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HIGHLY-FUNCTIONAL MUTANT OF HUMANIZED ANTI-EGFR ANTIBODY VARIABLE REGION

Foreign code F120006673
File No. S2010-0177
Posted date May 24, 2012
Country WIPO
International application number 2010JP070127
International publication number WO 2011062112
Date of international filing Nov 11, 2010
Date of international publication May 26, 2011
Priority data
  • P2009-263147 (Nov 18, 2009) JP
Title HIGHLY-FUNCTIONAL MUTANT OF HUMANIZED ANTI-EGFR ANTIBODY VARIABLE REGION
Abstract Disclosed is an antibody that exhibits excellent cytotoxicity and cell growth inhibition and that is based on an anti-human epithelial cell growth factor receptor (1) (Her1) antibody (528). Further disclosed is a method for producing same, and the like. The mutant of an H chain humanized variable region (5H) or an L chain humanized variable region (5L) of the anti-human epithelial cell growth factor receptor (1) (Her1) antibody (528) is the aforementioned antibody characterized by having one to a plurality (for example: 1 to 5, or 1 to 3) of amino acid mutations within CDR2. Further disclosed are antibody molecules containing said region, a nucleic acid molecule coding for these polypeptides, a method for producing said antibody molecules, and the like.
Outline of related art and contending technology BACKGROUND ART
Rheumatoid arthritis and cancer (malignant tumor) security against therapy, in recent years, immunotherapy has been used. Immunotherapy for cancer, cancer cytotoxic activity specific for the antibodies are used. Such antibodies from the antibody medicament is, with fewer side effects, with the manufacturing of high therapeutic effect can be seen that on the other hand, an established animal cell is used to generate high costs for manufacture must be in question.
For this reason, of an antibody VH and VL domains of a single polypeptide chain in the single-chain antibody (scFv) antibody of the fabrication of a low molecule is a worldwide trend. The small molecule antibodies may also be produced in E. coli is inexpensive, by lowering of the molecular weight is reduced in vivo half-life, efficacy duration is reduced there is a concern. In addition, is an intact antibody such as IgG, against an antigen in a multivalent target binding on the other hand, typically, a monovalent low-molecular-weight antibody valency and therefore, the decrease of the affinity in question. Further, the main mechanism of action of antibody pharmaceuticals Fc mediated antibody dependent cell-mediated cytoxicity (ADCC) since the lit, do not have scFv Fc is a concern that the effect is low. It should be noted that, for the scFv, the non-patent document 1 can be referred to.
For this reason, specific immune cells and the cancer cells with the small molecule bridge the bispecific antibody and the like have been developed, referred to as BiTE, one portion of the first scFv and 2 tandem scFv type low-molecular bispecific antibody (Science. 2008 Aug 15 advanced in clinical trials is the only; 321 (5891): 974-7.). However this BiTE is, since the prepared using animal cells, the problems inherent to the yield and the manufacturing cost. Such as BiTE tandem scFv type low-molecular double-specific antibody may be, of the soluble low molecular weight prepared from E. coli but is difficult and also reports (J Mol Biol. 2003 330 (1): 99-111.), using the fact that animal cells BiTE is prepared.
One of the multispecific antibody is bispecific antibody (Bispecific Antibody: BsAb) two different 2 is capable of binding to a specifically to an antigen and therefore, this making the specific anti-tumor having an effect as a therapeutic as enabling a utilization method, which has been actively carried out thereof. (Diabody: Db) diabody such bispecific antibody is a minimum unit, derived from the same parent antibody variable region of the heavy chain (H chain) (V region) and light chain ('VH' by each other) the variable region of (L chain) ('VL' by each) (V region) non-covalently to each other to form a heterodimer is devised utilizing a property that the (non-patent document 2) in.
Such a diabody-type bispecific antibody is characterized by a high, low-molecular-weight (molecular weight of about 60,000) and low immunogenic attributed to the fact that high-permeability to tumor tissue, and further, for example, using microorganisms such as Escherichia coli can be prepared by an inexpensive mass, also, the functions using the genetic engineering can be readily changed can be exemplified.
The present inventors have, thus far, anti-human epidermal growth factor receptor antibody 528 and anti-CD3 antibody 1 OKT3 (Her1) that is produced using a diabody-type bispecific antibodies and humanized antibody (Ex3) and the diabody-type bispecific antibody is extremely potent anti-tumor effect (hExh3) has a finding (Patent Document 1). Further, other antibody that is produced using a diabody-type bispecific antibody by comparison, the above diabody-type bispecific antibody exhibits a superior effect in order to, humanized 528 antibody and humanized OKT3 variable region of the humanized antibody of the structural stability of itself, and combinations thereof is very important to be estimated.
Further, the present inventors have humanized diabody-type bispecific antibodies is based on the control of diverse structure and are highly functional bispecific antibody (Patent Document 2) have developed.
It should be noted that, other than the diabody-type bispecific antibody in the preparation of a bispecific antibody and the like, and non-patent document Non-patent Documents 3 to 4 described.
1 Anti-human epidermal growth factor receptor antibody 528 (Her1) cancer cell growth inhibiting effect. However, as already described, in the case of binding to a monovalent divalent EGFR, a low affinity for the antigen, the effect is hardly known. Actually humanized 528 (scFv) is a single chain antibody of cancer cell growth inhibiting effect was observed. A scFv in order to overcome such a drawback, by modification of the linker scFv already has been tried is the multimerization of (non-patent document 5). In recent years, inducing apoptosis in lymphoma scFv dimer and the like have been reported (non-patent document 6). However, with respect to solid cancer and carcinoma positive EGFR, exhibit growth inhibitory effects of the multimeric scFv not reports.
Scope of claims (In Japanese)請求の範囲 [請求項1]
配列番号4で示されるアミノ酸配列から成る抗ヒト上皮細胞成長因子受容体1(Her1)抗体528のH鎖のヒト型化可変領域(5H)の変異体、又は、配列番号2で示されるアミノ酸配列から成る抗ヒト上皮細胞成長因子受容体1(Her1)抗体528のL鎖のヒト型化可変領域(5L)の変異体であって、CDR2内に1個~数個のアミノ酸変異を有することを特徴とする前記変異体。

[請求項2]
配列番号4で示されるアミノ酸配列から成る抗ヒト上皮細胞成長因子受容体1(Her1)抗体528のH鎖のヒト型化可変領域(5H)の変異体であって、該変異体を含むFv抗体の結合係数(Ka)が抗体528のH鎖のヒト型化可変領域(5H)を含むFv抗体の8~33倍となるような、前記変異体。

[請求項3]
5Hの52番目のチロシンがトリプトファンに置換している、請求項1又は2記載の変異体。

[請求項4]
更に、55番目のセリンが置換している、請求項3記載の変異体。

[請求項5]
55番目のセリンがトレオニン、リシン、アルギニン、アスパラギン及びグルタミンから成る群から選択されるアミノ酸に置換している、請求項4記載の変異体。

[請求項6]
更に、63番目のリシン、65番目のリシン、及び/又は66番目のアスパラギンが置換している、請求項4又は5記載の変異体。

[請求項7]
63番目のリシンがグルタミンに置換している、請求項6記載の変異体。

[請求項8]
65番目のリシンがグルタミンに置換している、請求項6記載の変異体。

[請求項9]
66番目のアスパラギンがグルタミン、リシン、又はセリンに置換している、請求項6記載の変異体。

[請求項10]
更に、97番目のアラニンが変異している、請求項6記載の変異体。

[請求項11]
97番目のアラニンがトレオニンに変異している、請求項10記載の変異体。

[請求項12]
配列番号2で示されるアミノ酸配列から成る抗ヒト上皮細胞成長因子受容体1(Her1)抗体528のL鎖のヒト型化可変領域(5L)の変異体であって、該変異体を含むFv抗体の結合係数(Ka)が抗体528のL鎖のヒト型化可変領域(5L)を含むFv抗体の50~200倍となるような、前記変異体。

[請求項13]
5Lにおける55番目のリシンがロイシンに、58番目のアスパラギン酸がアルギニンに、及び/又は、60番目のフェニルアラニンがセリン若しくはロイシンに置換している、請求項1又は2記載の変異体。

[請求項14]
請求項1~13のいずれか一項に記載の変異体を構成要素として含む抗体分子。

[請求項15]
IgG型抗体分子、ヒト型化ダイアボディ型二重特異性抗体、高機能性二重特異性抗体、抗体分子、及び、多量体化低分子抗体から成る群から選択される、請求項13記載の抗体分子。

[請求項16]
抗ヒト上皮細胞成長因子受容体1抗体528のL鎖のヒト型化可変領域(5L)、抗CD3抗体OKT3のL鎖のヒト型化可変領域(OL)、及び、抗CD3抗体OKT3のH鎖のヒト型化可変領域(OH)が、夫々、配列番号2,6及び8で示されるアミノ酸配列から成る、請求項14又は15記載の抗体分子。

[請求項17]
請求項14~16のいずれか一項に記載の抗体分子を構成する一本鎖ポリペプチド。

[請求項18]
請求項1~13のいずれか一項に記載の変異体、又は、請求項16に記載の一本鎖ポリペプチドをコードする核酸分子。

[請求項19]
請求項14~16のいずれか一項に記載の抗体分子を構成する2種類の一本鎖ポリペプチドを共にコードする核酸分子。

[請求項20]
請求項18又は19記載の核酸分子を含有する複製可能なクローニングベクター又は発現ベクター。

[請求項21]
共発現ベクターである、請求項20記載のベクター。

[請求項22]
プラスミドベクターである、請求項20又は21記載のベクター。

[請求項23]
請求項21ないし22のいずれか一項に記載のベクターで形質転換された宿主細胞。

[請求項24]
請求項14~16のいずれか一項に記載の抗体分子の製造方法であって、請求項21に記載の宿主細胞を培養し、該抗体分子を構成する2種類の一本鎖ポリペプチドを発現させ、該ポリペプチドを回収・精製し、該2種類の一本鎖ポリペプチドを会合させ、形成された抗体分子を分離・回収することから成る、前記方法。

[請求項25]
原核細胞が大腸菌であり、2種類の一本鎖ポリペプチドを大腸菌の培養培地上清、ペリズマ画分、菌体内可溶性画分、又は、菌体内不溶性画分から回収する、請求項24記載の製造方法。

[請求項26]
請求項14~16のいずれか一項に記載の抗体分子の製造方法であって、請求項21記載の共発現ベクターで形質転換された宿主細胞を培養し該抗体分子を構成する2種類の一本鎖ポリペプチドを発現させ、該形質転換菌内でダイアボディ型二重特異性抗体を形成せしめ、形成された二重特異性抗体を分離・回収することから成る、前記方法。

[請求項27]
請求項14~16のいずれか一項に記載の抗体分子を有効成分として含有することを特徴とする医薬組成物。

[請求項28]
腫瘍細胞を排除する、殺傷する、傷害する及び/又は減少せしめるためのものであることを特徴とする請求項26記載の医薬組成物。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • TOHOKU UNIVERSITY
  • Inventor
  • KUMAGAI Izumi
  • NAKANISHI Takeshi
  • ASANO Ryutaro
  • UMETSU Mitsuo
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 RO RS RU 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 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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