecule in the present invention can be produced by appropriately using a known method known in the technical field such as a chemical synthesis method, a cell culture method and the like, or a modification method thereof, as well as a genetic recombination technique. For example, FGF23 is the method of Shimada et al. {Endocrinology 143 , 3179 (2002)}. Similarly, the klotho protein in the present invention can also be produced by appropriately using a known method known in the technical field such as a chemical synthesis method, a cell culture method, etc., or a modification method thereof, in addition to the gene recombination technique. it can.
[0049]
The composite of the present invention can also be produced, for example, by the following production method. That is, it can be prepared by mixing the above-mentioned FGF23 and klotho protein and allowing to stand at 4 ° C. to room temperature for several minutes to several hours. In order to further strengthen the binding of the complex, it is possible to use a crosslinking agent such as Disuccinimidyl suberate and Bis (sulfosuccinimidyl) suberate. In addition to gene recombination technology, a gene binding to FGF23 and klotho protein can be produced by appropriately using known methods known in the technical field such as chemical synthesis methods, cell culture methods, etc., or modification methods thereof. Can do. It can also be produced by purifying the FGF23-klotho protein complex present in vivo using anti-FGF23 antibody-binding beads or anti-klotho protein antibody-binding beads.
[0050]
Pharmaceutical composition comprising the klotho protein of the present invention
The klotho protein of the present invention can be used as a pharmaceutical composition for diseases in which an increase in blood phosphate concentration and / or an increase in blood active vitamin D concentration is undesirable. Since the klotho protein or the pharmaceutical composition containing the klotho protein as in Example 13 has an action of changing blood phosphorus and blood active vitamin D concentration, blood phosphate concentration and blood active vitamin are used. Diseases that cause changes in D concentration, such as hyperphosphatemia, renal osteodystrophy, dialysis osteopathy, osteoporosis, tubular dysfunction, osteopenia, hypocalcemia, hyperparathyroidism, It can be used for the treatment of ectopic calcification, pruritus, osteosclerosis, Paget's disease, hypercalcemia, hypoparathyroidism, bone pain, muscle weakness, skeletal deformity, and growth disorder.
[0051]
Pharmaceutical composition comprising anti-klotho protein antibody of the present invention
As in Example 14, the antibody of the present invention has the effect of increasing blood phosphate concentration and / or blood active vitamin D concentration. Therefore, it can be used as a pharmaceutical composition for diseases considered to be affected by changes in blood phosphorus and / or blood active vitamin D levels, such as neoplastic osteomalacia, ADHR, XLH, etc. It can be expected to improve hypophosphatemia, bone mineralization failure, bone pain, muscle weakness, skeletal deformation, growth disorder, and low 1,25D blood disease commonly observed in these diseases. FGF-23 plays an important role under physiological conditions, and the antibody of the present invention controls FGF-23's phosphorus metabolism-regulating action and calcium metabolism-regulating action through vitamin D metabolism regulated by FGF-23. Depending on mineral metabolism and vitamins such as osteoporosis, Kur disease, hypercalcemia, hypocalcemia, ectopic calcification, osteosclerosis, Paget's disease, hyperparathyroidism, hypoparathyroidism, pruritus It can be used as a pharmaceutical composition therapeutically and prophylactically for diseases caused by abnormalities in D metabolism.
[0052]
Pharmaceutical composition comprising a complex of FGF23 and klotho protein of the present invention
FGF23 and solubilized klotho protein bind to each other (Example 12), and addition of FGF23 only in the presence of klotho protein causes an intracellular signal (increased ERK phosphorylation, increased EGR1 expression) (Examples 6, 7, 8) In addition, blood phosphorus and / or blood active vitamin D concentration decreases in transplantation experiments with solubilized klotho protein-expressing cells (Example 13). It is also possible that the concentration of active vitamin D in the blood is changed. Therefore, the complex of the Klotho protein of the present invention and FGF23 can be used as a pharmaceutical composition for diseases in which an increase in blood phosphate concentration and / or an increase in blood active vitamin D concentration is undesirable. That is, diseases that cause changes in blood phosphate concentration and blood active vitamin D concentration, such as hyperphosphatemia, renal osteodystrophy, dialysis osteopathy, osteoporosis, tubular dysfunction, osteopenia , Hypocalcemia, hyperparathyroidism, ectopic calcification, pruritus, osteosclerosis, Paget's disease, hypercalcemia, hypoparathyroidism, bone pain, muscle weakness, skeletal deformity, growth disorder, etc. It can be used for treatment.
[0053]
Use of the pharmaceutical composition of the present invention
The pharmaceutical composition comprising as an active ingredient the klotho protein, anti-klotho protein antibody, and complex of klotho protein and FGF23 of the present invention may contain a pharmaceutically acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, Gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, etc. It is done. The additive to be used is selected appropriately or in combination from the above according to the dosage form of the present invention.
[0054]
The prophylactic or therapeutic agent containing the klotho protein, anti-klotho protein antibody, complex of klotho protein and FGF23 of the present invention can be administered orally or parenterally.
Instead of administering a complex of Klotho protein and FGF23, it is also effective to administer each protein simultaneously.
[0055]
When administered orally, it may be a solid preparation such as a tablet, granule, powder, pill or the like, or a liquid preparation such as a liquid or syrup. In particular, granules and powders can be made into unit dosage forms as capsules, and in the case of liquid preparations, they may be dried products that are redissolved when used.
[0056]
Among these dosage forms, oral solid preparations usually contain additives such as binders, excipients, lubricants, disintegrants, wetting agents and the like that are commonly used in pharmaceutical compositions. Oral liquid preparations usually contain additives such as stabilizers, buffering agents, corrigents, preservatives, fragrances, coloring agents and the like that are generally used in the formulation.
When administered parenterally, it can be an injection, a suppository or the like.
[0057]
In the case of injection, it may be a powder which is usually provided in the form of a unit dose ampoule or a multi-dose container and redissolved in a suitable carrier such as sterile pyrogen-free water when used. These dosage forms usually contain additives such as emulsifiers and suspending agents that are commonly used in pharmaceutical compositions. Examples of the injection technique include intravenous drip injection, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intradermal injection. Further, the dose varies depending on the age of administration subject, administration route, and number of administrations, and can be varied over a wide range.
[0058]
In this case, the effective amount administered as a combination of an effective amount of the complex of klotho protein, anti-klotho protein antibody, klotho protein and FGF23 of the present invention and a suitable diluent and a pharmaceutically usable carrier is: In the case of klotho protein / complex, it is 0.01 to 100 μg, preferably 0.5 to 20 μg, per kg of body weight. In the case of an antibody, the dose is 0.1 μg to 2 mg, preferably 1 to 500 μg, per kg of body weight at a time.
[0059]
Screening system
Abnormal blood phosphorus levels and / or active vitamin D levels can cause various diseases, such as hyperphosphatemia, renal osteodystrophy, dialysis osteopathy, osteoporosis, hypophosphatemia, kulu disease, bone softening , Tubular dysfunction, osteopenia, hypocalcemia, hyperparathyroidism, ectopic calcification, pruritus, osteosclerosis, Paget's disease, hypercalcemia, hypoparathyroidism, bone pain , Muscle weakness, skeletal deformity, growth disorders and low 1,25D blood, etc., and substances that regulate blood phosphorus and / or blood active vitamin D levels are It is considered useful for treatment. The system utilizing the interaction between FGF23 and klotho protein according to the present invention searches for substances that treat the above diseases (eg, proteins, peptides, carbohydrates, lipids, non-peptide compounds, synthetic compounds, fermentation products, biological substances). Available to: As an index for the search for such substances, a system for detecting intracellular signals due to FGF23-klotho protein interaction (for example, phosphorylation of intracellular proteins, activation of early response genes, intracellular calcium, cAMP, cGMP, etc.) Changes in intracellular signaling molecule concentration, changes in cell membrane potential, changes in intracellular pH, release of extracellular signaling molecules, changes in cell morphology, and the like.
[0060]
For example, FGF23 is allowed to act on a cell line expressing the full-length klotho protein, and by using a system that confirms that intracellular ERK phosphorylation is enhanced, the interaction between FGF23 and klotho protein is inhibited or enhanced. New substances (low molecular compounds, antibodies, etc.) can be searched. In addition, by using a system that confirms that intracellular ERK phosphorylation is enhanced by adding solubilized klotho protein and FGF23 to various cell lines, FGF23-klotho interaction (for example, blood phosphorus concentration change or It is possible to search for substances that inhibit or enhance the active vitamin D level in the blood).
[0061]
For example, FGF23 is allowed to act on a cell line expressing the full-length klotho protein, and EGR1 promoter expression is detected using the EGR1 promoter-luciferase reporter gene. By using a system or the like, it is possible to search for substances (low molecular compounds, antibodies, etc.) that inhibit or enhance the interaction between FGF23 and klotho protein. In addition, by adding solubilized klotho protein and FGF23 to various cell lines, a system that confirms that EGR1 gene expression is increased, or that the EGR1 promoter is activated can be used using the EGR1 promoter-luciferase reporter gene. It is possible to search for substances that inhibit or enhance the FGF23-klotho protein interaction (for example, changes in blood phosphorus concentration or blood active vitamin D concentration). is there.
[0062]
In addition, it is considered that gene expression changes are caused by FGF23-klotho protein interaction, vitamin D1α hydroxylase gene, vitamin D24 hydroxylase gene, and other genes that are considered to cause gene change by FGF23-klotho protein interaction. And search for substances that inhibit or enhance FGF23-klotho protein interactions (for example, changes in blood phosphorus concentration or blood active vitamin D concentration) by using reporter genes that use the promoters of those genes Is possible.
[0063]
The present invention relates to a method for lowering blood phosphorus concentration and / or blood active vitamin D concentration by administering klotho protein into the body, and blood phosphorus concentration and / or blood by administering anti-klotho protein antibody into the body. A method for increasing the concentration of moderately active vitamin D is also included. Furthermore, the present invention relates to the use of klotho protein for producing a pharmaceutical composition for lowering blood phosphorus concentration and / or blood active vitamin D concentration, and blood phosphorus concentration and / or blood active vitamin D concentration. Also included is the use of an anti-klotho protein antibody for the manufacture of a pharmaceutical composition for raising
[0064]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[Example 1] Confirmation of egr1 expression increasing action in kidney by administration of FGF23 to mice
In order to search for genes whose expression changes in the kidney upon administration of human FGF23 to mice, the search was performed using membrane arrays Atlas mouse 1.2k and Atlas mouse 1.2kII (Clontech, USA). The membrane array was performed according to the attached protocol. The samples used were mouse kidney 1 hour after administration of human FGF23 (5 μg / head), mouse kidney 8 hours after administration of human FGF23, and vehicle administration mouse kidney. The production and purification of recombinant human FGF23 was performed by Shimada et al. {Endocrinology 143 , 3179 (2002)}. Mice were performed with BALB / c (male, 6 weeks old) at each n = 2. As a result, EGR1 was found as a gene whose expression increased 1 hour after human FGF23 administration.
[0065]
In order to confirm this phenomenon, first strand cDNA was prepared using the above three types of mouse kidney total RNA, and RT-PCR was performed. First strand cDNA was synthesized using First strand cDNA synthesis kit (Invitrogen, USA) according to the attached protocol. First strand cDNA was prepared using total RNA extracted from mouse kidney 1 hour after human FGF23 administration, mouse kidney 8 hours after human FGF23 administration, and medium administration mouse kidney. Synthesized.
[0066]
Using the first strand cDNA as a template, PCR was carried out for 25 cycles using the mouse EGR1-specific primer shown below (SEQ ID NOs: 4 and 5) as a cycle consisting of 94 degrees 20 seconds and 68 degrees 1 minute.
FW primer for mouse EGR1 RT-PCR: CTTAATACCACCTACCAATCCCAGC (SEQ ID NO: 4)
RV primer for mouse EGR1 RT-PCR: GTTGAGGTGCTGAAGGAGCTGCTGA (SEQ ID NO: 5)
[0067]
As shown in FIG. 1, when these PCR products were electrophoresed on a 2% agarose gel, an increase in EGR1 expression was confirmed only in the mouse kidney 1 hour after administration of human FGF23. On the other hand, when the target G3PDH primer was used, the same level of expression was observed in all samples. From this result, it was confirmed that the expression of EGR1 increased in the mouse kidney 1 hour after administration of FGF23.
[0068]
[Example 2] Enhanced ERK phosphorylation in the kidney by administration of FGF23 to rats
It is known that EGR1 expression increase is caused by activation of the MAP kinase cascade. Therefore, it was examined whether ERK phosphorylation was increased in the rat kidney when human FGF23 was administered. Rats (7 weeks old, SD, male) were administered 5 μg / head of human FGF23, and the kidneys were removed after 5 and 10 minutes. Only the kidney cortex was cut with a razor. The cut rat kidney cortex was frozen in liquid nitrogen. Frozen kidney is extracted buffer (20 mM Hepes (pH 7.5), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.1% SDS, 50 mM sodium fluoride, 2 mM sodium Vanadate, 1 tab./7 ml COMPLETE (Roche, USA)) Was homogenized with a Polytron homogenizer. The mixture was allowed to stand on ice for 2 hours and then centrifuged at 1000 G for 10 minutes, and the supernatant fraction was used for the subsequent experiments. The protein concentration of the supernatant fraction was measured with a BCA kit (PIERCE, USA), and all samples were prepared with an extraction buffer to a protein concentration of 22.7 mg / ml. This sample was subjected to Western blotting using an anti-phosphorylated ERK antibody (Santa Cruz, USA). As shown in FIG. 2, it was confirmed that in rats administered with human FGF23, ERK phosphorylation was enhanced after 5 minutes and 10 minutes.
[0069]
[Example 3] Cloning of mouse klotho gene
The full-length cDNA cloning of the mouse klotho gene was performed by PCR. The following primers were synthesized based on GeneBank data (Accession No. AB005141).
Mouse klotho gene cloning FW primer:
CCCGAATTCGCAGCATGCTAGCCCGCGCCCCTCCTC (SEQ ID NO: 6)
Mouse klotho gene cloning RV1 primer:
ATTTGCGGCCGCTTACTTATAACTTCTCTGGCCTTTCTT (SEQ ID NO: 7)
[0070]
The primer of SEQ ID NO: 6 is a primer on the 5 ′ side of the mouse klotho gene, and includes a start codon and a Kozak sequence portion of 5 bases upstream thereof. In addition, an EcoRI site has been added.
[0071]
The primer of SEQ ID NO: 7 is a primer on the 3 ′ side of the mouse klotho gene, which contains a stop codon and is further added with a Not I site. Mouse kidney cDNA was used as a template. Mouse kidney cDNA was prepared according to the method of Example 1. For the PCR reaction, LA Taq with GC Buffer (Takara, Japan) was used. That is, TaKaRa La Taq polymerase 0.5 μl, 2XGC buffer 25 μl, dNTP Mix 8 μl (2.5 mM), SEQ ID NO: 6 primer (1 μM), SEQ ID NO: 7 primer (1 μM), mouse kidney cDNA 1 μl described above, and water finally The PCR reaction was performed under the following conditions. The primary denaturation was performed at 94 ° C. for 5 minutes, followed by 35 cycles of 94 ° C. for 20 seconds, 55 ° C. for 30 seconds and 72 ° C. for 4 minutes, and finally the extension reaction was performed at 72 ° C. for 7 minutes. The amplified product by PCR was confirmed by 0.8% agarose electrophoresis. As a result, a single band was confirmed around 3kbp. The band was excised from the gel and MinElute Gel extraction kit (Qiagen, Germany) was used. Purification from the gel. The purified mouse klotho cDNA fragment was cleaved with EcoRI and NotI, and a ligation reaction was performed on PERI8 vector (Edge Biosystems, USA) cleaved with EcoRI and NotI. The ligation reaction was performed using TaKaRa Ligation Kit version 2 (Takara, Japan) according to the attached protocol. In order to confirm the base sequence of the cloned mouse klotho cDNA, the following primers for mouse klotho gene sequence confirmation were synthesized based on GeneBank data (Accession No. AB005141) of the mouse klotho gene sequence.
ACTACCGCTTCTCCATATCGTG (SEQ ID NO: 8)
TCGCCTTAAGCTCCCATTGGAT (SEQ ID NO: 9)
ATGGGGTCGACGTCATTGGGTACA (SEQ ID NO: 10)
TCTTGCCTCTGGGTAACCAGAC (SEQ ID NO: 11)
CTTGCAGGCTGACTGGATAGA (SEQ ID NO: 12)
AGCAAGACTCACTGAGGATCTA (SEQ ID NO: 13)
CACGATATGGAGAAGCGGTAGT (SEQ ID NO: 14)
ATCCAATGGGAGCTTAAGGCGA (SEQ ID NO: 15)
TGTACCCAATGACGTCGACCCCAT (SEQ ID NO: 16)
GTCTGGTTACCCAGAGGCAAGA (SEQ ID NO: 17)
TCTATCCAGTCAGCCTGCAAG (SEQ ID NO: 18)
TAGATCCTCAGTGAGTCTTGCT (SEQ ID NO: 19)
[0072]
As a result of the nucleotide sequence analysis of the mouse klotho cDNA cloned using these primers, the mouse klotho gene was completely matched and the cloning of the mouse klotho gene was completed. Hereinafter, this plasmid is referred to as mklotho / pEAK8.
[0073]
[Example 4] Cloning of a gene encoding mouse solubilized klotho protein
Solubilized klotho protein was prepared using the mouse klotho gene prepared in Example 3 as a template. The following primers were synthesized for production.
RV3 primer for cloning mouse-solubilized klotho protein-encoding gene:
ATTTGCGGCCGCTTAGGTTTGAAAAAATCCACATTCGGTGCA (SEQ ID NO: 20)
[0074]
The primer of SEQ ID NO: 20 contains up to 2940 bases of the mouse klotho gene, added with a TAA stop codon, and further added with a NotI site. PCR reaction was performed using the primers of SEQ ID NO: 8 and SEQ ID NO: 20 and Pyrobest Polymerase (Takara, Japan). That is, TaKaRa Pyrobest polymerase 0.5 μl, 10 × Pyrobest Buffer 5 μl, dNTP Mix 4 μl (1.25 mM), SEQ ID NO: 8 primer 1 μM, SEQ ID NO: 20 primer 1 μM, mouse klotho cDNA 10 ng were added, and water was added so that the final volume was 50 μl. added. PCR reaction was performed as follows. Primary denaturation was performed at 94 ° C. for 5 minutes, followed by 35 cycles of 94 ° C. for 20 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 4 minutes, and finally the extension reaction was performed at 72 ° C. for 7 minutes. The amplified product by PCR was confirmed by 0.8% agarose electrophoresis. As a result, a single band was confirmed at around 2.7kbp, so the band was excised from the gel and MinElute Gel extraction kit (Qiagen, Germany) was used. And purified from the gel. The purified mouse klotho cDNA fragment was digested with KpnI and NotI, and the mouse klotho-PEAK8 vector prepared in Example 3 was ligated to the KpnI and NotI digested. Confirmation of the base sequence was performed according to Example 3 to complete the preparation of the solubilized mouse klotho protein. Hereinafter, this plasmid is referred to as soluble-mklotho / pEAK8. This solubilized mouse klotho protein encodes amino acid numbers 1 to 980. In addition, the mouse solubilized klotho cDNA obtained was cleaved with EcoRI and NotI, and an intramolecular ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) were ligated to plasmid pEAK8 (Edge Biosystems, USA). Soluble-mklotho / IRES-EGFP-pEAK8 was obtained by inserting into the EcoRI and NotI sites of the prepared vector IRES-EGFP-pEAK8.
[0075]
[Example 5] Preparation of mouse-solubilized klotho protein stably expressing cells
Using TransIT LT1 (MIRUS, USA), the soluble-mklotho / IRES-EGFP-pEAK8 plasmid was introduced into CHOras clone1 cells according to the package insert. Drug-resistant cells were selected in MEMα medium containing 5 μg / ml puromycin and 10% FBS, and cells with strong luminescence intensity of EGFP (Green Fluorescent Protein) were cloned by sorting by FACS vantage (Becton Dickinson, USA). When the cloned cells became confluent, the cells were replaced with serum-free DF (DMEM / F-12) medium, and the supernatant was collected after 2 days. The recovered supernatant was subjected to Western blotting using an anti-klotho protein antibody (ALPHA DIAGNOSTIC, USA), and a clone with a strong klotho protein band around 130 kDa was selected.
[0076]
[Example 6] Enhanced ERK phosphorylation by FGF23 stimulation by transient expression of mouse full-length klotho protein
Confirmation of ERK phosphorylation enhancement
Intracellular ERK phosphorylation when the mouse full length klotho protein was transiently expressed in PEAKrapid cells (Edge Biosystems, USA) using the mouse full length klotho protein expression plasmid prepared in Example 3 and stimulated with FGF23 The enhancement of was verified. A 12-well plate was transiently transfected with the mklotho / pEAK8 plasmid prepared in Example 3 according to the protocol attached to PEAKrapid cells. 24 hours after transfection, the medium was replaced with serum-free DMEM medium, and CO was further added for 24 hours. ₂ Incubation was performed in an incubator. After adding heparin (Sigma, USA) to 10 μg / ml, FGF23, bFGF, and medium were added. After incubating at 37 ° C. for 10 minutes, the supernatant was aspirated and washed with PBS, and then the cells were lysed with SDS-PAGE sample buffer (Daiichi Kagaku, Japan). This sample was subjected to Western blotting in the same manner as in Example 2. As shown in FIG. 3, enhancement of ERK phosphorylation by FGF23 was confirmed only in mouse full-length klotho protein-expressing PEAKrapid cells.
[0077]
[Example 7] Confirmation of increased expression of egr1 by FGF23 stimulation by transient expression of mouse full-length klotho protein
When the mouse full-length klotho protein was transiently expressed in PEAKrapid cells (Edge Biosystems, USA), the increase in the expression of EGR1 by FGF23 stimulation was verified. PEAK rapid cells were transfected in the same manner as in Example 6. 48 hours after transfection, heparin was added to 10 μg / ml, and stimulation was further applied with human FGF23 (100 ng / ml). After 30 minutes of stimulation, the cells were washed with PBS and suspended using ISOGEN (Nippon Gene, Japan). Total RNA was prepared according to the package insert, and RT-PCR was performed in the same manner as in Example 1. Similar to the results of Example 1, in the mouse full-length klotho protein-expressing PEAKrapid cells, a significant increase in the expression of EGR1 was confirmed by FGF23 100 ng / ml stimulation.
[0078]
[Example 8] Confirmation of enhanced ERK phosphorylation in mouse solubilized klotho protein-expressing cell supernatant and CHO23 clone1 cells by addition of FGF23
8- (1) Preparation of culture supernatant of mouse-solubilized klotho protein-expressing cells
The mouse solubilized klotho protein-expressing CHOras clone1 cell prepared in Example 5 was placed in a tissue culture flask (225 cm). ² , Corning, USA). After culturing for 24 hours, the medium was replaced with serum-free MEMα medium, cultured at 37 ° C. for 5 days, and the culture supernatant was collected.
[0079]
8- (2) Confirmation of enhanced ERK phosphorylation in CHOras clone1 cells by adding culture supernatant of mouse solubilized klotho protein expressing cells and FGF23
CHOras clone1 cells and HEK293 cells were seeded in a 24-well plate (Corning, USA). After culturing for 24 hours, the mouse-solubilized klotho protein-expressing CHOras clone1 cell culture supernatant prepared in 8- (1) was substituted and further cultured for 4 hours. Subsequently, human FGF23 (100 ng / ml) was added after adding 10 μg / ml of heparin. After incubating at 37 ° C. for 10 minutes, the supernatant was aspirated and washed with PBS, and then the cells were lysed with SDS-PAGE sample buffer (Daiichi Kagaku, Japan). This sample was subjected to Western blotting in the same manner as in Example 2 using an anti-ERK antibody (Santa Cruz, USA). As shown in FIG. 5, enhancement of ERK phosphorylation by FGF23 was confirmed only in cells to which mouse-solubilized klotho protein was added.
[0080]
[Example 9] Assay on PEAK cells expressing klotho protein using EGR1 promoter luciferase
9- (1) Preparation of mouse EGR1 promoter-luciferase fusion gene
In order to investigate the increase in gene expression of mouse EGR1, a mouse EGR1 promoter / luciferase fusion gene was prepared. The mouse EGR1 promoter part was cloned by PCR. Primers were prepared as follows based on literature (Barbara, A. et al., Proc. Natl. Acad. Sci. USA Vpl. 85, pp. 7857-7861). FW primer for mouse EGR1 promoter cloning:
GGGGTACCAACAGATCCTGGCGGGGACTTAGGAC (SEQ ID NO: 21)
RV primer for mouse EGR1 promoter cloning:
CCCAAGCTTTCGCGACTCCCCGAATCGGCCTCTATT (SEQ ID NO: 22)
[0081]
The primer of SEQ ID NO: 21 is a primer starting from the -1023 base portion of the mouse EGR1 promoter portion, and a HindIII site is further added. The primer of SEQ ID NO: 22 is a primer containing the mouse EGR1 mRNA start site (+1) and has a KpnI site added thereto. Mouse genomic DNA (Clontech, USA) was used as a template. The PCR reaction was performed using LA Taq GC Buffer (Takara Shuzo, Japan) according to the attached manual. PCR reaction conditions were 94 ° C for 5 minutes with primary denaturation, followed by 30 cycles of 94 ° C for 20 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, and finally an extension reaction at 72 ° C for 7 minutes. It was. The amplified product by PCR was confirmed by electrophoresis on a 2% agarose gel. As a result, a single band was confirmed in the vicinity of about 1 kbp. The band was excised from the gel and purified with the GeneClean kit (Bio 101, USA). Then, cleavage was performed with HindIII and KpnI. Purification was performed again using the GeneClean kit (Bio 101, USA), and ligation was similarly performed on pGL3-Basic (Promega, USA) cut with HindIII and KpnI. The ligation reaction was performed using TaKaRa Ligation kid version 2 (Takara Shuzo, Japan) according to the attached protocol. The cloned mouse EGR1 promoter was confirmed in nucleotide sequence and the production of the mouse EGR1 promoter-luciferase fusion gene was completed. Hereinafter, this plasmid is referred to as mEGR1promoter-luc./pGL3Basic.
[0082]
9- (2) Increase in luciferase activity upon stimulation with human FGF23 in klotho-expressing PEAK cells using mouse EGR1 promoter luciferase reporter gene
Using the mouse EGR1 promoter / luciferase reporter gene prepared in 9- (1) above, changes in EGR1 expression upon FGF23 stimulation of EGR1 in mouse klotho-expressing PEAK cells were examined. PEK rapid cells (Edge Biosystems, USA) were transfected simultaneously with mklotho / pEAK8 prepared in Example 3 and mEGR1promoter-luc./pGL3Basic prepared in Example 9- (1) according to the attached protocol. The transfection was performed on a 10 cm diameter culture dish (Becton Dickinson, USA). Four hours after transfection, the cells were detached with 0.53 mM EDTA-containing PBS and seeded on white 96-well plates (Corning, USA). After culturing for 24 hours, human FGF23 was added at 0 to 100 ng / ml, and heparin (Sigma, USA) was also added at 10 μg / ml at the same time. Furthermore, after leaving still at 37 degreeC for 24 hours, Luciferase activity was measured according to the protocol using Steady-Glo Luciferase assay system (USA Promega). The measurement was performed using TopCount (Packard, USA). The results are shown in FIG.
[0083]
Example 10 Preparation of anti-mouse klotho protein partial peptide polyclonal antibody
10- (1) Synthesis of peptide corresponding to FGF-23 partial sequence
The hydrophobicity of the protein portion of SEQ ID NO: 23 (mouse full length klotho protein) was predicted using the calculation function of DNASIS Mac version 7.2, and a site suitable for peptide antibody production was predicted. A partial sequence that was considered to be appropriate for antibody production was extracted from the viewpoint of having a high degree of hydrophilicity and other than a site that could become a sugar chain modification or phosphorylation site. As a result, peptide mklotho-97 (SEQ ID NO: 24) in which a cysteine residue was added to the C-terminal of a peptide consisting of 16 amino acids starting from histidine at residue number 97 in SEQ ID NO: 23, 21 amino acids starting from 118th proline Peptide mklotho-118 (SEQ ID NO: 25) obtained by adding a cysteine residue to the N-terminal of a peptide consisting of residues, peptide mklotho-299 (SEQ ID NO: 26) consisting of 20 amino acid residues starting from the 299th alanine, and 941st proline A peptide mklotho-941 (SEQ ID NO: 27) consisting of 25 amino acid residues starting was selected as an antigen and chemically synthesized.
mklotho-97: HHSGAAPSDSPIVVAPC (SEQ ID NO: 24)
mklotho-118: CPPLSSTGDVASDSYNNVYRDT (SEQ ID NO: 25)
mklotho-299: ALSSHWINPRRMTDYNIREC (SEQ ID NO: 26)
mklotho-941: PSMKHYRKIIDSNGFLGSGTLGRFC (SEQ ID NO: 27)
[0084]
10- (2) Preparation of polyclonal antibody against anti-FGF-23 partial peptide
All the above peptides were conjugated to bovine thyroglobulin, which is a carrier protein, through their cysteine residues and subjected to immunization. Immunization used two rabbits against one antigenic peptide. In the first immunization, a peptide bound to 100 μg of carrier protein per bird was emulsified with Freud's complete adjuvant and administered intradermally or subcutaneously in rabbits. One week after the first immunization, a peptide bound to 100 μg of carrier protein was made into an emulsion with Freud's incomplete adjuvant and administered in the same manner. The same administration was performed 6 times at 2-week intervals, and after 1 week of the final administration, whole blood was collected to prepare antiserum.
[0085]
In order to prepare an affinity column for purifying an antibody against a partial peptide of anti-mouse klotho protein from rabbit serum, each peptide used for immunization was solid-phased on a gel using SulfoLink Kit (PIERCE, USA). Turned into. Antiserum was added to this column using PBS (-) as an adsorption buffer, and the antibody that binds to the peptide used for immunization was retained on the column. Next, the antibody bound to the column was eluted and collected using 0.1 M glycine buffer (pH 2.8) as an elution buffer. The elution fraction was adjusted to around pH 7.2 by adding 1M Tris-HCl (pH 9.0). The prepared antibody solution was applied to a gel filtration column NAP25 (Amersham Pharmacia Biotech, USA) and the buffer solution was replaced with PBS (-), and then the membrane filter MILLEX-GV (Millipore, USA) having a pore size of 0.22 μm was used. By sterilizing by filtration, antibodies against each peptide were obtained. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm and setting 1 mg / ml as 1.35 OD. Each purified antibody thus obtained will be indicated using the name of the peptide used for immunization. For example, an antibody obtained by immunization with the mklotho-97 peptide of SEQ ID NO: 24 is referred to as mklotho-97 antibody.
[0086]
10- (3) Recognition of mouse klotho protein by antibody against partial peptide of anti-mouse klotho protein
The solubilized mouse klotho protein transiently expressed PEAK rapid cell supernatant prepared in Example 8 using the mklotho-97 antibody, mklotho-118 antibody, mklotho-299 antibody and mklotho-941 antibody obtained by the method described above The solubilized mouse klotho protein was analyzed by Western blotting. As shown in FIG. 6, a solubilized mouse klotho protein protein around 130 kDa was detected with any antibody. Therefore, these four types of antibodies were found to be antibodies capable of recognizing mouse klotho.
[0087]
[Example 11] Preparation of biotin-labeled antibody
Biotin labeling was performed using a polyclonal antibody against the partial peptides of the four kinds of purified mouse klotho proteins described above. To 1 ml of antibody solution diluted to a concentration of 1 mg / ml with 50 mM sodium bicarbonate solution, 10 μl of a solution prepared by dissolving Biotin-AC5-Osu (Japan, Dojin Chemical) in dimethylformamide at a concentration of 1.82 mg / ml, Inverted at 4 ° C for 2 hours. Thereafter, this reaction solution was applied to a NAP10 column (Amersham Pharmacia Biotech, USA), unreacted Biotin-AC5-Osu was removed, the solvent was replaced with PBS (-), and four types of biotin-labeled anti-FGF23 partial peptide polyclonal Antibody was obtained.
[0088]
[Example 12] Binding experiment of FGF-23 and klotho protein
The binding of FGF-23 protein and klotho protein was confirmed by the following experiment.
12- (1) Preparation of FGF-23 immobilized resin
Production and purification of recombinant FGF-23RQH were performed according to Example 15 described below. This purified product was immobilized on NHS-activated Sepharose 4FF (Amersham Pharmacia Biotech, USA) according to a conventional method in an amount of 3.3 mg per 1 ml of resin.
[0089]
12- (2) Binding of solubilized klotho protein to FGF-23RQH immobilized resin
To 8 ml of the solubilized mouse klotho protein transiently expressed PEAK rapid cell supernatant prepared in Example 8 or the supernatant of the parental PEAK rapid cell as a control, 50 μl of the above FGF-23-immobilized resin was added. The solubilized mouse klotho protein and FGF-23 were reacted by inverting at 1 ° C. for 1 hour. Thereafter, the resin was washed 4 times with PBS to remove unreacted substances. Add 300 μl sample buffer (50 mM Tris-HCl pH 6.8, 0.4% SDS, 6% glycerol, 0.002% bromophenol blue, 2% β mercaptoethanol) to each resin, heat at 95 ° C for 5 minutes, The supernatant obtained by centrifugation was collected. These supernatants and cell supernatants were analyzed by Western blotting using the mklotho-118 antibody and mklotho-941 antibody obtained in Example 10. The result is shown in FIG. 7 shows the result of silver staining of a similar sample using 2D-silver staining reagent II “Daiichi” (Daiichi Chemicals Co., Ltd., Japan). From these results, it was shown that the solubilized mouse klotho protein bound to the FGF-23RQH-immobilized column and concentrated. Therefore, it was revealed that FGF-23 and klotho protein bind.
[0090]
[Example 13] Transplantation test of solubilized klotho protein-expressing CHO cells
13- (1) Examination of serum 1,25 dihydroxyvitamin D concentration
In order to examine the biological activity of the solubilized klotho protein recombinant, the solubilized klotho protein recombinant CHO cells obtained in Example 5 were transplanted subcutaneously into nude mice to make the solubilized klotho protein recombinant constant. High-solubilized klotho protein-expressing model mice retaining tumors derived from transplanted cells secreted into the liver were prepared. Seven-week-old male BALB / c nude mice were used as experimental animals, and cell transplantation was performed as follows. Mice were housed in plastic cages during the entire period from the arrival to dissection, and were allowed to freely ingest commercially available rodent solid food and tap water.
[0091]
1 × 10 each of CHO cells expressing solubilized klotho protein and parental CHO cells as controls ⁸ Disperse in PBS buffer to make cells / mL, and inject 0.1 mL each of these cell suspensions into the back of two nude mice (control CHO cell transplantation group n = 5, solubilized klotho protein-expressing CHO cell transplantation) Group n = 5). On day 14 after cell transplantation, blood was collected by blood sampling under ether anesthesia, and serum was prepared using a microtainer (Becton Dickinson, USA). The 1,25 dihydroxyvitamin D concentration in the obtained serum was measured using a 1,25 (OH) 2D RIA kit (TFB, USA).
As a result, the 1,25 dihydroxyvitamin D concentration in the solubilized klotho protein-expressing cell transplantation group was significantly lower than that in the control CHO cell transplantation group (FIG. 8).
[0092]
13- (2) Examination of serum phosphorus concentration
In the same manner as described above, solubilized klotho protein-expressing CHO cells and control CHO cells were subcutaneously transplanted into 5-week-old male BALB / c nude mice (control CHO cell transplant group n = 6, solubilized klotho protein expression). Serum phosphorus concentration at CHO cell transplantation group n = 6), 27th day was measured using P-test Wako (Wako Pure Chemicals, Japan).
As a result, as shown in FIG. 8, the serum phosphorus concentration in the solubilized klotho protein-expressing cell transplantation group was significantly lower than that in the control CHO cell transplantation group.
[0093]
From the above results, it was proved that the solubilized klotho protein recombinant had a physiological activity that reduces serum phosphorus concentration and 1,25 dihydroxyvitamin D concentration. This activity is similar to that of FGF-23 already reported.
[0094]
[Example 14] Phosphorus and vitamin D-elevating effect of mice administered with anti-klotho protein antibody
The anti-klotho protein antibody p118 obtained in Example 10 was administered intraperitoneally to BALB / c, male, 7-week-old normal mice at a single dose of 200 μg / 0.2 mL per mouse. To the control group, 0.2 mL of solvent (PBS) was intraperitoneally administered. 9 hours after administration, blood was collected from the orbit using a glass capillary, serum was separated using a microtainer (Becton Dickyson, USA), blood was collected from the abdominal vena cava 24 hours later, and serum was collected using the same microtainer. Separated. In the obtained serum after 9 hours, the 1,25-dihydroxyvitamin D concentration was measured using a 1,25 (OH) 2D RIA kit “TFB” (TEB, Inc., Japan). The serum concentration after 24 hours was determined using Phosphorus-Test Wako (Wako Pure Chemicals, Japan) according to the package insert. The anti-klotho protein antibody and the control group each consisted of 6 mice, and had free access to CE2 (Claire Japan, Japan) containing tap water and 1.03% inorganic phosphate and 1.18% calcium. .
As a result, 1,25-dihydroxyvitamin D 9 hours after administration of the p118 antibody and serum phosphate concentration 24 hours later were significantly increased compared to the vehicle administration group (FIG. 9).
From the above results, it was shown that the anti-klotho protein antibody can increase serum phosphorus concentration and serum active vitamin D concentration.
[0095]
[Example 15] Acquisition of FGF-23, FGF-23H, and FGF-23RQH protein proteins
15- (1) Construction of FGF-23H protein expression vector
The cDNA encoding FGF-23 is obtained by using the cDNA library of the tumor responsible for neoplastic osteomalacia as a template and using F1EcoRI primer (SEQ ID NO: 28), LHisNot primer (SEQ ID NO: 29) and LA-Taq DNA polymerase. After incubating at 1 ° C. for 1 minute, amplification was carried out by performing 35 cycles of PCR with one cycle consisting of 96 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds. The F1EcoRI primer anneals to a sequence further 5 ′ upstream of the base sequence encoding FGF-23 and adds an EcoRI restriction enzyme site to the 5 ′ side of the region encoding FGF-23 of the amplified fragment. The LHisNot primer consists of a sequence that anneals to the sequence 5 'of the stop codon of the sequence encoding FGF-23, and a stop codon that follows the sequence encoding the His6-tag sequence (His-His-His-His-His-His). Contains the NotI restriction enzyme sequence. As a result, the amplified fragment encodes the FGF-23 protein protein with a His6-tag sequence added to the C-terminus, and has a NotI restriction enzyme site downstream thereof. This amplified fragment was digested with EcoRI and NotI and ligated with pcDNA3.1Zeo (Invitrogen, USA), which is an animal cell expression vector similarly digested with EcoRI and NotI. The expression vector thus prepared was cloned, and the nucleotide sequence was determined to confirm that it encoded the FGF-23 protein protein to which the desired His6-tag sequence was added. This vector is referred to as pcDNAFGF-23H.
F1EcoRI: CCGGAATTCAGCCACTCAGAGCAGGGCACG (SEQ ID NO: 28)
LHisNot: ATAAGAATGCGGCCGCTCAATGGTGATGGTGATGATGGATGAACTTGGCGAA (SEQ ID NO: 29)
[0096]
15- (2) Construction of FGF-23 protein expression vector
Incubate at 94 ° C for 1 minute using F1EcoRI primer, LNot primer (SEQ ID NO: 30) and LA-Taq DNA polymerase using pcDNA / FGF-23H as a template, then at 94 ° C for 30 seconds, at 55 ° C for 30 seconds, 72 ° C Amplification was performed by carrying out 25 cycles of PCR, with a 1 minute step consisting of 1 minute. After completion of the reaction, the end of the PCR product is smoothed with T4 DNA polymerase (Roche, Switzerland), and then phosphorylated at the DNA end using Polynucleotide kinase (Roche, Switzerland) to encode the FGF-23 protein. A cDNA fragment was prepared. The expression vector pCAGGS (Niwa H, et al., Gene. 1991, 108: 193-199) was digested with EcoRI, the ends were blunted using Klenow fragment (Roche, Switzerland), and bovine intestinal alkaline phosphatase (Japan, Takara Shuzo). Was used for dephosphorylation. The cDNA fragment encoding FGF-23 prepared in this way and the pCAGGS vector were ligated. The expression vector thus prepared was cloned, the nucleotide sequence was determined, and it was confirmed that the sequence encoding the target FGF-23 protein was accurately inserted. This vector is referred to as pCAGGS / FGF-23.
[0097]
Further, a fragment encoding FGF-23 amplified with the above-mentioned F1EcoRI primer and LNot primer was digested with EcoRI and NotI and purified. This is applied to the EcoRI and NotI restriction enzyme sites of the pEAK8 / IRES / EGFP vector in which the intramolecular ribosome entry sequence (IRES) and enhanced green fluorescent protein (EGFP) are linked to the expression vector pEAK8 (Edge Biosystem, USA). Inserted and cloned. The nucleotide sequence of the obtained plasmid was determined and confirmed to encode FGF-23 protein. This vector is called pEAK8 / IRES / EGFP / FGF-23.
LNot: ATAAGAATGCGGCCGCTCAGATGAACTTGGCGAA (SEQ ID NO: 30)
After pCAGGS / FGF-23 was digested with EcoRI and linearized, the ends were blunted using a Klenow fragment (Roche, Switzerland). This was further digested with BamHI, and a DNA fragment containing FGF-23 cDNA was separated and purified by agarose electrophoresis. Further, after digesting the expression vector INPEP4 with BglII, the ends were blunted using a Klenow fragment (Roche, Switzerland). After further digestion with BamHI, the vector was purified by agarose electrophoresis. A fragment containing these FGF-23 cDNAs and a vector were ligated. The expression vector thus prepared was cloned, the nucleotide sequence was determined, and it was confirmed that the sequence encoding the target FGF-23 protein was accurately inserted. This vector is referred to as INPEP4 / FGF-23.
[0098]
15- (3) Construction of FGF-23RQH protein expression vector
The FGF-23 protein was found to be susceptible to protein cleavage between the 179th Arg residue and the 180th Ser residue. The N-terminal amino acid sequence of this cleavage site is Arg176-His177-Thr178-Arg179 (SEQ ID NO: 31) that matches the Arg-XX-Arg sequence that is the recognition sequence of protein converting enzyme, and this missense mutation in ADHR It is known to be a substitution mutation at the 176th or 178th Arg residue. Therefore, we show resistance to cleavage by protein converting enzyme, and have a His6-tag at the C-terminus as a model of mutant FGF-23 found in ADHR, and the 176th and 179th Arg residues are Gln residues. A vector was constructed to experimentally prepare a mutated FGF-23 protein (hereinafter referred to as FGF-23RQH) substituted for.
[0099]
As a preparation method, RQF (SEQ ID NO: 32) which is a forward primer containing a base substitution sequence for substituting Arg for Gln and RQR (SEQ ID NO: 33) which is a reverse primer were synthesized.
[0100]
Furthermore, ME1 (SEQ ID NO: 34) and HNt (SEQ ID NO: 35), which are primers for amplifying the FGF-23 sequences on the 5 ′ side and 3 ′ side of the mutation introduction site, were prepared in combination with this base substitution primer. ME1 is a forward primer containing the start codon encoded by FGF-23 cDNA and has an EcoRI restriction enzyme sequence. HNt is a codon encoding a His6-tag sequence before the stop codon encoded by FGF-23 cDNA. A reverse primer that can insert a sequence and can add a NotI restriction enzyme sequence.
RQF: ATACCACGGCAGCACACCCAGAGCGCCGAG (SEQ ID NO: 32)
RQR: CTCGGCGCTCTGGGTGTGCTGCCGTGGTAT (SEQ ID NO: 33)
ME1: ATGAATTCCACCATGTTGGGGGCCCGCCTCAGG (SEQ ID NO: 34)
HNt: ATGCGGCCGCCTAATGATGATGATGATGATGGATGAACTTGGCGAAGGG (SEQ ID NO: 35)
[0101]
PCR reaction was performed using 10 ng of pGAGGS / FGF-23 as a template and a primer (200 nM each) of a combination of RQF and HNt and a combination of ME1 and RRQ. The reaction was carried out using pfu DNA polymerase (Promega, USA) for 25 cycles with 1 cycle of 94 ° C for 30 minutes, 55 ° C for 30 seconds, and 72 ° C for 1 minute. did. Dilute the two types of reaction solutions obtained in this way 10-fold, mix 1 μl each of them into a template, and add 50 μl of the PCR reaction solution added to ME1 and HNt to a final concentration of 200 nM. After being prepared and kept at 94 ° C. for 1 minute, 25 cycles of PCR reaction were carried out, with one cycle consisting of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 1 minute. Here, LA Taq DNA polymerase (Takara Shuzo, Japan) was used. The obtained amplification product of about 800 bp was digested with EcoRI and NotI and then purified to obtain insert DNA. This is applied to the EcoRI and NotI restriction enzyme sites of the pEAK8 / IRES / EGFP vector in which the intramolecular ribosome entry sequence (IRES) and enhanced green fluorescent protein (EGFP) are linked to the expression vector pEAK8 (Edge Biosystem, USA). Inserted and cloned. The nucleotide sequence of the obtained plasmid was determined, and it was confirmed that the desired 176th and 179th Arg were converted to Gln and encoded a mutant FGF-23 protein with a His6-tag sequence added to the C-terminus. . This vector is referred to as pEAK8 / IRES / EGFP / FGF-23RQH.
[0102]
15- (4) Acquisition and purification of FGF-23H-expressing cells, FGF-23-expressing cells, and FGF-23RQH-expressing cells
(i) Acquisition of FGF-23H expressing cells
About 20 μg of pcDNAFGF-23H was cleaved at the FspI restriction enzyme site in the ampicillin resistance gene in the vector, linearized and purified. This was dissolved in 10 μl of pure water, mixed with 1 × 10 7 CHO Ras clone-1 cells (Shiragata, S., et al. Biosci Biotech Biochem, 59: 345-347, 1995) and Gene Pulser II ( The gene was introduced into the cells by electroporation using Bio Rad (USA). The cells were cultured in a MEMα medium (Gibco BRL, USA) containing 10% FCS for 24 hours, and then added with Zeocin (Invitrogen, USA) to a final concentration of 0.5 mg / ml and cultured for 1 week. Cells that adhered and proliferated were released with trypsin and cloned by limiting dilution in the presence of Zeocin at a final concentration of 0.3 mg / ml to obtain 35 types of cloned cells. Among these cells, the cells that best expressed the FGF-23H protein were identified by Western blotting as shown below. The culture supernatant of each cloned cell was collected and subjected to SDS-polyacrylamide electrophoresis, and then the protein was transferred to a PVDF membrane (Millipore, USA), and an anti-His-tag (C-terminal) antibody (USA, Invitrogen) and an ECL luminescence system (Amersham Pharmacia Biotech, USA), a signal derived from the FGF-23H protein of about 32 kDa was detected. As a result, the highest expression was found in the clone named # 20, which was named CHO-OST311, and was named as CHO-OST311, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1 1-1 Higashi 1-chome, Tsukuba, Ibaraki, Japan) Deposited at the center 6) (Accession number: FERM BP-7273). In this specification, CHO-OST311 is referred to as CHO-FGF23H.
[0103]
(ii) Acquisition of FGF-23 and FGF-23RQH expressing cells
The pEAK8 / IRES / EGFP / FGF-23 and pEAK8 / IRES / EGFP / FGF-23RQH vectors were introduced into CHO Ras clone-1 cells by a gene transfer method using membrane fusion lipid. CHO Ras clone-1 cells are cultured on a 6-well plate so that the cells cover about 60% of the bottom surface. Then, the culture solution is removed, and 1 ml of MEMα culture solution without serum is added. Mix 2.5 μg of the vector to be introduced and 10 μl of Transfectam (registered trademark) (Promega, USA) with 50 μl of MEMα culture medium without serum, mix both, and let stand for 10 minutes. And added to the prepared 6-well plate. After culturing for 2 hours, the culture medium containing this DNA was removed and replaced with a culture medium containing 10% FCS and cultured overnight. The next day, Puromycin (Sigma, USA) was added so that the final concentration was 5 μg / ml, and drug resistant cells were selected. The drug-resistant cells obtained in this way were cloned by the limiting dilution method in the same manner as the above-mentioned acquisition of FGF-23H-expressing cells. Furthermore, a cell line that best expresses the target protein was obtained by Western blotting. These cells are referred to as CHO-FGF23 and CHO-FGF23RQ, respectively.
[0104]
(iii) Purification of recombinant protein
When a recombinant in the culture supernatant of CHO-FGF23H was detected by Western blotting using an antibody against the C-terminal His6-tag sequence, a band around 32 kDa and a band around 10 kDa were observed as shown in FIG. 10A. It was. When these two bands were cut out from the gel and the amino acid sequence on the N-terminal side was determined, the amino acid sequence from the 25th position of SEQ ID NO: 36 was detected in the band with the larger molecular weight. Was thought to have been removed. On the other hand, the amino acid sequence from the 180th position of SEQ ID NO: 36 was confirmed from the band having a small molecular weight, and it was found that the fragment was generated by cleavage between the 179th and 180th positions. Moreover, the presence of a peptide considered to have a sequence up to the 179th position was also confirmed by detection using a polyclonal antibody that recognizes the N-terminal side of FGF-23.
[0105]
After centrifuging 1000 ml of culture supernatant of CHO-FGF23H cells at 16,200 g for 15 minutes at 4 ° C to remove floating cells, SP-sepharose FF packed in a 30 mm ID x 200 mm column supernatant (Registered trademark) (Amersham Pharmacia Biotech, USA), a peptide having His6-tag added to a peptide corresponding to positions 180 to 251 of SEQ ID NO: 36 passes through, and peptides 25 to 251 (hereinafter referred to as “peptides”) The His6-tag sequence added to the full-length FGF-23 protein was adsorbed to the column. When adsorbed substances were eluted from this column with a NaCl concentration gradient from 0 to 0.7 M in 50 mM sodium phosphate buffer (pH 6.7), His6-tag was added to the fraction eluted with about 0.3 M NaCl. The full-length FGF-23 protein added with was observed, and in the fraction eluted at about 0.4 M, a peptide considered to have a sequence on the N-terminal side from the 179th position of SEQ ID NO: 36 was confirmed. The fraction thus separated by the SP-Sepharose column could then be further separated by applying to a metal affinity column, Talon Superflow (registered trademark) (Clontech, USA). The N-terminal sequence from position 179 also had an affinity for the metal column and was effective for purification. Furthermore, purification was performed with an SP-Sepharose column, and full length FGF-23H was obtained as a single band by CBB staining. The result is shown in FIG. 10B.
[0106]
FGF-23 protein can also be purified by the same method. The culture supernatant of CHO-FGF23 was filtered through SuperCap (registered trademark) (Pall Gelman Laboratory, USA) with a membrane having a pore size of 0.2 μm, and the filtered solution was SP-Sepharose FF (Amersham Pharmacia Biotech, USA) Passed through. A substance having weak affinity with the column was washed and eluted with 50 mM sodium phosphate buffer, pH 6.7. When the protein retained in the column was eluted with a NaCl concentration gradient from 0 to 0.7M, full-length FGF-23 protein was observed in the fraction eluted with about 0.3M NaCl. This was adsorbed on a metal affinity column, Talon Superflow (registered trademark) (Clontech, USA), washed with 50 mM sodium phosphate buffer, pH 6.7, and added with varying concentrations of Imidazole. The protein was eluted and purified. Further, the fraction containing the target protein was purified by adsorption and elution on an SP Sepharose FF column. The full-length FGF-23RQ protein was purified from the CHO-FGF23RQ supernatant by the same method.
[0107]
[Example 16] Obtaining a human monoclonal antibody-producing hybridoma against human FGF-23
Monoclonal antibodies in this example were prepared according to a general method as described in the introduction to monoclonal antibody experiment procedures (authored by Ando Minhe et al., Kodansha 1991). BALB / c mice were used as immunized animals. Human FGF-23 was immunized by the following two methods depending on the immunogen.
[0108]
16- (1) Immunization by combination of vector administration and recombinant protein administration
Intravenous BALB / c mice were treated with INPEP4 / FGF-23 vector (10 or 50 μg / mouse) prepared in Example 15- (1) and Trans IT (registered trademark) In Vivo Gene Delivery System reagent (Takara Shuzo). The first immunization was performed after introduction. From the first immunization, the same vector was introduced once a week later for booster immunization. Furthermore, the FGF-23RQH protein (20-30 μg / animal) prepared in Example 15- (4) was suspended in a RIBI adjuvant (Corixa, USA) containing squalene, Tween 80, Monophosphoryl lipid A and Trehalose dimycolate to prepare an emulsion. After that, booster immunization was performed 4 or 5 times by intraperitoneal injection, and the FGF-23H protein (18 μg / animal) prepared in Example 15- (4) was injected into the tail vein 4 days before the acquisition of spleen cells described below. Immunized by injection.
[0109]
16- (2) Immunization with human FGF-23
BALB / c mice were first immunized by intraperitoneal injection with FGF-23 (22 μg / animal) prepared in Example 15- (4) suspended in the above-mentioned RIBI adjuvant. Further, the protein was boosted once per week by intraperitoneal injection for 4 weeks, and FGF-23 (10 μg / mouse) was immunized by tail vein injection 3 days before acquisition of the spleen cells described below.
[0110]
16- (3) Production and selection of hybridomas
The spleen is removed from the immunized mouse as described above, and the collected spleen cells are mixed with mouse myeloma SP2 / 0 (ATCC: CRL1581) 5: 1, and polyethylene glycol 1500 (Roche Diagnostics, Japan) as a fusion agent. The cells were fused using Stix) to prepare a hybridoma. Hybridomas were selected in HAT-containing DMEM medium (Gibco BRL, USA) containing 10% fetal calf serum (FCS), hypoxanthine (H), aminopterin (A), and thymidine (T). This was performed by culturing the cells. Furthermore, cloning was performed by the limiting dilution method using HT-containing DMEM medium. Cloned hybridomas derived from single cells were obtained.
[0111]
16- (4) Selection of cloned hybridomas producing anti-FGF-23 antibody
By examining the binding between the antibody produced by the hybridoma and the FGF-23 protein, the hybridoma producing an antibody that specifically recognizes the FGF-23 protein was selected. Selection of hybridomas obtained by immunization by the first method described above was performed as follows. 50 mM NaHCO _Three 50 μl of FGF-23H protein solution diluted to a concentration of 1 μg / ml is added to each well of an ELISA 96-well microplate (Maxisorp (registered trademark), Nunc, USA) for 30 minutes at 37 ° C. After incubation at 4 ° C. for 12 hours, the FGF-23H protein was adsorbed on the microplate. Next, after removing this solution, adding blocking reagent (SuperBlock® Blocking Buffer, PIERCE, USA) to each well and incubating at room temperature for 30 minutes, each well was tris-buffered containing 0.1% Tween20. The plate was washed twice with saline (TRIZMA pre-set crystals (registered trademark), Sigma, USA, containing 500 mM NaCl) (T-TBS). 50 μl of the culture supernatant of each hybridoma was added to each well of the microplate coated with the FGF-23H protein as described above, and reacted for 30 minutes, and then each well was washed twice with T-TBS. Next, peroxidase-labeled goat anti-mouse IgG antibody (Zymed laboratories, USA) diluted by 3000 times 50 μl was added to each well and incubated at room temperature for 30 minutes. After washing this with T-TBS three times, 50 μl of a substrate buffer containing tetramethylbenzidine (DAKO, Denmark) was added to each well and incubated at room temperature for 15 minutes. Subsequently, 50 μl of 0.5 M sulfuric acid was added to each well to stop the reaction. Absorbance at a wavelength of 450 nm was measured with a microplate reader (MTP-300, Japan, Corona Electric Co., Ltd.) with a reference wavelength of 570 nm. Here, hybridomas showing a clear increase in absorbance were selected, and a similar experiment was performed using FGF-23 protein to select clones whose binding to FGF-23 was reconfirmed. As a result, nine clones were obtained as hybridomas producing an antibody that recognizes the FGF-23 protein. This included 1C3H, 1D6A, 2A2B, 2C3B, and 2C5L described later. Selection of hybridomas obtained by immunization by the second method described above was performed as follows. 50 mM NaHCO _Three 50 μl of FGF-23 protein solution diluted to a concentration of 1 μg / ml is added to each well of a 96-well microplate for ELISA (Maxisorp (registered trademark), Nunc, USA) and incubated at 4 ° C. for 10 hours. Then, FGF-23 protein was adsorbed on the microplate. Next, after removing this solution and adding a blocking reagent (SuperBlock® Blocking Buffer, PIERCE, USA) to each well and incubating for 30 minutes at room temperature, each well was tris-buffered containing 0.1% Tween20. Washed twice with saline (T-TBS). Thus, 50 μl of the culture supernatant of each hybridoma was added to each well of the microplate coated with FGF-23 protein, reacted for 30 minutes, and then each well was added to Tris-buffered saline containing 0.1% Tween20. Washed twice with (T-TBS). Next, peroxidase-labeled goat anti-mouse IgG antibody (Zymed laboratories, USA) diluted by 3000 times 50 μl was added to each well and incubated at room temperature for 30 minutes. After washing this with T-TBS three times, 50 μl of a substrate buffer containing tetramethylbenzidine (DAKO, Denmark) was added to each well and incubated at room temperature for 15 minutes. Subsequently, 50 μl of 0.5 M sulfuric acid was added to each well to stop the reaction. The absorbance at a wavelength of 450 nm was measured with a microplate reader (MTP-300, Japan, Corona Electric Co., Ltd.) with a reference wavelength of 570 nm. Here, hybridomas showing a clear increase in absorbance were selected. As a result, four new clones were obtained as hybridomas producing antibodies that recognize the FGF-23 protein. This included 3C1E.
[0112]
The subclass of the antibody specifically recognizing the FGF-23 protein thus obtained was identified using the Iso Strip mouse monoclonal antibody isotyping kit (Roche, USA). The results are shown in Table 1.
[0113]
[Table 1]

[0114]
Of the above hybridoma clones, three hybridoma clones 2C3B, 3C1E, and 1D6A were obtained on December 26, 2001 from the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture). 6) was deposited internationally based on the Budapest Treaty. In addition, the hybridoma clone 2C5L was deposited internationally on January 6, 2003 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st-1, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture) based on the Budapest Treaty. The accession numbers are as follows.
2C3B: FERM BP-7838
3C1E: FERM BP-7839
1D6A: FERM BP-7840
2C5L: FERM BP-8268
[0115]
[Example 17] Preparation of monoclonal antibody
17- (1) Preparation of culture supernatant containing anti-FGF-23 antibody
Anti-FGF-23 antibody-producing hybridoma was prepared at 10 μg / ml bovine insulin (Sigma, USA), 5.5 μg / ml human transferrin (Sigma, USA), 0.01 mM ethanolamine (Sigma, USA), 5 ng / ml To eRDF medium (Japan, Far East Pharmaceutical Co., Ltd.) containing sodium selenite (Sigma, USA). Hybridoma cultures for antibody preparation were performed in spinner flasks. The culture solution was passed through a filter (Pall Gelman Laboratory, USA) having a pore size of 0.2 μm to remove miscellaneous wastes such as hybridomas, and the culture supernatant containing the antibody was collected.
[0116]
17- (2) Purification of monoclonal antibodies using protein G
The culture supernatant containing the anti-FGF-23 antibody was passed through a protein G Sepharose 4FF column (Amersham Pharmacia Biotech, USA) to adsorb the antibody to the column and eluted with 0.1 M glycine buffer (pH 2.8). The elution fraction was adjusted to pH 7.2 by adding 1 M Tris-HCl. The antibody solution thus prepared was dialyzed and replaced with PBS (-) using a dialysis membrane (Spectrum Laboratories, USA) with a molecular weight cut off of 10000, and a membrane filter MILLEX-GV (US, Millipore) with a pore size of 0.22 μm. The solution was sterilized by filtration to obtain a purified anti-FGF-23 antibody. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm and setting 1 mg / ml as 1.35 OD.
[0117]
17- (3) Purification of monoclonal antibodies using protein A
Using a protein A carrier column (Immunobiology Laboratories, Japan), anti-FGF-23 antibody using glycine buffer (pH 8.9) as the adsorption buffer and citrate buffer (pH 4.0) as the elution buffer The antibody was affinity purified from the culture supernatant. 1M Tris-HCl was added to the elution fraction containing the antibody to adjust the pH to around 7.2. Next, the solution containing this antibody was replaced with PBS (−) using a dialysis membrane, and further sterilized by filtration through a membrane filter having a pore size of 0.22 μm to obtain a purified anti-FGF-23 antibody. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm and setting 1 mg / ml as 1.35 OD.
[0118]
Each purified monoclonal antibody thus obtained will be indicated using the name of the hybridoma produced. For example, an antibody produced by hybridoma 1D6A is referred to as a 1D6A antibody.
[0119]
[Example 18] Effect of anti-FGF-23 antibody in an assay system on klotho protein-expressing PEAK cells using EGR1 promoter luciferase
In order to examine whether the neutralizing activity ability of the anti-FGF-23 antibody can be measured by the assay system of Example 9, the anti-FGF-23 antibody (2C3B) prepared in Example 15 was examined. The method was performed according to Example 9, and 2C3B antibody was added to a final concentration of 2.5 μg / ml immediately before the addition of FGF-23. As a result, the 2C3B antibody significantly suppressed the increase in luciferase by FGF-23 (FIG. 11). From this, it was shown that the assay system in the cell expressing klotho protein using EGR1 promoter luciferase is effective in searching for a substance that suppresses signal transduction of FGF-23 and klotho protein.
[0120]
[Example 19] Effect of anti-FGF-23 antibody on assay system of ERK phosphorylation enhancement of CHOras clone1 cells by FGF-23 and solubilized klotho protein
In order to examine whether the neutralizing activity ability of the anti-FGF-23 antibody can be measured by the assay system of Example 8, the anti-FGF-23 antibody (2C3B) prepared in Example 15 was examined.
[0121]
The method was performed according to Example 8, and FGF-23 and 2C3B antibody were added to the cells in a premixed state. As a result, as shown in FIG. 12, enhanced ERK phosphorylation by FGF-23 and solubilized klotho protein was suppressed in a 2C3B antibody concentration-dependent manner. From this, it was shown that the assay system for the detection of enhanced ERK phosphorylation using FGF-23 and klotho protein is effective in searching for substances that suppress FGF-23 and klotho protein signaling.
[0122]
[Example 20] Effect of anti-FGF-23 antibody "2C3B antibody" in mice
In order to examine the effect of anti-human FGF-23 monoclonal antibody on normal mice, the following experiment was conducted. Normal mice (BALB / c, male, 12 weeks old) were randomly divided into 5 groups each consisting of 4 animals, and as shown in FIG. 0.67 mg / ml of anti-human FGF-23 monoclonal antibody (2C3B) was administered to the third group as a control, and 0.67 mg / ml of anti-TPO monoclonal antibody was administered as a single dose of 0.15 ml each in the tail vein. Twenty-four hours after administration, heart blood was collected under ether anesthesia, and serum was separated using a microtainer (Becton Dickinson, USA). The phosphate concentration in the obtained serum was added using Phosphorus-Test Wako (Wako Pure Chemical Industries, Japan), and the serum 1,25D concentration was attached using 1,25 (OH) 2D RIA kit TFB (TAF, USA). Measured according to the document. Between administration and blood collection, each group was housed in a plastic cage, and a solid diet CE-2 containing 1% of phosphorus and calcium (Japan Claire, Japan) and tap water were freely ingested.
[0123]
The obtained result is shown in FIG. The measured values are expressed as the mean value +/- standard deviation in each group. The group marked with * was subjected to a significant difference test by student-t. As a result, the vehicle (PBS) administration group and the anti-TPO antibody administration group were compared with p. Groups representing <0.01 are represented.
[0124]
【The invention's effect】
As shown in the Examples, administration of klotho protein decreases blood 1,25 dihydroxyvitamin D concentration and serum phosphorus concentration. In addition, administration of anti-kolotho protein antibody increases blood 1,25 dihydroxyvitamin D concentration and serum phosphorus concentration. These examples show that klotho protein and anti-kolotho protein antibodies alter blood 1,25 dihydroxyvitamin D and serum phosphorus levels and are used to treat and / or prevent diseases involving phosphorus and / or vitamin D It shows you can.
[0125]
[Sequence Listing]

[0126]
[Sequence Listing Free Text]
SEQ ID NOs: 4-22, 28-30, 32-35: synthetic DNA
SEQ ID NOs: 24-27, 31: synthetic peptides
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows the results of analysis by RT-PCR of changes in the expression of EGR1 gene in mouse kidney 1 hour and 8 hours after a single administration of human FGF23 recombinant.
FIG. 2 shows the results of analyzing the phosphorylation change of ERK protein in mouse kidney 5 minutes and 10 minutes after single administration of human FGF23 recombinant by Western blotting.
FIG. 3 is a view showing the result of analyzing the phosphorylation change of ERK protein 10 minutes after adding human FGF23 recombinant to full-length klotho protein-expressing PEAK cells by Western blotting.
FIG. 4 shows changes in luciferase activity upon stimulation with human FGF23 in PEAK cells transfected with a klotho expression plasmid and a reporter gene linked to luciferase downstream of the EGR1 promoter.
FIG. 5 is a graph showing the ERK phosphorylation enhancement in PEAK cells when culture supernatant of CHOras clone 1 cells stably expressing solubilized mouse klotho protein in PEAK cells and human FGF23 are added.
[Figure 6] Solubilized mouse klotho protein transiently expressed PEAK rapid cell supernatant sample and PEAK rapid cell culture supernatant sample as mklotho-97 antibody, mklotho-118 antibody, mklotho-299 antibody and mklotho-941 It is the result of analyzing by Western blotting using an antibody.
[Fig. 7] Solubilized mouse klotho protein transiently expressed PEAK rapid cell supernatant sample, PEAK rapid cell culture supernatant sample as its control, and FGF-23 binding when they were subjected to FGF-23-immobilized resin It is the result of analyzing a product sample by Western blotting using mklotho-118 antibody or mklotho-941 antibody. On the other hand, FIG. YY2B shows the result of staining a similar sample by the silver staining method.
FIG. 8A shows 1,25 dihydroxyvitamin D (1, 2) of the solubilized klotho protein-expressing cell transplant group (klotho, n = 5) and control CHO cell transplant group (control, n = 5) on the 14th day after cell transplantation. 25D) is a graph showing the concentration. The results are expressed as mean values ± standard error, and significant difference test with the control group was performed by Student's t.
FIG. 8B shows the serum phosphorus concentration and serum calcium concentration of the solubilized klotho protein-expressing cell transplant group (klotho, n = 6) and control CHO cell transplant group (control, n = 6) on the 27th day after cell transplantation. It is a graph. The results are expressed as mean values ± standard error, and significant difference test with the control group was performed by Student's t.
FIG. 9 is a graph showing serum phosphorus concentration 24 hours after administration and serum active vitamin D concentration 9 hours after administration of the anti-klotho protein antibody (p118) group and the vehicle administration group. The results are expressed as mean values ± standard error, and significant difference test with the control group was performed by Student's t.
FIG. 10A is a diagram in which CHO-FGF23H cell culture supernatant was separated by SDS-polyacrylamide gel electrophoresis, and recombinant FGF-23 protein and its metabolite were detected by Western blotting with an anti-FGF-23 polyclonal antibody. . In the culture supernatant, there are a full-length FGF-23H protein and an N-terminal fragment and a C-terminal fragment generated by cleavage between SEQ ID NO: 179 and 180 of the amino acid sequence of SEQ ID NO: 35. Full length FGF-23H protein and N-terminal fragment peptide were recognized by hFGF23-48 antibody and hFGF23-148 antibody. In the N-terminal fragment, the presence of a small fragmented peptide is observed. With the anti-His6-tag antibody, the full-length protein and the C-terminal fragment were detected.
FIG. 10B is a diagram of FGF-23 full-length protein purified from CHO-FGF23 cell culture supernatant, N-terminal fragment and C-terminal fragment separated by SDS-polyacrylamide gel electrophoresis and detected by CBB staining. is there.
FIG. 11 Changes in luciferase activity when anti-FGF-23 antibody (2C3B) is added to PEAK cells transfected with a klotho expression plasmid and a reporter gene linked to luciferase downstream of the EGR1 promoter during human FGF-23 stimulation. FIG. It was shown that luciferase activity increased by FGF-23 stimulation was suppressed by simultaneous addition of 2C3B.
FIG. 12: It was examined whether anti-FGF23 antibody (2C3B) suppresses the enhanced phosphorylation of ERK caused by adding solubilized klotho protein-expressing CHOras clone1 cell culture supernatant and FGF23 to CHOras clone1 cells. FIG. It was shown that enhanced ERK phosphorylation when FGF23 and solubilized klotho protein-expressing CHOras clone1 cell culture supernatant were added simultaneously was dependent on 2C3B antibody concentration.
FIG. 13 is a graph showing serum phosphate and serum 1,25-dihydroxyvitamin D concentrations at 24 hours after administration of 2C3B antibody, anti-TPO antibody, and vehicle.

Claims (5)

  A pharmaceutical composition for reducing blood phosphorus concentration and / or blood active vitamin D concentration, comprising klotho protein as an active ingredient.   The pharmaceutical composition according to claim 1, wherein the klotho protein is a solubilized klotho protein.   A pharmaceutical composition for increasing blood phosphorus concentration and / or blood active vitamin D concentration, comprising an anti-klotho protein antibody as an active ingredient.   Neoplastic osteomalacia, ADHR, XLH, renal osteodystrophy, dialysis osteopathy, osteoporosis, hypophosphatemia, kulu disease, osteomalacia, tubular dysfunction, osteopenia, hypocalcemia, high Phosphoremia, hyperparathyroidism, ectopic calcification, pruritus, osteosclerosis, Paget's disease, hypercalcemia, hypoparathyroidism, bone pain, muscle weakness, skeletal deformity, growth disorder and low 1 The pharmaceutical composition according to any one of claims 1 to 3, which is used for treatment and / or prevention of a disease involving phosphorus and / or vitamin D selected from the group consisting of 25D blood.   A method for screening a substance that fluctuates blood phosphorus level and / or blood active vitamin D level, comprising contacting FGF23 and klotho protein and detecting an intracellular signal due to interaction between FGF23 and klotho protein By the screening method.

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