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CYCLIC SINGLE-CHAIN ANTIBODY

Foreign code F200010150
File No. (S2018-0765-N0)
Posted date Jun 2, 2020
Country WIPO
International application number 2019JP026983
International publication number WO 2020013126
Date of international filing Jul 8, 2019
Date of international publication Jan 16, 2020
Priority data
  • P2018-130203 (Jul 9, 2018) JP
Title CYCLIC SINGLE-CHAIN ANTIBODY
Abstract Provided is a cyclic scFv wherein the N-terminus of an scFv, in which the heavy chain variable region (VH) is linked to the light chain variable region via a first peptide linker, is linked to the C-terminus thereof via a second peptide linker.
Outline of related art and contending technology BACKGROUND ART
Monoclonal antibodies, oncology, chronic inflammatory diseases, transplant, infection, a cardiovascular physician, or ophthalmic diseases in various clinical use as therapeutic agents, in addition, the inspection agent, such as the sensor element, being used in various applications. The main function of an antibody specific for an antigen (target molecule) and binding, antigen, Fv are recognized by the domain.
From the heavy chain Fv antibody Fv domains of the Fv (VH) and a light chain derived from the domain of the domain from (VL). Fv antibody domain has been extracted from the fragment in the target antibody Fv fragment number and the ability to bind to, the minimum unit of an antibody antigen-binding function of the form. Single-chain antibody (scFv: single-chain Fv) VL and VH is connected by a peptide linker to the present invention. For example, advanced glycation end products (AGEs) scFv recognize has been reported (Non-Patent Document 1).
Such as CHO cells or hybridoma monoclonal antibody is typically a eukaryotic cell from the used in production, a great cost is required for the production. ScFv about 25kDa molecular weight, molecular weight as compared with the full-length antibodies are very small. Therefore, such as E. coli as a host prokaryotic or eukaryotic organism can be produced and used, as compared with the length of the scFv antibody production has advantages in terms of cost.
Also corresponds to a trimer of scFv annular stranded trispecific antibodies are also been reported (Patent Document 1). ScFv is generally associated with the other hand forming a multimer has the property, the scFv to form the dimer, trimer, and tetramer has been reported in relation to the (non-patent document 2-4). Thus easily forming a multimer scFv the property that, in terms of improvement in the stability of scFv has been a problem.
The stability of the antibody molecule include the meaning of the one 3. The first is the heat resistance or thermal stability and, the second is the stability and proteolytic enzymes, and the third is due to suppress the formation of an aggregate storage stability. ScFv is particularly a problem in storage stability if it has.
In a modification of the peptide sortase and have been reported a method using (Patent Document 2 and 3, and 6 and Non-Patent Document 5), using the methods for synthesis of the cyclic peptide sortase has been reported (Non-Patent Document 7) is.
Nintein, intein and the central portion, which sandwich (exteins) configured by the sequence. Nintein, intein from the cut portion of the self-host sequences, flanking the peptide bond cleavage by a reaction (exteins) connecting the internal protein factors. Results in the reaction, the enzyme does not require auxiliary or cofactors in the post-translational processes (non-patent document 8). Arranged upstream of an intein exteins sequence ' N- exteins' is referred to as, and disposed downstream of an intein exteins' C- exteins' is called. As the reaction product of an intein, one 2 of the mature protein cleavage can be obtained stable proteins.
Nintein, can be divided into two, and, 2 and two separate transcription and translation of the one encoded by the gene can be present as a fragment 2. Referred to as split intein Nintein divided, self-association, and linked by a peptide bond to N- C - exteins exteins protein splicing reaction to the catalyst. Split intein is, the division results in artificially can be manufactured. And also present in the natural, and archaebacterial plasfocjanin various been confirmed in the (non-patent document 9-14). Nostoc punctiforme DnaE (NpuDnaE) is derived from, have been reported in non-patent document is the highest reaction efficiency in the split intein Nintein (non-patent document 14 and 15). Cleavage reaction and a cyclization reaction of the protein of interest also can be used as has been reported (Patent Document 4 and 5).
Scope of claims (In Japanese)[請求項1]
 重鎖可変領域(VH)および軽鎖可変領域(VL)が第1のペプチドリンカーで連結された一本鎖抗体(scFv)において、そのN末端とC末端が第2のペプチドリンカーで連結されている、環状一本鎖抗体。

[請求項2]
 環状構造がペプチド結合のみで形成されている、請求項1に記載の環状一本鎖抗体。

[請求項3]
 VHとVLが分子内で会合して抗原結合部位を形成する、請求項1または2に記載の環状一本鎖抗体。

[請求項4]
 分子内に1つの抗原結合部位を有する、請求項1~3のいずれか1項に記載の環状一本鎖抗体。

[請求項5]
 第1のペプチドリンカーが15~27個のアミノ酸からなる、請求項1~4のいずれか1項に記載の環状一本鎖抗体。

[請求項6]
 第2のペプチドリンカーが15~28個のアミノ酸からなる、請求項1~5のいずれか1項に記載の環状一本鎖抗体。

[請求項7]
 VHおよびVLが同一で非環状の一本鎖抗体と比較して、凝集体形成が抑制されている、請求項1~6のいずれか1項に記載の環状一本鎖抗体。

[請求項8]
 第2のペプチドリンカーがトランスペプチダーゼにより形成される、請求項1~7のいずれか1項に記載の環状一本鎖抗体。

[請求項9]
 トランスペプチダーゼがソルターゼである、請求項8に記載の環状一本鎖抗体。

[請求項10]
 第2のペプチドリンカーがアミノ酸配列:LPXTG(ここで、Xは任意のアミノ酸残基を表す)を含む、請求項1~9のいずれか1項に記載の環状一本鎖抗体。

[請求項11]
 第2のペプチドリンカーがスプリットインテインによるトランス-スプライシング反応により形成される、請求項1~7のいずれか1項に記載の環状一本鎖抗体。

[請求項12]
 請求項1~7のいずれか1項に記載の環状一本鎖抗体の製造方法であって、
 1)重鎖可変領域(VH)および軽鎖可変領域(VL)が第1のペプチドリンカーで連結された一本鎖抗体(scFv)であって、N末端およびC末端にトランスペプチダーゼ認識配列を有する非環状ペプチドを調製する工程;
 2)トランスペプチダーゼを用いて前記一本鎖抗体のN末端およびC末端のトランスペプチダーゼ認識配列から第2のペプチドリンカーを形成し、前記一本鎖抗体を環化する工程
を含む、前記製造方法。

[請求項13]
 トランスペプチダーゼがソルターゼである、請求項8に記載の製造方法。

[請求項14]
 非環状ペプチドがN末端のトランスペプチダーゼ認識配列がLPXTG(ここで、Xは任意のアミノ酸残基を表す)を含む、請求項12または13に記載の製造方法。

[請求項15]
 非環状ペプチドがC末端のトランスペプチダーゼ認識配列がGGを含む、請求項12~14のいずれか1項に記載の製造方法。

[請求項16]
 請求項1~7のいずれか1項に記載の環状一本鎖抗体の製造方法であって、
 1)重鎖可変領域(VH)および軽鎖可変領域(VL)が第1のペプチドリンカーで連結された一本鎖抗体(scFv)であって、N末端にスプリットインテインのC末端側断片(Int-C)およびC末端にスプリットインテインのN末端側断片(Int-N)をそれぞれ有する非環状ペプチドを調製する工程;
 2)スプリットインテインによるトランス-スプライシング反応により第2のペプチドリンカーを形成し、前記一本鎖抗体を環化する工程
を含む、前記製造方法。

[請求項17]
 スプリットインテインのC末端側断片(Int-C)としてDnaE-Int-C、N末端側断片(Int-N)としてDnaE-Int-Nが用いられる、請求項16に記載の製造方法。

[請求項18]
 請求項16の工程1に記載の非環状ペプチドのアミノ酸配列をコードする塩基配列を含む核酸。

[請求項19]
 請求項18に記載の核酸を含有する組換えベクター。

[請求項20]
 請求項19に記載の組換えベクターを導入した形質転換体。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION KUMAMOTO UNIVERSITY
  • Inventor
  • MORIOKA HIROSHI
  • KOBASHIGAWA YOSHIHIRO
  • SATO TAKASHI
  • FUKUDA NATSUKI
  • YAMAUCHI SOICHIRO
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 DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS 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 ST TD TG
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