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CHOLESTEROL-DEPENDENT CYTOLYSIN VARIANT AND USE THEREOF IN DDS

Foreign code F120006881
File No. S2011-0448
Posted date Sep 26, 2012
Country WIPO
International application number 2012JP056203
International publication number WO 2012121395
Date of international filing Mar 9, 2012
Date of international publication Sep 13, 2012
Priority data
  • P2011-051295 (Mar 9, 2011) JP
Title CHOLESTEROL-DEPENDENT CYTOLYSIN VARIANT AND USE THEREOF IN DDS
Abstract The present invention provides a carrier whereby a drug capsule encapsulating a drug can be efficiently and very safely delivered to and incorporated into a variety of target cells or tissues, and also provides a drug delivery system in which the carrier is used. In the carrier, a cell- or tissue-specific antibody is bound to an antibody-binding domain of this cholesterol-dependent cytolysin variant. A cholesterol-containing microcapsule or functional cell packed with a medicinal ingredient or physiologically active substance serving as a carried material can be bound, via a domain (4) of the variant.
Outline of related art and contending technology BACKGROUND ART
Worldwide in recent years, from the viewpoint of cancer patients is important QOL(Quality of Life), cancer surgery, systemic dosage of anticancer agents, radiation irradiation or the like other than the main stream of cancer therapy, immune cells and cancer therapy has been studied in a missile therapy. For this purpose and efficiently with a specific cancer cell targeting therapeutic drugs with fewer side effects acting on the development of tools has been desired. Cancer such as for example monoclonal antibodies used as a targeting molecule to the targeting technology for the development of anti-cancer linked to it and also to many investigations of being made. However, in practice, the target targeting molecules thereof in cancer cells in a large amount is not enough integrated technology, further improvement is necessary.
Also a number of viruses such as AIDS and hepatitis infection, congenital genetic disease or gene expression is further based on a malfunction such as illnesses are a significant problem in recent years. Or suppressing the development in the treatment of these diseases, suppression of gene expression or by the method of the RNAi, gene therapy for restoration of a particular gene can be applied, the future important in medicine has been a problem. Is the current drug deliver RNAi atelocollagen nonspecific carrier is used, gene therapy is generally put into practical use, targeted cells or tissues and organs selectively delivering the gene expression modulation becomes essential.
In view of these circumstances, conventionally, such as anti-cancer therapeutic drugs or gene therapy agent is encapsulated in a liposome at a high concentration, in an efficient and high safety is delivered to the target cells and tissues for the treatment of a disease in which the construction of drug delivery system (DDS) has been demanded.
In addition, a technique similar to the present invention as described above is disclosed, the present application are known in the prior literature, patent document 1, non-patent document 1 and 2 can be exemplified.
However, Patent Document 1 is, specifically recognizing the human cell membrane of the cell membrane binding domain N inter-media ricin Cys residues introduced at the terminus of the human cells specific binding can be found out, this characteristic is utilized to membrane-bound human cells as an adapter not merely proposes to use. The adapter comprises a membrane-bound human cells, human cells binds to the membrane a variety of materials useful for the compatibility with the immobilized, of the agent into the cell, no function of intracellular release of a second.
Non-patent document 1 is also, to the cell membrane by the technology described in the above-mentioned Patent Document 1 binding domain of the remaining Cys N inter-media ricin group was introduced into the end of the domain to 4, further anti-carcinoembryonic antigen (CEA) cancer which is coupled to the antibody, the positive human CEA such as medullary thyroid cancer cells can be cancer cells, for the treatment of cancer and can be applied to the present invention. This technique is, in the sense that it can target cancer cells is useful but, in the same manner as described in the patent document 1, a drug such as anticancer agent into the cell, intracellular release of a second effect cannot be expected.
Non-patent document 2 is further, cholesterol dependent cell lysis in the same manner as the present invention (cholesterol-dependent cytolysin toxin: CDC) using the drug delivery system is disclosed, by the introduction of a pore-forming ability binding SS controlled CDC (CDC-SS) variant N-terminus of a peptide having high affinity to lung carcinoma cells (lung cancer targeting domain) may be conjugated to a, specific drug delivery to lung carcinoma cells only during transport technology. That is, not only the specificity of lung cancer cells, the target variable because there is no general-purpose properties deteriorate.
Scope of claims (In Japanese)請求の範囲 [請求項1]
(1)抗体結合ドメイン、
(2)コレステロール依存性細胞溶解毒素のドメイン1~3において、少なくとも2つの任意のアミノ酸残基がCys残基に置換されて、非還元条件下で互いにSS結合を形成してなる改変ドメイン1~3、及び
(3)コレステロール依存性細胞溶解毒素のドメイン4において、すべてのCys残基がAla、Ser、Gly及びThrから選択されるいずれかのアミノ酸残基に置換されてなる改変ドメイン4を有するコレステロール依存性細胞溶解毒素の変異体であって、
細胞膜またはコレステロール含有リポソーム膜に対して結合性を有し、且つ還元条件下でSS結合が開裂して膜孔形成能を発揮することを特徴とする、コレステロール依存性細胞溶解毒素の変異体。

[請求項2]
コレステロール依存性細胞溶解毒素がスイリシン及びインターメディリシンから選択される少なくとも1種である、請求項1に記載するコレステロール依存性細胞溶解毒素の変異体。

[請求項3]
コレステロール依存性細胞溶解毒素のドメイン1~3及びドメイン4がいずれもスイリシンに由来するものであるか、またはコレステロール依存性細胞溶解毒素のドメイン1~3はインターメディリシンに由来し、ドメイン4はスイリシンに由来するものである、請求項1または2に記載するコレステロール依存性細胞溶解毒素の変異体。

[請求項4]
抗体結合ドメインが黄色ブドウ球菌プロテインAのZドメイン、またはG群レンサ球菌に由来するプロテインGのBドメインである請求項1乃至3のいずれかに記載するコレステロール依存性細胞溶解毒素の変異体。

[請求項5]
上記還元条件下が細胞内のグルタチオンによる還元環境である請求項1乃至4のいずれかに記載するコレステロール依存性細胞溶解毒素の変異体。

[請求項6]
細胞内のグルタチオンによる還元環境が1~10mMの還元型グルタチオンを含むファゴリソソーム内または細胞質内の環境である請求項5に記載するコレステロール依存性細胞溶解毒素の変異体。

[請求項7]
請求項1乃至6のいずれかに記載するコレステロール依存性細胞溶解毒素の変異体に、その抗体結合ドメインを介して、細胞または組織特異的抗体が結合してなる、薬物運搬体。

[請求項8]
細胞または組織特異的抗体が、癌細胞に特異的に発現する蛋白質またはオリゴ糖を特異的に認識する抗体、ウイルス感染細胞の細胞膜上に出現するウイルスタンパク質を特異的に認識する抗体、及び免疫細胞に特異的に発現するCD抗原を特異的に認識する抗体である、請求項7に記載する薬物運搬体。

[請求項9]
請求項7または8に記載する薬物運搬体に、コレステロール依存性細胞溶解毒素の変異体のドメイン4を介して、薬効成分若しくは生理活性物質が充填されたコレステロール含有マイクロカプセルまたは機能性細胞が結合してなる、ドラッグデリバリーシステム。

[請求項10]
上記機能性細胞が免疫細胞または遺伝子組み換え細胞である、請求項9記載のドラッグデリバリーシステム。

[請求項11]
上記薬効成分または生理活性物質が、薬効や生理活性を発揮する化合物、ペプチド、タンパク質、核酸、及びドミナントネガティブ効果を有する物質からなる群から選択される少なくとも1つである、請求項9または10に記載するドラッグデリバリーシステム。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • TOKUSHIMA UNIVERSITY
  • Inventor
  • NAGAMUNE, Hideaki
  • TOMOYASU, Toshifumi
  • TABATA, Atsushi
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH 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 IS JP KE KG KM 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 PE PG PH PL PT QA RO RS RU RW 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 MD 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 ML MR NE SN TD TG
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