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NOVEL HIGH-FUNCTIONING ENZYME HAVING ALTERED HUMAN β-HEXOSAMINIDASE B SUBSTRATE SPECIFICITY, AND HAVING PROTEASE RESISTANCE APPLIED THERETO

Foreign code F140008026
File No. S2013-0022-C0
Posted date Nov 19, 2014
Country WIPO
International application number 2013JP078179
International publication number WO 2014061735
Date of international filing Oct 17, 2013
Date of international publication Apr 24, 2014
Priority data
  • P2012-232266 (Oct 19, 2012) JP
Title NOVEL HIGH-FUNCTIONING ENZYME HAVING ALTERED HUMAN β-HEXOSAMINIDASE B SUBSTRATE SPECIFICITY, AND HAVING PROTEASE RESISTANCE APPLIED THERETO
Abstract Provided is an improved β-subunit of human β-hexosaminidase, having activity derived from a α-subunit of wild-type human β-hexosaminidase, and having resistance to protease. A protein comprising, in the amino acid sequence for a β-subunit of wild-type human β-hexosaminidase, an amino acid sequence in which the 312th-318th amino acids have been substituted, in order, for glycine, serine, glutamic acid, proline, serine, glycine and threonine respectively.
Outline of related art and contending technology BACKGROUND ART
- Tailing directional zandohoffu saxophone disease and a disease that is, both, by a decrease in the activity of β - hexosaminidase A (Hex A), neural cell GM2 ganglioside is accumulated to the cranial nerves causing disease symptoms. Is Hex A, α - β - subunit of a heterodimeric subunits and, having an activity of enzymes that degrade the ganglioside GM2. - Tailing directional saxophone is a disease that is, based on the α - subunit Hex A deficiency and deficiency, is a disease that is zandohoffu, based on the subunit β - Hex A deficiency is deficient.
The present inventors discovered that this, with respect to the CHO cell lines and special yeast strain, α - β - subunit and the subunit encoded by the gene (each HEXA cDNA and HEXB cDNA) is inserted into the expression vector is introduced, the wild-type recombinant Hex A to establish a cell line which expresses constantly are. In this method the production of wild-type recombinant Hex A, was administered to mice zandohoffu disease models, GM2 accumulated in the cranial nervous system gangliosides showed an improvement of diarrhea and weight loss, disease - or tailing directional saxophone zandohoffu to check the validity of the disease and enzyme replacement therapies (Patent Document 1; non-patent document 1).
However, in general, an enzyme deficient patient and repeatedly administering the therapeutic agent, therapeutic agent in many cases the enzymes in the body and recognized as foreign, resulting in an antibody is produced, as a result, adverse side reactions such as anaphylactic reactions and allergic reaction appears. Therefore, it is wild-type recombinant Hex A saxophone - tailing directional zandohoffu disease when administered to a patient's disease is, in the same manner as described above, to possibly result in harmful side reactions. In addition, the wild-type recombinant Hex A, low stability in blood (in plasma), organ failure (neural cell) to the cells is a problem of low efficiency of incorporation of the.
In order to solve these problems, the inventors have found that α - β - β - hexosaminidase subunit and of the dimensional structure of the subunit based on the information, the active site of the α - subunit β - subunit was substituted with the active site of a modified β - subunit was produced. Then, this modified β - constitute a sub-unit as a component, a modified homodimer β - hexosaminidase B type (hereinafter, referred to as' ModB ') was prepared, the recombinant enzyme has activity in gangliosides GM2 was confirmed to be (Patent Document 2; non-patent document 2).
Scope of claims (In Japanese)請求の範囲 [請求項1]
 野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットのアミノ酸配列において、第312番目~第318番目のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸、プロリン、セリン、グリシン、及び、トレオニンに置換されているアミノ酸配列を含む、タンパク質。

[請求項2]
 さらに、第452番目のアミノ酸がアスパラギンに、及び/又は、第453番目のアミノ酸がアルギニンに置換されている、請求項1に記載のタンパク質。

[請求項3]
 以下の(i)~(iii)より選択されるいずれかのアミノ酸配列を含む、請求項2に記載のタンパク質:
 (i)配列番号6で示されるアミノ酸配列;
 (ii)配列番号6で示されるアミノ酸配列において、前記置換部位のアミノ酸を除く1から数個のアミノ酸が欠失、置換、又は、付加されたアミノ酸配列であって、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有し、且つ、プロテアーゼに対する抵抗性を有するタンパク質をコードするアミノ酸配列;あるいは
 (iii)配列番号6で示されるアミノ酸配列と少なくとも90%以上の配列同一性を有するアミノ酸配列であって、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有し、且つ、プロテアーゼに対する抵抗性を有するタンパク質をコードするアミノ酸配列(ただし、前記置換部位のアミノ酸は配列番号6で示されるアミノ酸配列と同一である)。

[請求項4]
 請求項1~3のいずれか1項に記載のタンパク質のホモ二量体からなる、タンパク質。

[請求項5]
 請求項1~3のいずれか1項に記載のタンパク質をコードする遺伝子。

[請求項6]
 請求項5に記載の遺伝子を含む組換えベクター。

[請求項7]
 請求項6に記載の組換えベクターを含む形質転換体。

[請求項8]
 請求項7に記載の形質転換体を培養する工程と、得られる培養物から野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有し、且つ、プロテアーゼに対する抵抗性を有するタンパク質を採取する工程とを含む、該タンパク質の製造方法。

[請求項9]
 請求項1~4のいずれか1項に記載のタンパク質を含むことを特徴とする、テイ-サックス病治療用医薬組成物。

[請求項10]
 請求項5に記載の遺伝子を含むことを特徴とする、テイ-サックス病治療用医薬組成物。

[請求項11]
 請求項1~4のいずれか1項に記載のタンパク質を含むことを特徴とする、ザンドホッフ病治療用医薬組成物。

[請求項12]
 請求項5に記載の遺伝子を含むことを特徴とする、ザンドホッフ病治療用医薬組成物。

[請求項13]
 請求項1~4のいずれか1項に記載のタンパク質、請求項5に記載の遺伝子、又は、請求項9若しくは10に記載のテイ-サックス病治療用医薬組成物を、テイ-サックス病の患者に投与することを含む、テイ-サックス病の治療方法。

[請求項14]
 請求項1~4のいずれか1項に記載のタンパク質、請求項5に記載の遺伝子、又は、請求項11若しくは12に記載のザンドホッフ病治療用医薬組成物を、ザンドホッフ病の患者に投与することを含む、ザンドホッフ病の治療方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • TOKUSHIMA UNIVERSITY
  • MEIJI PHARMACEUTICAL UNIVERSITY
  • Inventor
  • ITO Koji
  • SAKURABA Hitoshi
  • TSUJI Daisuke
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN TD TG
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