RU2012156248A - Полученные с помощью генной инженерии антитела с цистеиновыми заменами и их конъюгаты - Google Patents
Полученные с помощью генной инженерии антитела с цистеиновыми заменами и их конъюгаты Download PDFInfo
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- RU2012156248A RU2012156248A RU2012156248/10A RU2012156248A RU2012156248A RU 2012156248 A RU2012156248 A RU 2012156248A RU 2012156248/10 A RU2012156248/10 A RU 2012156248/10A RU 2012156248 A RU2012156248 A RU 2012156248A RU 2012156248 A RU2012156248 A RU 2012156248A
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Classifications
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- C—CHEMISTRY; METALLURGY
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
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- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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Abstract
1. Выделенное, полученное с помощью генной инженерии антитело с цистеиновыми заменами, содержащее свободную цистеиновую аминокислоту и последовательность тяжелой цепи, выбранную из SEQ ID NO:50-98, или последовательность легкой цепи, выбранную из SEQ ID NO:99-147, где цистеин в последовательности представляет собой свободную цистеиновую аминокислоту.2. Выделенное, полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.1, полученное с помощью способа, включающего:(i) мутагенез последовательности нуклеиновых кислот исходного антитела посредством замены одного или нескольких аминокислотных остатков цистеином для кодирования полученного с помощью генной инженерии антитела с цистеиновыми заменами;(ii) экспрессирование полученного с помощью генной инженерии антитела с цистеиновыми заменами и(iii) изолирование полученного с помощью генной инженерии антитела с цистеиновыми заменами.3. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.2, где мутагенез включает сайт-направленный мутагенез.4. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.2, где полученное с помощью генной инженерии антитело с цистеиновыми заменами экспрессируют на вирусной частице, выбранной из фага или частицы фагмида.5. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.2, дополнительно включающее:(i) взаимодействие полученного с помощью генной инженерии антитела с цистеиновыми заменами с реакционноспособным по отношению к тиолу аффинным реагентом, для генерирования аффинно меченого, полученного с помощью генной инженерии антитела с цистеиновыми заменами
Claims (28)
1. Выделенное, полученное с помощью генной инженерии антитело с цистеиновыми заменами, содержащее свободную цистеиновую аминокислоту и последовательность тяжелой цепи, выбранную из SEQ ID NO:50-98, или последовательность легкой цепи, выбранную из SEQ ID NO:99-147, где цистеин в последовательности представляет собой свободную цистеиновую аминокислоту.
2. Выделенное, полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.1, полученное с помощью способа, включающего:
(i) мутагенез последовательности нуклеиновых кислот исходного антитела посредством замены одного или нескольких аминокислотных остатков цистеином для кодирования полученного с помощью генной инженерии антитела с цистеиновыми заменами;
(ii) экспрессирование полученного с помощью генной инженерии антитела с цистеиновыми заменами и
(iii) изолирование полученного с помощью генной инженерии антитела с цистеиновыми заменами.
3. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.2, где мутагенез включает сайт-направленный мутагенез.
4. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.2, где полученное с помощью генной инженерии антитело с цистеиновыми заменами экспрессируют на вирусной частице, выбранной из фага или частицы фагмида.
5. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.2, дополнительно включающее:
(i) взаимодействие полученного с помощью генной инженерии антитела с цистеиновыми заменами с реакционноспособным по отношению к тиолу аффинным реагентом, для генерирования аффинно меченого, полученного с помощью генной инженерии антитела с цистеиновыми заменами; и
(ii) измерение связывания аффинно меченого, полученного с помощью генной инженерии антитела с цистеиновыми заменами со средами для захвата.
6. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.5, где реакционноспособный по отношению к тиолу аффинный реагент содержит биотиновый остаток.
7. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.5, где реакционноспособный по отношению к тиолу реагент содержит малеимидный остаток.
8. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.5, где среды для захвата содержат стрептавидин.
9. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.1, где полученное с помощью генной инженерии антитело с цистеиновыми заменами представляет собой слитый белок, содержащий пептид, связывающий альбумин (ABP).
10. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.9, где ABP содержит последовательность, выбранную из SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 и SEQ ID NO:5.
11. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.1, где полученное с помощью генной инженерии антитело с цистеиновыми заменами выбирают из моноклонального антитела, фрагмента антитела, биспецифического антитела, химерного антитела, антитела человека и гуманизированного антитела.
12. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.11, где фрагмент антитела представляет собой Fab фрагмент.
13. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.11, где полученное с помощью генной инженерии антитело с цистеиновыми заменами представляет собой антитело анти-HER2.
14. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.1, где полученное с помощью генной инженерии антитело с цистеиновыми заменами связывается с одним или несколькими из рецепторов (1)-(36):
(1) BMPR1B (рецептор морфогенетического белка костей типа IB);
(2) E16 (LAT1, SLC7A5);
(3) STEAP1 (шестой трансмембранный эпителиальный антиген простаты);
(4) 0772P (CA125, MUC16);
(5) MPF (MPF, MSLN, SMR, фактор потенцирования мегакариоцитов, мезотелин);
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, семейство 34 переносчиков растворенных веществ (фосфат натрия), элемент 2, натрий-зависимый фосфатный переносчик 3b);
(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, семафорин 5b Hlog, домен sema, семь повторяющихся единиц тромбоспондина (тип 1 и подобный типу 1), трансмембранный домен (TM) и короткий цитоплазматический домен, (семафорин) 5B);
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, ген RIKEN cDNA 2700050C12);
(9) ETBR (рецептор эндотелина типа B);
(10) MSG783 (RNF124, гипотетический белок FLJ20315);
(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, ген 1, связанный с раком простаты, белок 1, ассоциируемый с раком простаты, шестой трансмембранный эпителиальный антиген простаты 2, шестой трансмембранный белок простаты);
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, катионный канал с транзиторным рецепторным потенциалом, подсемейство M, элемент 4);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, фактор роста, полученный из тератокарциномы);
(14) CD21 (CR2 (рецептор комплемента 2) или C3DR (C3d/рецептор вируса Эпштейна-Барра) или Hs.73792);
(15) CD79b (CD79B, CD79β, IGb (связанный с иммуноглобулином бета), B29);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (якорный белок фосфатазы 1a, содержащий домен SH2);
(17) HER2;
(18) NCA;
(19) MDP;
(20) IL20Rα;
(21) Brevican;
(22) EphB2R;
(23) ASLG659;
(24) PSCA;
(25) GEDA;
(26) BAFF-R (рецептор фактора активирования B лимфоцитов, рецептор 3 BLyS, BR3);
(27) CD22 (изоформа CD22-B рецептора B лимфоцитов);
(28) CD79a (CD79A, CD79α, связанный с иммуноглобулином альфа, белок, специфичный к B лимфоцитам);
(29) CXCR5 (рецептор 1 лимфомы Беркитта, рецептор, связанный с белком G);
(30) HLA-DOB (бета субъединица молекулы MHC класса II (антигена 1a);
(31) P2X5 (ионный канал 5, управляемый лигандом пуринергического рецептора P2X);
(32) CD72 (CD72 дифференциации B лимфоцитов, Lyb-2);
(33) LY64 (антиген 64 лимфоцитов (RP105), тип I, мембранный белок семейства, богатого повторяющимися единицами лейцина (LRR);
(34) FcRH1 (белок 1, подобный Fc рецептору);
(35) IRTA2 (ген, связанный с транслокацией рецептора суперсемейства иммуноглобулинов 2); и
(36) TENB2 (вероятный трансмембранный протеогликан).
15. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.1, где антитело ковалентно присоединяется к метке для захвата, к метке для обнаружения, к остатку лекарственного средства или к твердой подложке.
16. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.15, где антитело ковалентно присоединяется к биотиновой метке для захвата.
17. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.15, где антитело ковалентно присоединяется к метке для обнаружения на основе флуоресцентного красителя.
18. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.17, где флуоресцентный краситель выбирают из типа флуоресцеина, типа родамина, данзила, лиссамина, цианина, фикоэритрина, техасского красного и их аналогов.
19. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.15, где антитело ковалентно присоединяется к радионуклидной метке для обнаружения, выбранной из 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Ga, 86Y, 89Zr, 99Tc, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At и 213Bi.
20. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.15, где антитело ковалентно присоединяется к метке для обнаружения с помощью хелатирующего лиганда.
21. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.20, где хелатирующий лиганд выбирают из DOTA, DOTP, DOTMA, DTPA и TETA.
22. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.15, где антитело ковалентно присоединяется к остатку лекарственного средства, выбранного из майтансиноида, ауристатина, доластатина, трихотецена, CC1065, калихимицина, энедииновых антибиотиков, таксана и антрациклина с образованием конъюгата антитело-лекарственное средство, имеющего Формулу I:
где Ab представляет собой антитело, L представляет собой линкер, D представляет собой остаток лекарственного средства и p равно 1, 2, 3, или 4.
24. Полученное с помощью генной инженерии антитело с цистеиновыми заменами по п.22, где D представляет собой майтансиноид, имеющий структуру:
где волнистая линия показывает ковалентное присоединение атома серы D к линкеру;
R независимо выбирают из H, метила, этила, 1-пропила, 2-пропила, 1-бутила, 2-метил-1-пропила, 2-бутила, 2-метил-2-пропила, 1-пентила, 2-пентила, 3-пентила, 2-метил-2-бутила, 3-метил-2-бутила, 3-метил-1-бутила, 2-метил-1-бутила, 1-гексила, 2-гексила, 3-гексила, 2-метил-2-пентила, 3-метил-2-пентила, 4-метил-2-пентила, 3-метил-3-пентила, 2-метил-3-пентила, 2,3-диметил-2-бутила и 3,3-диметил-2-бутила и
m равно 1, 2 или 3.
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