JP6370637B2 - Treatment and / or prevention drug for type 2 diabetes - Google Patents
Treatment and / or prevention drug for type 2 diabetes Download PDFInfo
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- JP6370637B2 JP6370637B2 JP2014165695A JP2014165695A JP6370637B2 JP 6370637 B2 JP6370637 B2 JP 6370637B2 JP 2014165695 A JP2014165695 A JP 2014165695A JP 2014165695 A JP2014165695 A JP 2014165695A JP 6370637 B2 JP6370637 B2 JP 6370637B2
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Description
本発明は、２型糖尿病の治療及び／又は予防薬に関する The present invention relates to a therapeutic and / or prophylactic agent for type 2 diabetes.
セレンは、生体にとって重要な役割を果たしている。例えば、中国の克山（ケシャン）地方は土壌中のセレンが少なく、これに起因してこの地区の住民の多くがセレン欠乏状態となり、延いては重篤な心筋症を生じることが知られている。また、土壌中のセレン濃度が低い地域の住民は、それによって血中のセレン濃度が低下し、動脈硬化・ガン等の発症率が増加することも知られている。そして、ＨＩＶ感染者は血中のセレン濃度が低下する傾向となり、これによってＨＩＶ感染者の生存率が低下することが知られている。 Selenium plays an important role for the living body. For example, the Keshan region in China is known to have low selenium in the soil, resulting in many selenium deficiencies in the area, resulting in severe cardiomyopathy. Yes. It is also known that inhabitants in areas where the selenium concentration in the soil is low, the selenium concentration in the blood decreases, and the incidence of arteriosclerosis and cancer increases. And it is known that the HIV infected person tends to decrease the selenium concentration in the blood, thereby reducing the survival rate of the HIV infected person.
また、セレン濃度の異なる餌を用いた動物実験の結果から、免疫機能、***形成といった雄性生殖機能等に影響を及ぼすことも知られている。更に、セレン含有タンパク質（セレノプロテイン）形成不全トランスジェニックマウスを創出したところ、これが初期胚性致死のフェノタイプを示すことも知られている。 In addition, it is also known from the results of animal experiments using foods with different selenium concentrations that it affects the immune function, male reproductive functions such as spermatogenesis, and the like. Furthermore, when a selenium-containing protein (selenoprotein) hypoplasia transgenic mouse was created, it is also known that this exhibits an early embryonic lethal phenotype.
日本人のセレンの平均摂取量は１００μｇ／日と言われ、３０μｇ／日を摂取することが推奨されている。セレンを多く含む食材としては、カツオ、カキ、ホタテ貝、タラ、イワシなどの魚介類及び肉類等が知られている。 The average selenium intake of Japanese is said to be 100 μg / day, and it is recommended to take 30 μg / day. As foods rich in selenium, fish and shellfish such as bonito, oysters, scallops, cod and sardines are known.
ところが、８００μｇ／日以上の摂取量となると、これが過剰摂取の域に達し、結果として下痢、脱毛、末梢神経障害等といった、水銀と同レベルの中毒症状を引き起こすと言われている。すなわち、セレンの適正摂取量の範囲は非常に狭いことが知られている。 However, when the intake is 800 μg / day or more, it reaches the overdose range, and as a result, it is said that toxic symptoms such as diarrhea, hair loss, and peripheral neuropathy are caused. That is, it is known that the range of proper selenium intake is very narrow.
このようなセレンのサプリメント効果に関して、１３１２人が参加する大規模な臨床試験（ｔｈｅ Ｎｕｔｒｉｔｉｏｎａｌ Ｐｒｅｖｅｎｔｉｏｎ Ｃａｎｃｅｒ Ｔｒｉａｌ：ＮＰＣ試験）が行われ、１日のセレン摂取量を２００μｇとした場合に、糖尿病を発症するリスクが高まるとの報告がされている（非特許文献１）。このような報告に関し、今もなお、セレン及び抗酸化物質であるビタミンＥのサプリメントとしての有用性評価（３万５千人が参加する、Ｓｅｌｅｎｉｕｍ ａｎｄ Ｖｉｔａｍｉｎ Ｅ Ｃａｎｃｅｒ Ｐｒｅｖｅｎｔｉｏｎ Ｔｒｉａｌ：ＳＥＬＥＣＴ試験）が検証されている。 With regard to the supplement effect of selenium, a large-scale clinical trial (the NPC trial) involving 1312 people is conducted, and diabetes develops when the daily selenium intake is 200 μg. It has been reported that the risk increases (Non-patent Document 1). With regard to such reports, evaluation of the usefulness of selenium and antioxidant vitamin E as a supplement (Selenium and Vitamin E Cancer Prevention Trial: SELECT test), which includes 35,000 people, is still being verified. ing.
セレノプロテインは、システイン残基の硫黄がセレンに置き換わったセレノシステイン残基を含むタンパク質の総称である。哺乳類のセレノプロテインとして、グルタチオンペルオキシダーゼ（ＧＰｘ）、チオレドキシンレダクターゼ（ＴＲ）、ヨードチロニン脱ヨード酵素、セレノホスフェートシンセターゼ、セレノプロテインＰ（ＳｅＰ）、セレノプロテインＷ、１５ｋＤａセレノプロテイン等が知られ、これらは細胞内の酸化還元に関与するタンパク質であることも知られている。 Selenoprotein is a general term for proteins containing a selenocysteine residue in which the sulfur of the cysteine residue is replaced with selenium. As mammalian selenoproteins, glutathione peroxidase (GPx), thioredoxin reductase (TR), iodothyronine deiodinase, selenophosphate synthetase, selenoprotein P (SeP), selenoprotein W, 15 kDa selenoprotein, etc. are known. It is also known that it is a protein involved in the redox.
中でも、主に肝臓で生合成されるセレノプロテインＰは、血漿中の主要なセレノプロテインで（血漿中の濃度は５．３μｇ／ｍｌ）、６９ｋＤａ（３６２アミノ酸）の糖タンパク質であることが知られている。セレノプロテインＰのアミノ酸一次構造と機能との関係が詳細に研究されており、Ｎ末端側に酵素活性部位を有し、セレノシステイン残基に富むＣ末端側が細胞へセレンを供給する機能を発揮していると考えられている。また、Ｎ末端でもＣ末端でもない領域にヒスチジンリッチドメインを有していることも知られている（非特許文献２〜５）。 Among them, selenoprotein P, which is mainly biosynthesized in the liver, is a major selenoprotein in plasma (the concentration in plasma is 5.3 μg / ml) and is known to be a 69 kDa (362 amino acid) glycoprotein. ing. The relationship between amino acid primary structure and function of selenoprotein P has been studied in detail, and the C-terminal side, which has an enzyme active site on the N-terminal side and is rich in selenocysteine residues, exhibits the function of supplying selenium to cells. It is thought that It is also known to have a histidine-rich domain in a region that is neither the N-terminus nor the C-terminus (Non-Patent Documents 2 to 5).
そしてセレノプロテインＰは、その肝臓での発現量、血漿中における濃度、並びに糖尿病病態との間で、相関関係が存在する研究結果が報告されている。より詳細には、セレノプロテインＰが増加することによって、インスリン抵抗性が増大することが明らかとなっている（非特許文献６）。 And selenoprotein P has been reported to have a correlation with the expression level in the liver, the concentration in plasma, and the diabetic condition. More specifically, it has been clarified that insulin resistance increases as selenoprotein P increases (Non-patent Document 6).
上述のように、セレノプロテインＰは、糖尿病に関連することが知られているにもかかわらず、セレノプロテインＰとの関係で、どのような化合物が糖尿病の治療薬の候補となるかという知見すら見当たらない。然るに、本発明は糖尿病、特に２型糖尿病の治療及び／又は予防剤の有効成分を提供する事である。 As described above, even though selenoprotein P is known to be related to diabetes, it has been found that what compounds are candidates for diabetes treatment in relation to selenoprotein P. I can't find it. However, the present invention is to provide an active ingredient of a therapeutic and / or prophylactic agent for diabetes, particularly type 2 diabetes.
上記課題を解決すべく、発明者らは鋭意研究を重ねた結果、セレノプロテインＰの特定の領域をエピトープとする抗体が、セレノプロテインＰの細胞内への取り込みを阻害することを見出した。また、斯かる抗体が、細胞内におけるグルタチオンペルオキシダーゼの増加を抑制することも見出した。本発明はこれらの知見に基づいて完成されたものであり、以下に示す広い態様の発明を含むものである。
［項１］
セレノプロテインＰの２０４〜２６１番目のアミノ酸配列内に存在するエピトープに特異的に結合する抗体。
［項２］
エピトープが、セレノプロテインＰの２０４〜２５４番目のアミノ酸配列内に存在する、項１に記載の抗体。
［項３］
エピトープが、セレノプロテインＰの２０４〜２１７番目のアミノ酸配列内に存在する、項１又は項２に記載の抗体。
［項４］
ポリクローナル抗体又はモノクローナル抗体である項１〜項３の何れか一項に記載の抗体。
［項５］
イムノグロブリン、Ｆ（ａｂ’）２、Ｆａｂ、Ｆｖ、ｓｃＦｖ、ｓｃＦｖ−Ｆｃ、テトラボディー及びミニボディーからなる群より選択される少なくとも１つの構造を有する、項１〜項４に記載の抗体。
［項６］
前記抗体が、
配列番号３又は１３に示すアミノ酸配列からなる重鎖ＣＤＲ１、
配列番号４又は１４に示すアミノ酸配列からなる重鎖ＣＤＲ２、
及び
配列番号５又は１５に示すアミノ酸配列からなる重鎖ＣＤＲ３
を含む重鎖可変領域、
及び／又は
配列番号８又は１８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、
配列番号９又は１９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、
及び
配列番号１０又は２０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域を有する、項１〜項５の何れか１項に記載の抗体。
［項７］
前記抗体が、
配列番号３に示すアミノ酸配列からなる重鎖ＣＤＲ１、
配列番号４に示すアミノ酸配列からなる重鎖ＣＤＲ２、及び
配列番号５に示すアミノ酸配列からなる重鎖ＣＤＲ３
を含む重鎖可変領域、又は
配列番号１３に示すアミノ酸配列からなる重鎖ＣＤＲ１、
配列番号１４に示すアミノ酸配列からなる重鎖ＣＤＲ２、及び
配列番号１５に示すアミノ酸配列からなる重鎖ＣＤＲ３
を含む重鎖可変領域及び／又は
配列番号８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、
配列番号９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、及び
配列番号１０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域、又は
配列番号１８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、
配列番号１９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、及び
配列番号２０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域
を有する、項１〜項６の何れか１項に記載の抗体。
［項８］
前記抗体が、
配列番号３に示すアミノ酸配列からなる重鎖ＣＤＲ１、
配列番号４に示すアミノ酸配列からなる重鎖ＣＤＲ２、及び
配列番号５に示すアミノ酸配列からなる重鎖ＣＤＲ３
を含む重鎖可変領域、及び
配列番号８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、
配列番号９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、及び
配列番号１０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域；又は
配列番号１３に示すアミノ酸配列からなる重鎖ＣＤＲ１、
配列番号１４に示すアミノ酸配列からなる重鎖ＣＤＲ２、及び
配列番号１５に示すアミノ酸配列からなる重鎖ＣＤＲ３
を含む重鎖可変領域、及び
配列番号１８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、
配列番号１９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、及び
配列番号２０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域を有する、項１〜項７の何れか１項に記載の抗体。
［項９］
前記抗体が、
配列番号２又は１２に示すアミノ酸配列を含む重鎖可変領域、及び／又は
配列番号７又は１７に示すアミノ酸配列を含む軽鎖可変領域
を有する、項１〜項８の何れか１項に記載の抗体。
［項１０］
前記抗体が、
配列番号２に示すアミノ酸配列を含む重鎖可変領域、及び
配列番号７に示すアミノ酸配列を含む軽鎖可変領域；又は
配列番号１２に示すアミノ酸配列を含む重鎖可変領域、及び
配列番号１７に示すアミノ酸配列を含む軽鎖可変領域
を有する、項１〜項９の何れか１項に記載の抗体。
［項１１］
前記抗体が定常領域を含む、項１〜項１０の何れか１項に記載の抗体。
［項１２］
前記抗体がキメラ抗体である、項１〜項１１の何れか１項に記載の抗体。
［項１３］
前記抗体がヒト化抗体である、項１〜項１２の何れか１項に記載の抗体。
［項１４］
前記抗体が、
配列番号１又は１１に示すアミノ酸配列を含む重鎖、及び／又は配列番号６又は１６に示すアミノ酸配列を含む軽鎖を有する、項１〜項１３の何れか１項に記載の抗体。
［項１５］
前記抗体が、
配列番号１に示すアミノ酸配列を含む重鎖、及び
配列番号６に示すアミノ酸配列を含む軽鎖、又は
配列番号１１に示すアミノ酸配列を含む重鎖、及び
配列番号１６に示すアミノ酸配列を含む軽鎖
を有する、項１〜項１４の何れか１項に記載の抗体。
［項１６］
項１〜項１５の何れか１項に記載の抗体を含む、セレノプロテインＰの細胞内取り込み阻害剤。
［項１７］
項１〜項１５の何れか１項に記載の抗体を含む、細胞内のグルタチオンペルオキシダーゼの誘導抑制剤。
［項１８］
項１〜項１５の何れか１項に記載の抗体を含む、２型糖尿病の治療薬。
［項１９］
項１〜項１５の何れか１項に記載の抗体を生体に投与する工程を含む、セレノプロテインＰの細胞内取り込みを阻害する方法。
［項２０］
項１〜項１５の何れか１項に記載の抗体を生体に投与する工程を含む、細胞内のグルタチオンペルオキシダーゼの誘導を抑制する方法。
［項２１］
項１〜項１５の何れか１項に記載の抗体を生体に投与する工程を含む、２型糖尿病の治療方法。
In order to solve the above-mentioned problems, the inventors have conducted extensive research and found that an antibody having a specific region of selenoprotein P as an epitope inhibits the uptake of selenoprotein P into cells. It was also found that such an antibody suppresses an increase in glutathione peroxidase in cells. The present invention has been completed based on these findings, and includes the following broad aspects of the invention.
[Claim 1]
An antibody that specifically binds to an epitope existing within the amino acid sequence of amino acids 204 to 261 of selenoprotein P.
[Section 2]
Item 2. The antibody according to Item 1, wherein the epitope is present in the amino acid sequence at positions 204 to 254 of selenoprotein P.
[Section 3]
Item 3. The antibody according to Item 1 or Item 2, wherein the epitope is present in the amino acid sequence at positions 204 to 217 of selenoprotein P.
[Claim 4]
Item 4. The antibody according to any one of Items 1 to 3, which is a polyclonal antibody or a monoclonal antibody.
[Section 5]
Item 5. The antibody according to Item 1, wherein the antibody has at least one structure selected from the group consisting of immunoglobulin, F (ab ′) 2 , Fab, Fv, scFv, scFv-Fc, tetrabodies and minibodies.
[Claim 6]
The antibody is
Heavy chain CDR1, consisting of the amino acid sequence shown in SEQ ID NO: 3 or 13,
Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4 or 14,
And a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5 or 15
A heavy chain variable region comprising,
And / or a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 8 or 18,
A light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9 or 19,
And light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10 or 20
Item 6. The antibody according to any one of Items 1 to 5, which has a light chain variable region comprising:
[Claim 7]
The antibody is
Heavy chain CDR1, consisting of the amino acid sequence shown in SEQ ID NO: 3,
Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4 and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5
A heavy chain variable region comprising or a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13,
Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14 and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15
A heavy chain variable region comprising: and / or a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8,
Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9 and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10
A light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18,
Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 19 and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 20
Item 7. The antibody according to any one of Items 1 to 6, which has a light chain variable region comprising
[Section 8]
The antibody is
Heavy chain CDR1, consisting of the amino acid sequence shown in SEQ ID NO: 3,
Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4 and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5
A light chain CDR1 comprising the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8,
Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9 and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10
A light chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13,
Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14 and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15
A light chain CDR1 consisting of the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18,
Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 19 and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 20
Item 8. The antibody according to any one of Items 1 to 7, which has a light chain variable region comprising
[Claim 9]
The antibody is
Item 10. The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2 or 12 and / or the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 or 17, antibody.
[Section 10]
The antibody is
A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7; or a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 12, and shown in SEQ ID NO: 17 Item 10. The antibody according to any one of Items 1 to 9, which has a light chain variable region comprising an amino acid sequence.
[Section 11]
Item 11. The antibody according to any one of Items 1 to 10, wherein the antibody comprises a constant region.
[Claim 12]
Item 12. The antibody according to any one of Items 1 to 11, wherein the antibody is a chimeric antibody.
[Claim 13]
Item 13. The antibody according to any one of Items 1 to 12, wherein the antibody is a humanized antibody.
[Section 14]
The antibody is
Item 14. The antibody according to any one of Items 1 to 13, which has a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 1 or 11, and / or a light chain comprising the amino acid sequence represented by SEQ ID NO: 6 or 16.
[Section 15]
The antibody is
A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain containing the amino acid sequence shown in SEQ ID NO: 6, or a heavy chain containing the amino acid sequence shown in SEQ ID NO: 11, and a light chain containing the amino acid sequence shown in SEQ ID NO: 16 Item 15. The antibody according to any one of Items 1 to 14, which has
[Section 16]
Item 18. An inhibitor of intracellular uptake of selenoprotein P, comprising the antibody according to any one of items 1 to 15.
[Section 17]
Item 16. An inhibitor of intracellular glutathione peroxidase induction, comprising the antibody according to any one of Items 1 to 15.
[Section 18]
Item 20. A therapeutic agent for type 2 diabetes, comprising the antibody according to any one of Items 1 to 15.
[Section 19]
Item 16. A method for inhibiting intracellular uptake of selenoprotein P, comprising a step of administering the antibody according to any one of Items 1 to 15 to a living body.
[Section 20]
Item 16. A method for suppressing intracellular glutathione peroxidase induction, comprising a step of administering the antibody according to any one of Items 1 to 15 to a living body.
[Claim 21]
Item 16. A method for treating type 2 diabetes, comprising a step of administering the antibody according to any one of items 1 to 15 to a living body.
以下に、本発明の効果を説明するが、本発明は以下に示すすべての効果を発揮する発明に限定されないのは言うまでもない。 The effects of the present invention will be described below, but it goes without saying that the present invention is not limited to the invention that exhibits all the effects described below.
本発明のモノクローナル抗体は、セレノプロテインＰの細胞内取り込み阻害剤の有効成分として効果を発揮する。 The monoclonal antibody of the present invention is effective as an active ingredient of a selenoprotein P intracellular uptake inhibitor.
本発明のモノクローナル抗体は、細胞内のグルタチオンペルオキシダーゼの誘導抑制剤の有効成分として効果を発揮する。 The monoclonal antibody of the present invention is effective as an active ingredient of an intracellular glutathione peroxidase induction inhibitor.
本発明のモノクローナル抗体は、２型糖尿病の治療薬の有効成分として効果を発揮する。 The monoclonal antibody of the present invention is effective as an active ingredient of a therapeutic agent for type 2 diabetes.
本発明を実施するために使用する様々な技術は、特にその出典を明示した技術を除いては、公知の文献等に基づいて当業者であれば容易かつ確実に実施可能である。例えば、遺伝子工学及び分子生物学的技術であれば、Ｓａｍｂｒｏｏｋ ａｎｄ Ｒｕｓｓｅｌｌ，“Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ Ａ ＬＡＢＯＲＡＴＯＲＹ ＭＡＮＵＡＬ”，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，Ｎｅｗ Ｙｏｒｋ，（２００１）；Ａｕｓｕｂｅｌ，Ｆ．Ｍ．ｅｔ ａｌ．“Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ”，Ｊｏｈｎ Ｗｉｌｅｙ＆Ｓｏｎｓ，Ｎｅｗ Ｙｏｒｋ，．ＮＹ等の文献を参照
すればよい。
Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known documents and the like, except for a technique that clearly indicates the source. For example, in the case of genetic engineering and molecular biological techniques, Sambrook and Russell, “Molecular Cloning A LABORATORY MANUAL”, Cold Spring Harbor Laboratory Press, New York, (2001); Ausubel, F .; M.M. et al. “Current Protocols in Molecular Biology”, John Wiley & Sons, New York,. References such as NY may be referred to.
また、抗体工学的技術であれば、Ｋａｂａｔ ｅｔ ａｌ．，”Ｓｅｑｕｅｎｃｅｓ ｏｆ Ｐｒｏｔｅｉｎｓ ｏｆ Ｉｍｍｕｎｏｌｏｇｉｃａｌ Ｉｎｔｅｒｅｓｔ，”Ｕ．Ｓ．Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｈｅａｌｔｈ ａｎｄ ｈｕｍａｎ Ｓｅｒｖｉｃｅｓ，（１９８３），Ｋｏｎｔｅｒｍａｎ ａｎｄ Ｄuｂｅｌ，“Ａｎｔｉｂｏｄｙ Ｅｎｇｉｎｅｅｒｉｎｇ”，Ｓｐｒｉｎｇｅｒ等の文献を参照すればよい。 Also, antibody engineering techniques include Kabat et al. "Sequences of Proteins of Immunological Interest," U.S. S. Reference may be made to documents such as Department of Health and human Services, (1983), Konterman and Dubel, “Antibody Engineering”, Springer, and the like.
〔抗体〕
本発明に係る抗体は、セレノプロテインＰの２０４〜２６１番目のアミノ酸配列内に存在するエピトープに特異的に結合する抗体を含む。より好ましくは２０４〜２５４番目のアミノ酸配列内に存在するエピトープに、更に好ましくは、２０４〜２１７番目のアミノ酸配列内に存在するエピトープに特異的に結合する抗体である。
〔antibody〕
The antibody according to the present invention includes an antibody that specifically binds to an epitope present in amino acid sequence 204-261 of selenoprotein P. More preferred is an antibody that specifically binds to an epitope present in the 204th to 254th amino acid sequence, and more preferred to an epitope present in the 204th to 217th amino acid sequence.
セレノプロテインＰのアミノ酸配列は、例えばＮＣＢＩのウェブサイト（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／）において、Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＮＰ＿００１０７８９５５ ＶＥＲＳＩＯＮ ＮＰ＿００１０７８９５５．１ ＧＩ：１４８２７７０１８として収載されている。より具体的には、配列番号２５に示すものである。 The amino acid sequence of selenoprotein P can be obtained from, for example, Accession No. on the NCBI website (http://www.ncbi.nlm.nih.gov/). NP_001078955 VERSION NP_001078955.1 GI: 148277018 is listed. More specifically, it is shown in SEQ ID NO: 25.
上述の用語“特異的な結合”とは、セレノプロテインＰの例えば２０４〜２６１番目のアミノ酸配列内に存在するエピトープに選択的に結合することに限定されず、セレノプロテインＰの２０４〜２６１番目のアミノ酸配列以外のアミノ酸配列に、特にセレノプロテインＰのホモログ等といった、セレノプロテインＰと一次又は高次構造的に類似する分子がセレノプロテインＰと共存している場合に、セレノプロテインＰの２０４〜２６１番目のアミノ酸配列内に存在するエピトープと優先的に結合することで説明される。 The term “specific binding” as described above is not limited to selectively binding to an epitope present in, for example, the 204th to 261st amino acid sequences of selenoprotein P, but the 204th to 261nd of selenoprotein P. Selenoprotein P 204-261 when a molecule similar to selenoprotein P, such as a homologue of selenoprotein P, has a primary or higher-order structure similar to selenoprotein P in addition to amino acid sequence. This is explained by preferential binding to an epitope present in the second amino acid sequence.
なお、上述のような本発明の抗体が、セレノプロテインＰの２０４〜２６１番目のアミノ酸配列内に存在するエピトープに特異的に結合するということは、上述したセレノプロテインＰのホモログ等の特定のアミノ酸配列内に存在するエピトープとの結合が排除されるものではない。 In addition, the fact that the antibody of the present invention as described above specifically binds to an epitope present in the 204th to 261st amino acid sequence of selenoprotein P means that a specific amino acid such as the above-mentioned selenoprotein P homolog or the like. Binding to an epitope present in the sequence is not excluded.
このような上述の抗体の、セレノプロテインＰの例えば２０４〜２６１番目のアミノ酸配列内に存在するエピトープとの特異的な結合の程度は、Ｋｄ値、Ｋｏｆｆ値、又はＫｏｎ値といった反応速度定数でも評価される。なお、Ｋｄ値とは、Ｋｏｆｆ値をＫｏｎ値で除して得られる値である。 The degree of specific binding of the above-mentioned antibody to an epitope present in, for example, the 204th to 261st amino acid sequences of selenoprotein P is also evaluated by a reaction rate constant such as a Kd value, a Koff value, or a Kon value. Is done. The Kd value is a value obtained by dividing the Koff value by the Kon value.
このような本発明に係る抗体のセレノプロテインＰの２０４〜２６１番目のアミノ酸配列内に存在するエピトープとの結合に関する反応速度定数は特に限定されないが、例えばＫｄ値であれば、通常０．０１〜１００ｎＭ程度である。 The reaction rate constant related to the binding with the epitope present in the 204th to 261st amino acid sequences of the selenoprotein P of the antibody according to the present invention is not particularly limited. It is about 100 nM.
本発明に係る抗体は、モノクローナル抗体であってもポリクローナル抗体であってもよい。また、抗体の由来も特に限定はされない。 The antibody according to the present invention may be a monoclonal antibody or a polyclonal antibody. Further, the origin of the antibody is not particularly limited.
本発明に係る抗体の構造は、イムノグロブリン（Ｉｇ）分子に限定はされず、その断片であってもよい。この様な断片としては重鎖及び／又は軽鎖可変領域を含んでいればよく、斯かる断片を適宜再構成した構造であってもよい。 The structure of the antibody according to the present invention is not limited to an immunoglobulin (Ig) molecule, and may be a fragment thereof. Such a fragment may contain a heavy chain and / or light chain variable region, and may have a structure in which such a fragment is appropriately reconstituted.
このような本発明の抗体の具体的な構造として、例えばＦ（ａｂ’）２、Ｆａｂ、Ｆｖ、ｓｃＦｖ、ｓｃＦｖ−Ｆｃ、テトラボディー、ミニボディー等が挙げられる。 Specific examples of such an antibody of the present invention include F (ab ′) 2 , Fab, Fv, scFv, scFv-Fc, tetrabodies, minibodies and the like.
また、上記イムノグロブリンも、そのアイソタイプは限定されず、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＧ、ＩｇＭ、ＩｇＹ等が挙げられる。そして、ＩｇＧのサブクラスも特に限定はされず、ＩｇＧ１、ＩｇＧ２、ＩｇＧ２ａ、ＩｇＧ２ｂ、ＩｇＧ３、ＩｇＧ４等が挙げられる。 The isotype of the immunoglobulin is not limited, and examples include IgA, IgD, IgE, IgG, IgM, IgY. The IgG subclass is not particularly limited, and examples thereof include IgG1, IgG2, IgG2a, IgG2b, IgG3, and IgG4.
本発明の抗体は、特定のアミノ酸配列を含む重鎖可変領域及び／又は軽鎖可変領域を有する態様であることができる。具体的には、
配列番号３又は１３に示すアミノ酸配列からなる重鎖ＣＤＲ１、
配列番号４又は１４に示すアミノ酸配列からなる重鎖ＣＤＲ２、及び
配列番号５又は１５に示すアミノ酸配列からなる重鎖ＣＤＲ３
を含む重鎖可変領域、及び／又は
配列番号８又は１８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、
配列番号９又は１９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、及び
配列番号１０又は２０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域
を有する抗体が挙げられる。
The antibody of the present invention can be in an embodiment having a heavy chain variable region and / or a light chain variable region comprising a specific amino acid sequence. In particular,
Heavy chain CDR1, consisting of the amino acid sequence shown in SEQ ID NO: 3 or 13,
Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4 or 14 and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5 or 15
And / or a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 8 or 18,
Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9 or 19 and Light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10 or 20
An antibody having a light chain variable region comprising
更に好ましくは、
配列番号３に示すアミノ酸配列からなる重鎖ＣＤＲ１、
配列番号４に示すアミノ酸配列からなる重鎖ＣＤＲ２、及び
配列番号５に示すアミノ酸配列からなる重鎖ＣＤＲ３
を含む重鎖可変領域、又は
配列番号１３に示すアミノ酸配列からなる重鎖ＣＤＲ１、
配列番号１４に示すアミノ酸配列からなる重鎖ＣＤＲ２、及び
配列番号１５に示すアミノ酸配列からなる重鎖ＣＤＲ３
を含む重鎖可変領域及び／又は
配列番号８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、
配列番号９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、及び
配列番号１０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域、又は
配列番号１８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、
配列番号１９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、及び
配列番号２０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域
を有する抗体が挙げられる。
More preferably,
Heavy chain CDR1, consisting of the amino acid sequence shown in SEQ ID NO: 3,
Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4 and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5
A heavy chain variable region comprising or a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13,
Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14 and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15
A heavy chain variable region comprising: and / or a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8,
Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9 and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10
A light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18,
Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 19 and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 20
An antibody having a light chain variable region comprising
更に好ましくは、
配列番号３に示すアミノ酸配列からなる重鎖ＣＤＲ１、
配列番号４に示すアミノ酸配列からなる重鎖ＣＤＲ２、及び
配列番号５に示すアミノ酸配列からなる重鎖ＣＤＲ３
を含む重鎖可変領域、及び
配列番号８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、
配列番号９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、及び
配列番号１０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域；又は
配列番号１３に示すアミノ酸配列からなる重鎖ＣＤＲ１、
配列番号１４に示すアミノ酸配列からなる重鎖ＣＤＲ２、及び
配列番号１５に示すアミノ酸配列からなる重鎖ＣＤＲ３
を含む重鎖可変領域、及び
配列番号１８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、
配列番号１９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、及び
配列番号２０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域
を有する抗体が挙げられる。
More preferably,
Heavy chain CDR1, consisting of the amino acid sequence shown in SEQ ID NO: 3,
Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4 and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5
A light chain CDR1 comprising the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8,
Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9 and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10
A light chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13,
Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14 and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15
A light chain CDR1 consisting of the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18,
Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 19 and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 20
An antibody having a light chain variable region comprising
上述の重鎖可変領域及び／又は軽鎖可変領域を有する抗体は、更にフレームワーク領域（ＦＲ）又はその準領域を含んでいてもよい。ＦＲを構成するアミノ酸配列は、公知の方法によって適宜決定することができる。具体的には、Ｔｈｅ Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）のウェブサイト（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／）に記載の情報を参照すればよい。 The above-mentioned antibody having a heavy chain variable region and / or a light chain variable region may further contain a framework region (FR) or a subregion thereof. The amino acid sequence constituting the FR can be appropriately determined by a known method. Specifically, the information described in the website of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) may be referred to.
ヒト由来の機能的生殖細胞から得られるＦＲとして、例えばＫＯＬ、ＮＥＷＭ、ＲＥＩ、ＥＵ、ＴＵＲ、ＴＥＩ、ＬＡＹ、ＰＯＭ等が挙げられる。これらのヒト型のＦＲの例は、Ｋａｂａｔ，ｅｔ．ａｌ．”Ｓｅｑｕｅｎｃｅｓ ｏｆ Ｐｒｏｔｅｉｎｓ ｏｆ Ｉｍｍｕｎｏｌｏｇｉｃａｌ Ｉｎｔｅｒｅｓｔ”：ＵＳ Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｈｅａｌｔｈ ＡＮＤ ｈｕｍａｎ Ｓｅｒｖｉｃｅｓ、ＮＩＨ（１９９１） ＵＳＡ、又はＷｕ ＴＴ，Ｋａｂａｔ ＥＡ．“Ａｎ ａｎａｌｙｓｉｓ ｏｆ ｔｈｅ ｓｅｑｕｅｎｃｅｓ ｏｆ ｔｈｅ ｖａｒｉａｂｌｅ ｒｅｇｉｏｎｓ ｏｆ Ｂｅｎｃｅ Ｊｏｎｅｓ ｐｒｏｔｅｉｎｓ ａｎｄ ｍｙｅｌｏｍａ ｌｉｇｈｔ ｃｈａｉｎｓ ａｎｄ ｔｈｅｉｒ ｉｍｐｌｉｃａｔｉｏｎｓ ｆｏｒ ａｎｔｉｂｏｄｙ ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ．”Ｊ Ｅｘｐ Ｍｅｄ．１３２：２１１−５０（１９７０）等を参照すればよい。 Examples of FRs obtained from functional germ cells derived from humans include KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM. Examples of these human-type FRs are described in Kabat, et. al. “Sequences of Proteins of Immunological Interest”: US Department of Health AND human Services, NIH (1991) USA, or Wu TT, Kabat EA. “An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and the empirical fort. 132: 211-50 (1970).
このようなＦＲを含む重鎖可変領域及び／又は軽鎖可変領域を有する抗体として、例えば
配列番号２又は１２に示すアミノ酸配列を含む重鎖可変領域、及び／又は
配列番号７又は１７に示すアミノ酸配列を含む軽鎖可変領域
を有する抗体が挙げられる。
As an antibody having such a heavy chain variable region and / or light chain variable region containing FR, for example, a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2 or 12, and / or an amino acid shown in SEQ ID NO: 7 or 17 An antibody having a light chain variable region comprising a sequence can be mentioned.
より好ましくは、
配列番号２に示すアミノ酸配列を含む重鎖可変領域、及び
配列番号７に示すアミノ酸配列を含む軽鎖可変領域；又は
配列番号１２に示すアミノ酸配列を含む重鎖可変領域、及び
配列番号１７に示すアミノ酸配列を含む軽鎖可変領域
を有する抗体が挙げられる。
More preferably,
A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7; or a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 12, and shown in SEQ ID NO: 17 An antibody having a light chain variable region containing an amino acid sequence can be mentioned.
上述重鎖可変領域及び／又は軽鎖可変領域を有する抗体は、更に定常領域を有していてもよい。また、これらの抗体は、ヒト化されていても、キメラ化されていてもよい。 The above-mentioned antibody having a heavy chain variable region and / or a light chain variable region may further have a constant region. These antibodies may be humanized or chimerized.
ヒト化抗体とは、定常領域及び可変領域のＦＲがヒト由来のアミノ酸配列であり、それ以外の部分がヒト以外の生物種に由来するアミノ酸配列である抗体を意味する。また、キメラ化とは、定常領域がヒト由来のアミノ酸配列であり、可変領域がヒト以外の生物種に由来するアミノ酸配列である。 The humanized antibody means an antibody in which the FRs of the constant region and variable region are amino acid sequences derived from humans, and the other part is an amino acid sequence derived from a biological species other than humans. Chimerization is an amino acid sequence in which the constant region is derived from a human and the variable region is derived from a biological species other than human.
このようなヒト以外の生物種とは、特に限定はされないが、例えばマウス、ラット、ウサギ、ダチョウ、サル、チンパンジー、ウマ、ロバ、ハムスター、モルモット等が挙げられる。 Examples of such non-human species include, but are not limited to, mouse, rat, rabbit, ostrich, monkey, chimpanzee, horse, donkey, hamster, guinea pig and the like.
このような定常領域は、例えば重鎖定常領域であれば、Ｎ末端から順にＥＴＴを有するアミノ酸配列、より好ましくはＮ末端から順にＥＴＴＡを有するアミノ酸配列を含む定常領域が挙げられる。また、軽鎖定常領域であれば、Ｎ末端から順にＲＡを有するアミノ酸配列、より好ましくはＮ末端から順にＲＡＡを有するアミノ酸配列を含む定常領域が挙げられる。 For example, if such a constant region is a heavy chain constant region, an amino acid sequence having ETT in order from the N-terminus, more preferably a constant region comprising an amino acid sequence having ETTA in order from the N-terminus. Moreover, in the case of the light chain constant region, there may be mentioned a constant region comprising an amino acid sequence having RA in order from the N-terminus, more preferably an amino acid sequence having RAA in order from the N-terminus.
上述の定常領域を有する抗体として、例えば、
配列番号１又は１１に示すアミノ酸配列を含む重鎖及び／又は
配列番号６又は１６に示すアミノ酸配列を含む軽鎖
を有する抗体が挙げられる。
As an antibody having the above constant region, for example,
An antibody having a heavy chain containing the amino acid sequence shown in SEQ ID NO: 1 or 11 and / or a light chain containing the amino acid sequence shown in SEQ ID NO: 6 or 16 can be mentioned.
好ましくは、
配列番号１に示すアミノ酸配列を含む重鎖及び
配列番号６に示すアミノ酸配列を含む軽鎖又は
配列番号１１に示すアミノ酸配列を含む重鎖及び
配列番号１６に示すアミノ酸配列を含む軽鎖
を有する抗体が挙げられる。
Preferably,
An antibody having a heavy chain containing the amino acid sequence shown in SEQ ID NO: 1 and a light chain containing the amino acid sequence shown in SEQ ID NO: 6 or a heavy chain containing the amino acid sequence shown in SEQ ID NO: 11 and a light chain containing the amino acid sequence shown in SEQ ID NO: 16 Is mentioned.
なお、上記のアミノ酸配列には、本発明の抗体の機能、効果等を減衰させない範囲に限り、適宜変異が施されていてもよい。具体的な変異導入の数は、共に特に限定はされないが、通常は変異前のアミノ酸配列と８５％以上、好ましくは９０％以上、より好ましくは９５％以上、最も好ましくは９９％以上の同一性を有する変異体となるような変異導入数とすればよい。 The amino acid sequence may be appropriately mutated as long as the function, effect, etc. of the antibody of the present invention are not attenuated. The specific number of mutagenesis is not particularly limited, but is usually 85% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more identity with the amino acid sequence before mutation. The number of mutation introductions may be such that a mutant having
変異の導入箇所は、特に限定はされないが、本発明の抗体の機能、効果等を減衰させないことに鑑みて、例えば、上述したＦＲ領域、定常領域等に変異を導入することが好ましい。 The site for introducing the mutation is not particularly limited, but it is preferable to introduce the mutation into the above-mentioned FR region, constant region, etc., for example, in view of not diminishing the function, effect, etc. of the antibody of the present invention.
本明細書にて使用する用語、『同一性』とは、２以上の対比可能なアミノ酸配列又は塩基配列の、お互いに対する同一のアミノ酸配列又は塩基配列の程度をいう。従って、ある２つのアミノ酸配列又は塩基配列の同一性が高いほど、それらの配列の同一性または類似性は高い。アミノ酸配列又は塩基配列の同一性のレベルは、通常は、配列分析用ツールであるＦＡＳＴＡを用い、デフォルトパラメーターを用いて決定される。 As used herein, the term “identity” refers to the degree of two or more comparable amino acid sequences or base sequences that are identical to each other. Therefore, the higher the identity of a certain two amino acid sequences or base sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence or base sequence identity is usually determined using FASTA, a sequence analysis tool, using default parameters.
若しくは、ＫａｒｌｉｎおよびＡｌｔｓｃｈｕｌによるアルゴリズムＢＬＡＳＴ（例えば、Ｋａｒｌｉｎ Ｓ，Ａｌｔｓｃｈｕｌ ＳＦ．Ｐｒｏｃ．Ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ．８７：２２６４−２２６８（１９９０）、Ｋａｒｌｉｎ Ｓ，Ａｌｔｓｃｈｕｌ ＳＦ．Ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ．９０：５８７３−７（１９９３）等）を用いて決定できる。このようなＢＬＡＳＴのアルゴリズムに基づいたＢＬＡＳＴＮやＢＬＡＳＴＸと呼ばれるプログラムが開発されている（例えば、Ａｌｔｓｃｈｕｌ ＳＦ，ＧｉｓｈＷ，Ｍｉｌｌｅｒ Ｗ，Ｍｙｅｒｓ ＥＷ，Ｌｉｐｍａｎ ＤＪ．Ｊ Ｍｏｌ Ｂｉｏｌ．２１５：４０３−１０（１９９０）等）。これらの解析方法の具体的な手法は公知であり、ＮＣＢＩのウェブサイトが提供しているＩｇＢＬＡＳＴ（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｉｇｂｌａｓｔ／）を参照すればよい。 Alternatively, the algorithm BLAST by Karlin and Altschul (for example, Karlin S, Altschul SF. Proc. Natl Acad Sci USA. 87: 2264-2268 (1990), Karlin S, Altschul SF. Natl Acad Sci 73: 90). 1993) etc.). Programs called BLASTN and BLASTX based on such BLAST algorithms have been developed (for example, Altschul SF, GishW, Miller W, Myers EW, Lipman DJ. J Mol Biol. 215: 403-10 (1990)). ). Specific methods of these analysis methods are known, and IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/) provided by the NCBI website may be referred to.
上述の変異導入とは、置換、欠失、挿入等である。具体的な変異導入については、公知の方法を採用することができ、特に限定はされないが、例えば置換であれば保存的な置換技術を採用すればよい。 The above-described mutation introduction includes substitution, deletion, insertion and the like. For specific mutagenesis, a known method can be employed, and is not particularly limited. For example, a conservative substitution technique may be employed for substitution.
本明細書にて使用する用語『保存的な置換技術』とは、アミノ酸残基が類似の側鎖を有するアミノ酸残基に置換される技術を意味する。 As used herein, the term “conservative substitution technique” means a technique in which an amino acid residue is substituted with an amino acid residue having a similar side chain.
例えば、リジン、アルギニン、ヒスチジンといった塩基性側鎖を有するアミノ酸残基同士で置換されることが、保存的な置換技術にあたる。その他、アスパラギン酸、グルタミン酸といった酸性側鎖を有するアミノ酸残基；グリシン、アスパラギン、グルタミン、セリン、スレオニン、チロシン、システインといった非帯電性極性側鎖を有するアミノ酸残基；アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファンといった非極性側鎖を有するアミノ酸残基；スレオニン、バリン、イソロイシンといったβ−分枝側鎖を有するアミノ酸残基、チロシン、フェニルアラニン、トリプトファン、ヒスチジンといった芳香族側鎖を有するアミノ酸残基同士での置換も同様に、保存的な置換技術にあたる。 For example, substitution with amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution technique. In addition, amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains such as threonine, valine, and isoleucine, and aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine Similarly, substitution between amino acid residues is a conservative substitution technique.
本発明に係る抗体の製造方法は公知の方法を用いて作成すればよい。例えば、上述のセレノプロテインＰの全長又は少なくともセレノプロテインＰの２０４〜２６１番目のアミノ酸配列を有するペプチド断片を、動物に免疫付与した後に斯かる動物から抗体を回収し、次いで回収した抗体の中からセレノプロテインＰの２０４〜２６１番目のアミノ酸配列内に存在するエピトープと特異的に結合する抗体を選択又はスクリーニングすればよい。 What is necessary is just to produce the manufacturing method of the antibody which concerns on this invention using a well-known method. For example, after immunizing an animal with the above-mentioned full-length selenoprotein P or at least a peptide fragment having the amino acid sequence 204-261 of selenoprotein P, the antibody is recovered from the animal, and then the recovered antibody An antibody that specifically binds to an epitope present in the 204th to 261nd amino acid sequences of selenoprotein P may be selected or screened.
なお、セレノシステインＰの全長又はそのペプチド断片の作成方法は、以下の実施例にて示す方法を採用すればよい。 In addition, what is necessary is just to employ | adopt the method shown in the following Examples as the preparation method of the full length of selenocysteine P or its peptide fragment.
具体的な選択方法又はスクリーニング方法は特に限定はされないが、例えばＥＬＩＳＡ法等といった公知の免疫科学的手段を適宜改変して用いればよい。 A specific selection method or screening method is not particularly limited, and a known immunoscientific means such as an ELISA method may be appropriately modified and used.
本発明に係る抗体がモノクローナル抗体であれば、上述の免疫付与の後に抗体産生Ｂ細胞をリンパ節、脾臓等から採取し、これとミエローマ細胞等といった公知の細胞を融合させたハイブリドーマを作成し、斯かるハイブリドーマが産生する抗体を回収した後に、上述のような選択手段又はスクリーニング手段に供すればよい。 If the antibody according to the present invention is a monoclonal antibody, antibody-producing B cells are collected from the lymph nodes, spleen, etc. after the immunization described above, and a hybridoma is prepared by fusing this with known cells such as myeloma cells, After collecting the antibody produced by such a hybridoma, it may be used for the selection means or screening means as described above.
他の方法としては、本発明に係る抗体のアミノ酸配列コードする塩基配列を、例えば上述のハイブリドーマ細胞から解析し、これをコードする塩基配列を含む核酸を作成した後に、斯かる核酸を含む遺伝子断片を抗体産生に適した公知のＣＨＯ等の細胞に導入して、これを発現させるといった方法が挙げられる。 As another method, for example, the nucleotide sequence encoding the amino acid sequence of the antibody according to the present invention is analyzed from the above-mentioned hybridoma cells, and a nucleic acid containing the nucleotide sequence encoding this is prepared, and then a gene fragment containing such nucleic acid is included. May be introduced into cells such as known CHO suitable for antibody production and expressed.
また、上述のアミノ酸配列を基にペプチド合成機を用いて製造することも可能である。 It can also be produced using a peptide synthesizer based on the amino acid sequence described above.
本発明に係る抗体は、セレノプロテインＰのＰの２０４〜２６１番目のアミノ酸配列内に存在するエピトープに特異的に結合することによって、セレノプロテインＰの細胞内取り込みを阻害する効果を発揮する。従って、本発明に係る抗体は、セレノプロテインＰの細胞内への取り込み阻害剤の有効成分とすることができる。また、本発明に係る抗体を、ヒトのみならず生体に投与することによって、セレノプロテインＰの細胞内への取り込みを阻害することができる。 The antibody according to the present invention exerts an effect of inhibiting intracellular uptake of selenoprotein P by specifically binding to an epitope existing in the amino acid sequence 204-261 of P of selenoprotein P. Therefore, the antibody according to the present invention can be used as an active ingredient of an inhibitor of selenoprotein P uptake into cells. Moreover, the uptake | capture of the selenoprotein P in the cell can be inhibited by administering the antibody which concerns on this invention not only to a human but to a biological body.
また、本発明に係る抗体は、細胞内のグルタチオンペルオキシダーゼの誘導を抑制する効果を発揮する。従って、本発明に係る抗体は、細胞内のグルタチオンペルオキシダーゼの誘導抑制剤の有効成分とすることができる。そして、本発明に係る抗体を、ヒトのみならず生体に投与することによって、細胞内のグルタチオンペルオキシダーゼの誘導を抑制することができる。 In addition, the antibody according to the present invention exhibits an effect of suppressing the induction of intracellular glutathione peroxidase. Therefore, the antibody according to the present invention can be used as an active ingredient of an intracellular glutathione peroxidase induction inhibitor. And the induction | guidance | derivation of intracellular glutathione peroxidase can be suppressed by administering the antibody which concerns on this invention not only to a human but to a biological body.
セレノプロテインＰの細胞内への取り込み上昇や、細胞内のグルタチオンペルオキシダーゼの誘導は、２型糖尿病の発症機序に深く関与するものである。これらの作用を阻害する効果を発揮する、本発明に係る抗体は２型糖尿病の治療剤の有効成分として優れた効果を発揮することが期待される。そして、本発明に係る抗体を、ヒトのみならず生体に投与することによって、２型糖尿病の治療することが期待される。 Increased uptake of selenoprotein P into cells and induction of intracellular glutathione peroxidase are deeply involved in the pathogenesis of type 2 diabetes. The antibody according to the present invention that exhibits the effect of inhibiting these actions is expected to exhibit an excellent effect as an active ingredient of a therapeutic agent for type 2 diabetes. And it is expected to treat type 2 diabetes by administering the antibody according to the present invention not only to humans but also to living bodies.
〔セレノプロテインＰの細胞内取り込み阻害剤〕
本発明に係るセレノプロテインＰの細胞内取り込み阻害剤は、上述の抗体を有効成分として含む。セレノプロテインＰの細胞内への取り込みを阻害する態様には、細胞表面上に存在するセレノプロテインＰを選択的に通過させる機能を有するレセプターを通過させないのみならず、細胞表面に結合する事も阻害する態様も包含する。
[Inhibitor of cellular uptake of selenoprotein P]
The intracellular uptake inhibitor of selenoprotein P according to the present invention contains the above-mentioned antibody as an active ingredient. In the mode of inhibiting the uptake of selenoprotein P into the cell, not only the receptor having the function of selectively passing selenoprotein P existing on the cell surface but also the binding to the cell surface is inhibited. This embodiment is also included.
本発明に係るセレノプロテインＰの細胞内取り込み阻害剤における、上述の抗体の含有量は、セレノプロテインＰの細胞内取り込み阻害剤１００重量部に対して、通常は０．００１〜１００重量部程度とすればよい。すなわち、上述の抗体そのものを、本発明に係るセレノプロテインＰの細胞内取り込み阻害剤としてもよい。 In the selenoprotein P intracellular uptake inhibitor according to the present invention, the content of the antibody described above is usually about 0.001 to 100 parts by weight with respect to 100 parts by weight of the selenoprotein P intracellular uptake inhibitor. do it. That is, the above-described antibody itself may be used as an inhibitor of intracellular uptake of selenoprotein P according to the present invention.
本発明に係るセレノプロテインＰの細胞内取り込み阻害剤の使用対象動物は、生体であればヒトに限らずあらゆる動物個体を対象とできる。例えば、マウス、ラット、ウサギ、ハムスター、モルモット、サル、チンパンジー等の実験動物；イヌ、ネコ等の愛玩動物；その他保護を必要とするあらゆる動物種などが挙げられる。 The animal to be used for the selenoprotein P intracellular uptake inhibitor according to the present invention is not limited to a human being as long as it is a living body, and can be any animal individual. Examples include laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, monkeys, chimpanzees; companion animals such as dogs and cats; and other animal species that require protection.
本発明に係るセレノプロテインＰの細胞内取り込み阻害剤は、上述の生体から取出した組織及び／又は細胞に対して使用してもよい。具体的な細胞とは、特に限定はされないが、例えば筋芽細胞、心筋細胞、平滑筋細胞、筋管細胞等の筋肉細胞、Ｔ細胞等が挙げられる。そして、細胞とは初代培養細胞であっても、不死化された細胞であってもよい。 The intracellular uptake inhibitor of selenoprotein P according to the present invention may be used for the tissues and / or cells extracted from the above-mentioned living body. Specific cells are not particularly limited, and examples thereof include myoblasts, cardiomyocytes, smooth muscle cells, muscle cells such as myotube cells, and T cells. The cell may be a primary cultured cell or an immortalized cell.
本発明に係るセレノプロテインＰの細胞内取り込み阻害剤の使用量は、例えば生体に対して使用する場合、上述の抗体の量に換算して、通常であれば０．５〜５０ｍｇ／ｋｇ（個体体重）程度の量で使用すればよい。細胞に対して使用する場合も、上述の抗体の量に換算して、通常であれば、１μｇ〜１ｍｇ／ｍｌ（培地）程度の量で使用すればよい。 The amount of the selenoprotein P intracellular uptake inhibitor according to the present invention used is, for example, 0.5 to 50 mg / kg (individual) in terms of the amount of the above antibody when used for a living body. It may be used in an amount of about (weight). When used for cells, it is usually used in an amount of about 1 μg to 1 mg / ml (medium) in terms of the above-mentioned antibody amount.
〔細胞内のグルタチオンペルオキシダーゼの誘導抑制剤〕
本発明に係るグルタチオンペルオキシダーゼの誘導抑制剤は、上述の抗体を有効成分として含む。誘導抑制には、単にグルタチオンペルオキシダーゼの発現量の抑制のみならず、細胞においてグルタチオンペルオキシダーゼの活性の低下も意味する。
[Inhibitor of intracellular glutathione peroxidase induction]
The induction inhibitor of glutathione peroxidase according to the present invention contains the above-mentioned antibody as an active ingredient. Inhibition of induction means not only suppression of the expression level of glutathione peroxidase but also reduction of the activity of glutathione peroxidase in cells.
用語、「誘導」とは、グルタチオンペルオキシダーゼの発現誘導することも包含する。 The term “induction” also includes inducing expression of glutathione peroxidase.
グルタチオンペルオキシダーゼ（ＧＰｘ）とは、還元型グルタチオンを利用して、過酸化水素、過酸化脂質等を基質として、これを水、ヒドロキシ型脂質を生成する酵素である。また、活性中心にセレン含有タンパク質であるセレノシステインを有するタンパク質である。 Glutathione peroxidase (GPx) is an enzyme that uses reduced glutathione to produce hydrogen and hydroxy lipid using hydrogen peroxide, lipid peroxide, etc. as a substrate. Moreover, it is a protein which has selenocysteine which is a selenium containing protein in an active center.
グルタチオンペルオキシダーゼは、ＧＰｘ１、ＧＰｘ２、ＧＰｘ３、ＧＰｘ４、ＧＰｘ５、ＧＰｘ６、ＧＰｘ７、ＧＰｘ８等の何れにも限定はされないが、好ましくはＧＰｘ１である。 The glutathione peroxidase is not limited to GPx1, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8, etc., but is preferably GPx1.
例えば、ＧＰｘ１の具体的なアミノ酸配列は、ＮＣＢＩのウェブサイト（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／）において、Ａｃｃｅｓｓｉｏｎ Ｎｏ．Ｐ０７２０３ ＶＥＲＳＩＯＮ Ｐ０７２０３．４ ＧＩ：３１１０３３４８１として収載されている。より具体的には、配列番号２６に示すものである。 For example, the specific amino acid sequence of GPx1 can be found in the Accession No. on the NCBI website (http://www.ncbi.nlm.nih.gov/). P07203 VERSION P07203.4 GI: 311033481 More specifically, it is shown in SEQ ID NO: 26.
本発明に係るグルタチオンペルオキシダーゼの誘導抑制剤における、上述の抗体の含有量は、グルタチオンペルオキシダーゼの誘導抑制剤１００重量部に対して、通常は０．００１〜１００重量程度とすればよい。すなわち、上述の抗体そのものを、本発明に係るグルタチオンペルオキシダーゼの誘導抑制剤としてもよい。 The content of the above-mentioned antibody in the glutathione peroxidase induction inhibitor according to the present invention is usually about 0.001 to 100 weights per 100 parts by weight of the glutathione peroxidase induction inhibitor. That is, the above-described antibody itself may be used as the glutathione peroxidase induction inhibitor according to the present invention.
本発明に係るグルタチオンペルオキシダーゼの誘導抑制剤の使用対象及び使用量は、上述の同様とすればよい。 The usage target and amount of the glutathione peroxidase induction inhibitor according to the present invention may be the same as described above.
〔２型糖尿病の治療薬〕
本発明に係る２型糖尿病の治療薬は、上述の抗体を有効成分として含む。
[Therapeutic agent for type 2 diabetes]
The therapeutic agent for type 2 diabetes according to the present invention contains the above-mentioned antibody as an active ingredient.
糖尿病とは、血糖値、ヘモグロビンＡ１ｃ（ＨｂＡ１ｃ）の値が一定の基準値を超えていることを診断基準とする疾患であり、中でも２型糖尿病はインスリンの抵抗性が高い状態、膵臓のランゲルハンス島からのインスリンの分泌能が低下していることを主な原因とする疾患である。この意味で、膵臓のランゲルハンス島におけるインスリン分泌細胞であるβ細胞が死滅することを原因とする１型糖尿病とは区別される。より詳細には、『糖尿病の分類と診断基準に関する委員会報告（国際標準化対応版）』糖尿病 第５５巻７号（２０１２）を参照すればよい。 Diabetes is a disease whose diagnostic standard is that the blood glucose level and hemoglobin A1c (HbA1c) value exceed a certain reference value. Among them, type 2 diabetes is a state in which insulin resistance is high, pancreatic islets of Langerhans It is a disease mainly caused by a decrease in the secretory ability of insulin. In this sense, it is distinguished from type 1 diabetes caused by the death of β-cells, which are insulin-secreting cells in the pancreatic islets of Langerhans. For more details, refer to “Committee Report on Diabetes Classification and Diagnosis Criteria (International Standardization Version)” Diabetes Volume 55 No. 7 (2012).
２型糖尿病治療薬のメトフォルミンは、ＡＭＰＫ／ＦｏｘＯ３ａを介してセレノプロテインＰの発現量を低下させることが知られるので、メトフォルミンがインスリン抵抗性を改善する作用には、セレノプロテインＰ発現量の低下が関与する可能性が考えられる（Ｊ Ｂｉｏｌ Ｃｈｅｍ．２０１４．２８９．３３５−３４５）。 Metformin, a therapeutic agent for type 2 diabetes, is known to decrease the expression level of selenoprotein P via AMPK / FoxO3a. Therefore, the reduction of selenoprotein P expression level is an effect of metformin on improving insulin resistance. Possible involvement (J Biol Chem. 2014.289.335-345).
したがって、上述の２型糖尿病の中でも、セレノプロテインＰによってインスリンの抵抗性が高い状態にある２型糖尿病が、本発明に係る治療薬の対象疾患として好ましい。 Therefore, among the above-mentioned type 2 diabetes, type 2 diabetes in which insulin resistance is high by selenoprotein P is preferable as a target disease of the therapeutic agent according to the present invention.
用語『治療』とは、所望の薬理学的効果及び／又は生理学的効果を得ることを意味する。この効果は、疾病及び／又は疾病に起因する悪影響（病態、症状等）を、部分的又は完全に治癒することを含む。また、上記効果には、疾病及び／又は疾病に起因する悪影響（病態、症状等）の進行を阻止又は遅延する効果、病態や症状を緩和する（疾病、症状等の後退、又は進行の逆転を引き起こす）効果、再発を阻止する効果等が含まれる。 The term “treatment” means obtaining a desired pharmacological and / or physiological effect. This effect includes partial or complete cure of the disease and / or adverse effects (pathology, symptoms, etc.) resulting from the disease. In addition, the above effects include the effect of preventing or delaying the progression of the disease and / or adverse effects (pathology, symptoms, etc.) caused by the disease, and the relief of the disease state, symptoms (reversal of the disease, symptoms, etc., or reversal of the progression). Effects), effects to prevent recurrence, etc.
また、上記効果には、疾病及び／又は疾病に起因する悪影響（病態、症状等）の素因を持ち得るが、まだ持っていると診断されていない個体において、疾病及び／又は疾病に起因する悪影響（病態、症状等）が起こることを部分的又は完全に防止する効果が含まれる。従って、『治療』なる用語には、『緩解』、『再発防止』、『予防』等の意味も含まれる。 In addition, the above effects may have a predisposition to a disease and / or an adverse effect due to the disease (such as a disease state or symptom), but in an individual who has not yet been diagnosed as having an adverse effect due to the disease and / or the disease. It includes the effect of partially or completely preventing (pathology, symptoms, etc.) from occurring. Therefore, the term “treatment” includes meanings such as “remission”, “prevention of recurrence”, and “prevention”.
本発明に係る２型糖尿病の治療薬における、上述の抗体の含有量は、２型糖尿病の治療薬１００重量部に対して、通常は０．００１〜１００重量程度とすればよい。すなわち、上述の抗体そのものを、本発明に係る２型糖尿病の治療薬としてもよい。 In the therapeutic agent for type 2 diabetes according to the present invention, the content of the above-described antibody is usually about 0.001 to 100 weights per 100 parts by weight of the therapeutic agent for type 2 diabetes. That is, the above antibody itself may be used as a therapeutic agent for type 2 diabetes according to the present invention.
本発明に係る２型糖尿病の治療剤には、薬学分野の組成物を製造する際に使用される薬学的に許容可能な公知の担体又は添加物を配合してもよい。この様な担体或いは添加物の具体例として、任意の担体、希釈剤、賦形剤、懸濁剤、潤滑剤、アジュバント、媒体、送達システム、乳化剤、錠剤分解物質、吸収剤、保存剤、界面活性剤、着色剤、香料、または甘味料等が挙げられる。 The therapeutic agent for type 2 diabetes according to the present invention may contain a known pharmaceutically acceptable carrier or additive used for producing a composition in the pharmaceutical field. Specific examples of such carriers or additives include any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, tablet disintegrant, absorbent, preservative, interface. An active agent, a coloring agent, a fragrance | flavor, a sweetener, etc. are mentioned.
本発明に係る２型糖尿病の治療剤は上述の担体又は配合物を適宜組み合わせてあらゆる剤形とすることができる。具体的には、具体的な剤形としては、輸液剤、埋め込み注射剤、持続性注射剤等の注射剤；腹膜透析用剤、血液透析用剤等を含む透析用剤；口腔内崩壊錠、チュアブル錠、発泡錠、分散錠、溶解錠等等の錠剤；硬カプセル錠、軟カプセル錠等のカプセル剤；発泡顆粒剤、徐放性顆粒剤、腸溶性顆粒剤等を含む顆粒剤；散剤；エリキシル剤、懸濁剤、乳剤、リモナーデ剤等の経口液剤；シロップ用剤等のシロップ剤；経口ゼリー剤；トローチ剤、舌下錠、バッカル錠、付着錠、ガム剤等の口腔用錠剤；口腔用スプレー剤；口腔用半固形剤；含嗽剤；吸入粉末剤、吸入液剤、吸入エアゾール剤等の吸入剤；眼軟膏剤等の点眼剤；点耳剤；点鼻粉末剤、点鼻液剤等の点鼻剤；坐剤；直腸用半固形剤；注腸剤；膣錠；膣用坐剤；外用散剤等の外用固形剤；リニメント剤、ローション剤等の外用液剤；外用エアゾール剤、ポンプスプレー剤等のスプレー剤；軟膏剤；クリーム剤；ゲル剤；テープ剤、パップ剤等の貼付剤等が挙げられる。 The therapeutic agent for type 2 diabetes according to the present invention can be made into any dosage form by appropriately combining the above carriers or blends. Specifically, specific dosage forms include infusions such as infusions, implantable injections, and continuous injections; dialysis agents including peritoneal dialysis agents, hemodialysis agents, and the like; orally disintegrating tablets; Tablets such as chewable tablets, effervescent tablets, dispersible tablets, dissolving tablets; capsules such as hard capsules and soft capsules; granules including effervescent granules, sustained-release granules, enteric granules; powders; Oral liquids such as elixirs, suspensions, emulsions and limonades; syrups such as syrups; oral jelly agents; oral tablets such as troches, sublingual tablets, buccal tablets, adhesive tablets, gums; Sprays for oral use; semisolid preparations for oral cavity; mouthwashes; inhalants such as inhalation powders, inhalation liquids, inhalation aerosols; eye drops such as eye ointments; ear drops; nasal powders, nasal liquids Nasal sprays; suppositories; rectal semi-solid preparations; enema; vaginal tablets; vaginal suppositories; ; External aerosols, sprays, such as pump sprays; liniments, liquids for external use such as lotions ointments; creams; gels; tape, adhesive agent such as poultices and the like.
これらの剤形は、第１６改正日本薬局方解説書等の公知の文献に基づいて製造することができる。 These dosage forms can be produced based on known documents such as the 16th revised Japanese Pharmacopoeia Manual.
本発明に係る２型糖尿病の治療薬の投与対象動物は、上述のセレノプロテインＰの細胞内取り込み阻害剤の使用対象とそれぞれ同様とすればよい。 The target animal for administration of the therapeutic agent for type 2 diabetes according to the present invention may be the same as the target for use of the above-described selenoprotein P intracellular uptake inhibitor.
本発明に係る２型糖尿病の治療薬の投与方法は、特に限定されず、上述の投与対象、剤形等を適宜勘案して公知の投与方法を採用すればよい。具体的には、経口、筋肉内、静脈内、動脈内、くも膜下腔内、皮内、腹腔内、鼻腔内、肺内、眼内、腟内、頸部内、直腸内、皮下等へ投与する方法が挙げられる。 The administration method of the therapeutic agent for type 2 diabetes according to the present invention is not particularly limited, and a known administration method may be adopted in consideration of the above-mentioned administration subject, dosage form and the like as appropriate. Specifically, oral, intramuscular, intravenous, intraarterial, intrathecal, intradermal, intraperitoneal, intranasal, intrapulmonary, intraocular, intravaginal, intracervical, intrarectal, subcutaneous, etc. The method of doing is mentioned.
本発明に係る２型糖尿病の治療薬の投与量は、投与対象動物がヒトであれば、通常は１〜１０ｍｇ／ｋｇ程度とすればよく、投与対象動物がマウスであれば、通常は１〜１０ｍｇ／ｋｇ程度とすればよい。その他の投与対象動物であれば、上述のヒト及びマウスにおける投与量を基に適宜設定することができる。 The dosage of the therapeutic agent for type 2 diabetes according to the present invention is usually about 1 to 10 mg / kg if the animal to be administered is a human, and usually 1 to 3 if the animal to be administered is a mouse. What is necessary is just to be about 10 mg / kg. Any other animal to be administered can be appropriately set based on the above-mentioned doses in humans and mice.
本発明に係る２型糖尿病の治療薬の投与は、上記の量を一日に一度に投与してもよく、数回に分けて投与してもよい。また、上記疾患に対する治療効果を有する範囲において、投与間隔は、毎日、隔日、毎週、隔週、２〜３週毎、毎月、隔月または２〜３ヶ月毎でもよい。 The therapeutic agent for type 2 diabetes according to the present invention may be administered in the above amount once a day or may be divided into several times. In addition, the administration interval may be daily, every other day, every week, every other week, every two to three weeks, every month, every other month, or every two to three months as long as it has a therapeutic effect on the above diseases.
〔セレノプロテインの細胞内取り込みを阻害する方法〕
本発明に係るセレノプロテインの細胞内取り込みを阻害する方法は、上述の抗体を生体に投与する工程を含む方法である。
[Method for inhibiting intracellular uptake of selenoprotein]
The method for inhibiting intracellular uptake of selenoprotein according to the present invention is a method comprising a step of administering the above-mentioned antibody to a living body.
上記〔セレノプロテインＰの細胞内取り込み阻害剤〕に関する各種説明を参照することで、本発明に係るセレノプロテインの細胞内取り込みを阻害する方法が実施できる。 By referring to the various explanations regarding the above [Selenoprotein P intracellular uptake inhibitor], the method of inhibiting selenoprotein intracellular uptake according to the present invention can be carried out.
〔細胞内のグルタチオンペルオキシダーゼの誘導を抑制する方法〕
本発明に係る細胞内のグルタチオンペルオキシダーゼの誘導を抑制は、上述の抗体を生体に投与する工程を含む方法である。
[Method for inhibiting the induction of intracellular glutathione peroxidase]
Inhibiting the induction of intracellular glutathione peroxidase according to the present invention is a method comprising the step of administering the above-mentioned antibody to a living body.
上記〔細胞内のグルタチオンペルオキシダーゼの誘導抑制剤〕に関する各種説明を参照することで、本発明に係るセレノプロテインの細胞内取り込みを阻害する方法が実施できる。 By referring to various explanations regarding the above [inhibitor of induction of intracellular glutathione peroxidase], the method for inhibiting intracellular uptake of selenoprotein according to the present invention can be carried out.
〔２型糖尿病の治療方法〕
本発明に係る２型糖尿病の治療方法は、上述の抗体を生体に投与する工程を含む方法である。
[Method for treating type 2 diabetes]
The method for treating type 2 diabetes according to the present invention is a method including the step of administering the above-described antibody to a living body.
上記〔２型糖尿病の治療剤〕に関する各種説明を参照することで、本発明に係る２型糖尿病の治療方法が実施できる。 The method for treating type 2 diabetes according to the present invention can be carried out by referring to the various descriptions related to the above [therapeutic agent for type 2 diabetes].
以下に、本発明を実施例に基づいてより詳細に説明するが、本発明がこれらの実施例に限定されないことは言うまでもない。 Hereinafter, the present invention will be described in more detail based on examples, but it is needless to say that the present invention is not limited to these examples.
＜実験例１：抗体の作製＞
８週齢のメスのＳＤラット（１５０〜２００ｇ）に、ヒト血漿から精製した２５０μｇのセレノプロテインＰ（ＳｅＰ）を免疫付与し、二週間後にリンパ節を回収した。回収したリンパ節とマウス由来のメラノーマを融合させてハイブリドーマを作成し、斯かるハイブリドーマが産生する抗体の中で、ＳｅＰと結合する１１クローンを、ＥＬＩＳＡ法を用いてスクリーニングした。
<Experimental Example 1: Production of antibody>
Eight week old female SD rats (150-200 g) were immunized with 250 μg of selenoprotein P (SeP) purified from human plasma, and lymph nodes were collected two weeks later. Hybridomas were prepared by fusing the recovered lymph nodes and mouse-derived melanoma, and among the antibodies produced by such hybridomas, 11 clones that bound to SeP were screened using the ELISA method.
得られた１１クローンが産生する抗ＳｅＰモノクローナル抗体をそれぞれ、ＡＡ３、ＡＢ１、ＡＥ２、ＡＨ５、ＢＤ１、ＢＤ３、ＢＦ２、ＤＨ６、ＤＨ７、ＤＨ９、ＤＣ１２と命名した。 The anti-SeP monoclonal antibodies produced by the 11 clones obtained were named AA3, AB1, AE2, AH5, BD1, BD3, BF2, DH6, DH7, DH9, and DC12, respectively.
得られた各種抗ＳｅＰモノクローナル抗体のアミノ酸配列を検討した。各種モノクローナル抗体を産生するハイブリドーマ細胞よりＮｕｃｌｅｏＳｐｉｎ ＲＮＡ ｋｉｔ（ＴＡＫＡＲＡ）を用いてＲＮＡを抽出した。濃度測定後、ＳＭＡＲＴｅｒ ＲＡＣＥ ｃＤＮＡ Ａｍｐｌｉｆｉｃａｔｉｏｎ Ｋｉｔを用いてｃＤＮＡを合成し、Ｐｒｉｍｅｓｔａｒ ＧＸＬ（ＴＡＫＡＲＡ）を用いて抗体の可変領域の増幅を行った。増幅したＤＮＡをＧｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔ（Ｑｉａｇｅｎ）を用いてゲルから抽出し、ｔａｑ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（ＴＡＫＡＲＡ）を用いてＡ付加を行った後にＴＯＰＯ ＴＡ Ｃｌｏｎｉｎｇ ｋｉｔ（Ｉｎｖｉｔｒｏｇｅｎ）を用いてｃｌｏｎｉｎｇを行った。単離したＰｌａｓｍｉｄの塩基配列はＢｉｇＤｙｅＴｅｒｍｉｎａｔｏｒ ｖ３．１ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ ＫｉｔとＡＢＩ ３１００ ＤＮＡ Ｓｅｑｕｅｎｃｅｒを用いて特定した。特定された塩基配列をＮＣＢＩが提供しているＩｇＢＬＡＳＴ（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｉｇｂｌａｓｔ／）で解析することによりＦＲ部位、ＣＤＲ部位を決定した。 The amino acid sequences of the various anti-SeP monoclonal antibodies obtained were examined. RNA was extracted from hybridoma cells producing various monoclonal antibodies using NucleoSpin RNA kit (TAKARA). After concentration measurement, cDNA was synthesized using SMARTER RACE cDNA Amplification Kit, and the variable region of the antibody was amplified using Primestar GXL (TAKARA). The amplified DNA was extracted from the gel using Gel Extraction kit (Qiagen), A was added using taq DNA polymerase (TAKARA), and then cloned using TOPO TA Cloning kit (Invitrogen). The base sequence of the isolated plasmid was specified using BigDyeTerminator v3.1 Cycle Sequencing Kit and ABI 3100 DNA Sequencer. The FR and CDR sites were determined by analyzing the identified nucleotide sequence with IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/) provided by NCBI.
各種モノクローナル抗体の可変領域中のＣＤＲ１〜３、ＦＲ１〜３及びＪ領域を図１及び図２に示す。また、各種モノクローナル抗体のアミノ酸及び核酸配列を表１に示す。 CDR1 to FR3, FR1 to FR3, and J region in the variable regions of various monoclonal antibodies are shown in FIGS. In addition, Table 1 shows amino acid and nucleic acid sequences of various monoclonal antibodies.
表１中の※について、配列番号５は、Ｎ末端から順にＳＲＨ、配列番号１０は、Ｎ末端から順にＱ ＱＹ、配列番号１５は、Ｎ末端から順にＱＱＹ、配列番号１９はＮ末端から 順にＣＳＳである。また、表中の§について、「全長」とは、「可変領域」の全てと、一部の定常領域を含む配列である。 Regarding * in Table 1, SEQ ID NO: 5 is SRH sequentially from the N terminus, SEQ ID NO: 10 is Q QY sequentially from the N terminus, SEQ ID NO: 15 is QQY sequentially from the N terminus, and SEQ ID NO: 19 is CSS sequentially from the N terminus. It is. In addition, regarding the § in the table, “full length” is a sequence including all of the “variable region” and a part of the constant region.
＜実験例２：エピトープの探索＞
次いで、１１種類のモノクローナル抗体に関して、ＳｅＰのどの部位をエピトープとするか確認する実験を行った。以下の（１）〜（８）に示す８種類のＣ末端にＥＧＦＰタグを有するＳｅＰ変異体を、ＨＥＫ２９３細胞を用いて発現させた。
<Experimental example 2: Search for epitope>
Next, for 11 types of monoclonal antibodies, an experiment was conducted to confirm which part of SeP is an epitope. The following eight types of SeP mutants having EGFP tags at the C-terminus shown in (1) to (8) were expressed using HEK293 cells.
ＳｅＰ変異体
（１）は示す変異体は、ＳｅＰの６０〜２９９番目のアミノ酸配列を含む。
（２）に示す変異体は、ＳｅＰの６０〜１０７番目のアミノ酸配列を含む。
（３）に示す変異体は、ＳｅＰの１０８〜１５５番目のアミノ酸配列を含む。
（４）に示す変異体は、ＳｅＰの１５６〜２０３番目のアミノ酸配列を含む。
（５）に示す変異体は、ＳｅＰの１５６〜２１７番目のアミノ酸配列を含む。
（６）に示す変異体は、ＳｅＰの２０４〜２５４のアミノ酸配列を含む。
（７）に示す変異体は、ＳｅＰの２０４〜２６１のアミノ酸配列を含む。
（８）に示す変異体は、ＳｅＰの２６２〜２９９番目のアミノ酸配列を含む。
The variant shown by the SeP variant (1) contains the 60-299th amino acid sequence of SeP.
The mutant shown in (2) contains the 60-107th amino acid sequence of SeP.
The variant shown in (3) includes the 108-155th amino acid sequence of SeP.
The mutant shown in (4) contains the 156th to 203rd amino acid sequence of SeP.
The mutant shown in (5) includes the amino acid sequence of positions 156 to 217 of SeP.
The mutant shown in (6) contains the amino acid sequence of SeP 204-254.
The variant shown in (7) includes the amino acid sequence of SeP 204-261.
The mutant shown in (8) includes the 262-299th amino acid sequence of SeP.
なお、２０４番目〜２１７番目及び２４４番目〜２４９番目のアミノ酸配列はヒスチジンリッチ領域であり、２５４番目〜２５５番目及び２６１〜２６２番目のアミノ酸配列は、カリクレイン認識サイトである。また、上記の各種ＳｅＰ変異体はＧＦＰタグを含んでいる。 The 204th to 217th and 244th to 249th amino acid sequences are histidine-rich regions, and the 254th to 255th and 261st to 262nd amino acid sequences are kallikrein recognition sites. The various SeP mutants described above contain a GFP tag.
各種ＳｅＰ変異体コードするプラスミドを作成し、これをＨＥＫ２９３細胞に導入して発現させた。次いで、斯かる細胞を溶解させ、その細胞溶解液をＳＤＳ−ＰＡＧＥし、次いでウエスタンブロッティングに供した。使用した抗体は、上述の１１個のモノクローナル抗体であり、二次抗体にはＨＰＲ結合抗マウスＩｇＧ抗体を用いた。また、発現の確認には抗ＧＦＰ抗体を用いた。ウエスタンブロッティングの結果を図３〜図４に示す。 Plasmids encoding various SeP mutants were prepared and introduced into HEK293 cells for expression. Subsequently, such cells were lysed, the cell lysate was subjected to SDS-PAGE, and then subjected to Western blotting. The antibodies used were the 11 monoclonal antibodies described above, and HPR-conjugated anti-mouse IgG antibody was used as the secondary antibody. An anti-GFP antibody was used for confirmation of expression. The results of Western blotting are shown in FIGS.
以上の結果から、上述のモノクローナル抗体は、ＳｅＰの２０４〜２６１、詳細には２０４〜２５４番目、さらに詳細には２０４〜２１７番目のアミノ酸配列をエピトープとすることが強く示唆された。 From the above results, it was strongly suggested that the above-mentioned monoclonal antibody has the epitope of the amino acid sequence of SeP 204-261, specifically 204-254, more specifically 204-217.
＜実験例３：細胞内へのＳｅＰ取り込み阻害実験・ＧＰｘ誘導阻害実験＞
セレノプロテインＰ及び上述の各種モノクローナル抗体をそれぞれ終濃度が０．５μｇ／ｍｌ及び１０μｇ／ｍｌとなるように０．５％非働化処理ウマ血清を含むＤＭＥＭ培地中で混合し、室温で２時間反応させた。
<Experimental Example 3: SeP uptake inhibition experiment into cells / GPx induction inhibition experiment>
Selenoprotein P and the various monoclonal antibodies described above were mixed in DMEM medium containing 0.5% inactivated horse serum so that the final concentrations were 0.5 μg / ml and 10 μg / ml, respectively, and reacted at room temperature for 2 hours. I let you.
これを筋肉細胞に分化誘導させたＣ２Ｃ１２細胞の培養培地（０．５％非働化処理ウマ血清を含むＤＭＥＭ培地）にそのまま添加して、３７℃で２４時間培養を行った。 This was directly added to the culture medium of C2C12 cells induced to differentiate into muscle cells (DMEM medium containing 0.5% inactivated horse serum) and cultured at 37 ° C. for 24 hours.
次いで、培養後のＣ２Ｃ１２細胞を回収し、この細胞の溶解液をウエスタンブロッティングに供して、細胞内のＳｅＰ及びＧＰｘ１の量を確認した。用いた抗体はそれぞれ上述の各種抗ＳｅＰモノクローナル抗体及び抗ＧＰｘ１抗体（ａｂ２２６０４（アブカム社）Ａｎｔｉ−Ｇｌｕｔａｔｈｉｏｎｅ Ｐｅｒｏｘｉｄａｓｅ １ ａｎｔｉｂｏｄｙ）である。また、内部コントロールとして、抗βアクチン抗体（Ｋ４８００（シグマ社）ａｎｔｉ−β−ａｃｔｉｎ ａｎｔｉｂｏｄｙ）を用いてβアクチンの発現量を検討した。結果を図５〜図８に示す。 Next, C2C12 cells after culture were collected, and the lysate of the cells was subjected to Western blotting to confirm the amount of intracellular SeP and GPx1. The antibodies used were the above-mentioned various anti-SeP monoclonal antibodies and anti-GPx1 antibodies (ab22604 (Abcam) Anti-Glutathione Peroxidase 1 antibody). Further, as an internal control, the expression level of β-actin was examined using an anti-β-actin antibody (K4800 (Sigma) anti-β-actin antibody). The results are shown in FIGS.
図中のＳｅＰは全長のＳｅＰを示し、Ｎ−ＳｅＰは全長ＳｅＰをカリクレイン処理して得られるＮ末端側の断片（２５４番目以降）を示す。各種モノクローナル抗体の中でも、ＡＥ２、ＡＡ３、及びＢＤ１はＣ２Ｃ１２細胞内へのＳｅＰ取り込みが阻害されることを示すことが明らかとなった。また、これらの抗体では、Ｃ２Ｃ１２細胞内のＧＰｘ１の量が少なくなることも明らかとなった。従って、上記３種類のモノクローナル抗体は、細胞内のＧＰｘ１の誘導を阻害することが強く示唆された。 In the figure, SeP represents the full-length SeP, and N-SeP represents an N-terminal fragment (254th and subsequent) obtained by treating the full-length SeP with kallikrein. Among various monoclonal antibodies, AE2, AA3, and BD1 have been shown to show that SeP uptake into C2C12 cells is inhibited. It was also revealed that these antibodies reduce the amount of GPx1 in C2C12 cells. Therefore, it was strongly suggested that the above three kinds of monoclonal antibodies inhibit the induction of GPx1 in cells.
次いで、ＳｅＰの濃度を１０μｇ／ｍＬと、２型糖尿病患者の血中濃度と同様の高濃度として、上述と同様の実験を行った。抗ＳｅＰ抗体はＡＥ２を用いた。結果を図９に示す。この結果、ＡＥ３は１０μｇ／ｍＬもの高い濃度のＳｅＰであっても、十分にＳｅＰの細胞内の取り込みも、細胞内でのＧＰｘ１の誘導も阻害することが明らかとなった。これは、少なくともＡＥ２抗体が、２型糖尿病の治療効果を発揮することを強く示唆している。 Subsequently, the experiment similar to the above was performed by setting the concentration of SeP to 10 μg / mL and a high concentration similar to the blood concentration of patients with type 2 diabetes. AE2 was used as the anti-SeP antibody. The results are shown in FIG. As a result, it has been clarified that AE3 sufficiently inhibits the uptake of SeP into the cell and the induction of GPx1 within the cell even when the concentration of SeP is as high as 10 μg / mL. This strongly suggests that at least the AE2 antibody exerts a therapeutic effect for type 2 diabetes.
さらに、細胞をＪｕｒｋａｔ細胞に代え、ＳｅＰの濃度を２７０ｎｇ／ｍＬ、とし、室温で１時間反応させたものを、Ｓｅ欠乏無血清培地で培養する上記Ｊｕｒｋａｔ細胞に添加した。引き続き３７℃で１２時間培養後に、細胞を回収し、細胞内のＳｅＰ及びＧＰｘ１の量をウエスタンブロッティングに供した。結果を図１０に示す。 Furthermore, the cells were replaced with Jurkat cells, and the concentration of SeP was 270 ng / mL and the reaction was performed at room temperature for 1 hour was added to the Jurkat cells cultured in Se-deficient serum-free medium. Subsequently, after culturing at 37 ° C. for 12 hours, the cells were collected, and the amounts of SeP and GPx1 in the cells were subjected to Western blotting. The results are shown in FIG.
この結果、ＤＣ１２及びＡＥ２は、上記Ｃ２Ｃ１２細胞の場合と同様に共に細胞内へのＳｅＰの取り込みを阻害することが明らかとなった。 As a result, it was revealed that DC12 and AE2 both inhibit the uptake of SeP into the cells as in the case of the C2C12 cells.
〔インビボ実験〕
ＡＥ２を５ｍｇ／ｋｇの濃度でＣ５７ＢＬ／６Ｊマウス（８週齢、雌に投与し、その後１、３、６、１２、２４時間後の血中での濃度を測定した。測定方法は、直接ＥＬＩＳＡ法に基づいて行った。また、投与方法は腹腔内投与と眼静脈投与の２種類の投与方法を試みた。結果を図１１に示す。
[In vivo experiment]
AE2 was administered to a C57BL / 6J mouse (8 weeks old, female at a concentration of 5 mg / kg, and the blood concentration was measured after 1, 3, 6, 12, and 24 hours thereafter. The measurement method was determined by direct ELISA. In addition, two administration methods, i.e., intraperitoneal administration and ocular vein administration, were tried, and the results are shown in FIG.
図１１に示すように、ＩＰ（腹腔内投与）であっても、ＩＶ（眼静脈投与）であっても、同様の血中濃度パターンを示し、共に投与から２４時間後もＡＥ２は血中に存在することが明らかとなった。 As shown in FIG. 11, both IP (intraperitoneal administration) and IV (ophthalmic vein administration) show similar blood concentration patterns, and AE2 remains in the blood even 24 hours after administration. It became clear that it existed.
次いで、ＳｅＰによるインスリン抵抗性の誘導をＡＥ２が阻害するかどうかを確認する実験を行った。先ず、Ｃ５７ＢＬ／６Ｊマウス（８週齢、雌）マウスにＡＥ２を１０ｍｇ／ｋｇの量の濃度で腹腔内投与し、２時間後に絶食環境下におくと共に１ｍｇ／ｋｇの量濃度でＳｅＰを腹腔内投与した。用いたＳｅＰは上述の全長のＳｅＰである。Ｓｅｐの投与から１０時間後に再度同量のＳｅＰを投与し、その２時間後に糖負荷行った。糖負荷は、マウスに１．５ｇ／ｋｇとなる量でグルコースを投与した。 Next, an experiment was conducted to confirm whether AE2 inhibits the induction of insulin resistance by SeP. First, AE2 was intraperitoneally administered to C57BL / 6J mice (8-week-old, female) mice at a concentration of 10 mg / kg. After 2 hours, the mice were placed in a fasting environment, and SeP was intraperitoneally administered at a concentration of 1 mg / kg. Administered. The SeP used is the above-mentioned full length SeP. Ten hours after administration of Sep, the same amount of SeP was administered again, and glucose loading was performed 2 hours later. Glucose load was administered to mice in an amount of 1.5 g / kg.
そして、糖負荷後の血中グルコース濃度を測定した。なお、コントロールとして、ＳｅＰを加えなかった実験群及びＡＥ２に代えてコントロール抗体であるラットＩｇＧを１０ｍｇ／ｋｇの量を投与した実験群を用意した。結果を図１２及び図１３に示す。 And the blood glucose level after a glucose load was measured. As controls, an experimental group in which SeP was not added and an experimental group in which rat IgG as a control antibody was administered in an amount of 10 mg / kg instead of AE2 were prepared. The results are shown in FIGS.
図１２（Ａ）に示すように、ＳｅＰ非投与群ではＡＥ２の投与、コントロール抗体の投与の間で糖負荷をかけても顕著な差は見られなかったが、ＳｅＰを投与してインスリン抵抗性が誘導されたマウス個体に糖負荷をかけた場合、ＡＥ２はコントロール抗体と比べて血中グルコース濃度を減少させることが明らかとなった。このような結果は、図１３に示す図１２の（Ａ）及び（Ｂ）のグラフから算出されたＡＵＣ値からも明らかである。これらの結果から、ＡＥ２はＳｅＰ誘導性のインスリン抵抗性を改善し、血糖値を下げる効果を発揮することが明らかとなった。従って、２型糖尿病の治療薬としてＡＥ２が有用であることが明らかとなった。 As shown in FIG. 12 (A), in the SeP non-administered group, there was no significant difference between the administration of AE2 and the administration of the control antibody even when a glucose load was applied. It was revealed that AE2 reduces blood glucose concentration compared to the control antibody when a glucose load is applied to a mouse individual in which is induced. Such a result is clear from the AUC value calculated from the graphs of FIGS. 12A and 12B shown in FIG. From these results, it has been clarified that AE2 improves the SeP-induced insulin resistance and exhibits the effect of lowering the blood glucose level. Therefore, it was revealed that AE2 is useful as a therapeutic agent for type 2 diabetes.
Claims (9)
配列番号３に示すアミノ酸配列からなる重鎖ＣＤＲ１、配列番号４に示すアミノ酸配列からなる重鎖ＣＤＲ２、及び配列番号５に示すアミノ酸配列からなる重鎖ＣＤＲ３を含む重鎖可変領域、並びに配列番号８に示すアミノ酸配列からなる軽鎖ＣＤＲ１、配列番号９に示すアミノ酸配列からなる軽鎖ＣＤＲ２、及び配列番号１０に示すアミノ酸配列からなる軽鎖ＣＤＲ３
を含む軽鎖可変領域を有する、抗体。 An antibody that specifically binds to an epitope present in amino acid sequence 204-261 of selenoprotein P , the antibody comprising:
A heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 3, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5, and SEQ ID NO: 8 A light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 9, and a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 10.
An antibody having a light chain variable region comprising
配列番号２に示すアミノ酸配列を含む重鎖可変領域、及び
配列番号７に示すアミノ酸配列を含む軽鎖可変領域
を有する、請求項１又は２に記載の抗体。 The antibody is
The antibody according to claim 1 or 2 , which has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 .
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