RU2005120240A - Белковый комплекс, полученный с использованием фрагмента иммуноглобулина, и способ получения такого комплекса - Google Patents
Белковый комплекс, полученный с использованием фрагмента иммуноглобулина, и способ получения такого комплекса Download PDFInfo
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- RU2005120240A RU2005120240A RU2005120240/13A RU2005120240A RU2005120240A RU 2005120240 A RU2005120240 A RU 2005120240A RU 2005120240/13 A RU2005120240/13 A RU 2005120240/13A RU 2005120240 A RU2005120240 A RU 2005120240A RU 2005120240 A RU2005120240 A RU 2005120240A
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Claims (45)
1. Белковый конъюгат, содержащий физиологически активный полипептид, непептидный полимер, и Fc-фрагмент иммуноглобулина, ковалентно связанные друг с другом.
2. Белковый конъюгат по п.1, где указанный непептидный полимер, на обоих своих концах, ковалентно связан через реакционноспособную группу с физиологически активным полипептидом и Fc-фрагментом иммуноглобулина.
3. Белковый конъюгат по п.2, где один или несколько комплексов физиологически активного полипептида и непептидного полимера ковалентно связаны с одной молекулой Fc-фрагмента иммуноглобулина.
4. Белковый конъюгат по п.1, где указанный Fc-фрагмент иммуноглобулина является негликозилированным.
5. Белковый конъюгат по п.1, где указанный Fc-фрагмент иммуноглобулина состоит из 1-4 доменов, выбранных из группы, включающей домены СН1, СН2, СН3 и СН4.
6. Белковый конъюгат по п.5, где указанный Fc-фрагмент иммуноглобулина, кроме того, содержит шарнирную область.
7. Белковый конъюгат по п.1, где указанный Fc-фрагмент иммуноглобулина выбран из группы, включающей Fc-фрагменты IgG, IgA, IgD, IgE, IgM и их комбинации и гибриды.
8. Белковый конъюгат по п.7, где указанный Fc-фрагмент иммуноглобулина выбран из группы, включающей Fc-фрагменты IgG1, IgG2, IgG3, IgG4 и их комбинации и гибриды.
9. Белковый конъюгат по п.8, где указанным Fc-фрагментом иммуноглобулина является Fc-фрагмент IgG4.
10. Белковый конъюгат по п.9, где указанным Fc-фрагментом иммуноглобулина является Fc-фрагмент человеческого агликозилированного IgG4.
11. Белковый конъюгат по п.2, где указанная реакционноспособная группа не-пептидного полимера выбрана из группы, включающей альдегидную группу, пропиональдегидную группу, бутилальдегидную группу, малеимидную группу и сукцинимидное производное.
12. Белковый конъюгат по п.11, где указанным сукцинимидным производным является сукцинимидилпропионат, сукцинимидилкарбоксиметил, гидроксисукцинимидил или сукцинимидилкарбонат.
13. Белковый конъюгат по п.12, где указанный непептидный полимер имеет на обоих концах реакционноспособую альдегидную группу в качестве реакционноспособной группы.
14. Белковый конъюгат по п.1, где указанный непептидный полимер каждым из своих концов посредством свободной реакционноспособной группы присоединен у аминоконца к лизиновому остатку, гистидиновому остатку или цистеиновому остатку Fc-фрагмента иммуноглобулина и физиологически активного полипептида.
15. Белковый конъюгат по п.1, где указанный непептидный полимер выбран из группы, включающей линейные полимеры полиэтиленгликоля, линейные полимеры полипропиленгликоля, сополимеры этиленгликоля и пропиленгликоля, полиоксиэтилированные полиолы, поливиниловые спирты, полисахариды, декстраны, поливинилэтиловые эфиры, биологически разлагаемые полимеры, липидные полимеры, хитины, гиалуроновые кислоты и их комбинации.
16. Белковый конъюгат по п.15, где указанным непептидным полимером является полиэтиленгликоль.
17. Белковый конъюгат по п.1, где указанный физиологически активный полипептид выбран из группы, состоящей из гормонов, цитокинов, ферментов, антител, факторов роста, факторов регуляции транскрипции, факторов свертывания крови, вакцин, структурных белков, белков-лигандов и рецепторов.
18. Белковый конъюгат по п.17, где указанный физиологически активный полипептид выбран из группы, включающей человеческий гормон роста, релизинг-фактор гормона роста, пептид, высвобождающий гормон роста, интерфероны, рецепторы интерферона, колониестимулирующие факторы, глюкагонподобные пептиды, рецептор, связанный с G-белком, интерлейкины, рецепторы интерлейкинов, ферменты, интерлейкинсвязывающих белков, цитокинсвязывающие белки, фактор активации макрофагов, пептид макрофага, В-клеточный фактор, Т-клеточный фактор, белок А, ингибитор аллергии, гликопротеины некроза клеток, иммунотоксин, лимофтоксин, фактор некроза опухоли, супрессоры опухолей, метастатический фактор роста, антитрипсин альфа-1, альбумин, альфа-лактальбумин, аполипопротеин-Е, эритропоэтин, высокогликозилированный эритропоэтин, ангиопоэтины, гемоглобин, тромбин, пептид, активирующий рецептор тромбина, тромбомодулин, фактор VII, фактор VIIa, фактор VIII, фактор IX, фактор XIII, фактор активации плазминогена, фибринсвязывающий пептид, урокиназу, стрептокиназу, гирудин, белок С, С-реактивный белок, ингибитор ренина, ингибитор коллагеназы, супероксиддисмутазу, лептин, тромбоцитарный фактор роста, эпителиальный фактор роста, эпидермальный фактор роста, ангиостатин, ангиотензин, фактор роста костей, белок, стимулирующий рост костей, кальцитонин, инсулин, атриопептин, фактор, индуцирующий образование хряща, элкатонин, фактор активации соединительной ткани, ингибитор пути тканевого фактора, фолликулостимулирующий гормон, лютеинизирующий гормон, релизинг-фактор лютеинизирующего гормона, факторы роста нервной ткани, паратиреоидный гормон, релаксин, секретин, соматомедин, инсулинподобный фактор роста, адренокортикоидный гормон, глюкагон, холецистокинин, панкреатический полипептид, пептид, высвобождающий гастрин, релизинг-фактор кортикотропина, тиреоид-стимулирующий гормон, аутотаксин, лактоферрин, миостатин, рецепторы, антагонисты рецепторов, антигены клеточной поверхности, антигены вирусных вакцин, моноклональные антитела, поликлональные антитела и фрагменты антител.
19. Белковый конъюгат по п.18, где указанным физиологически активным полипептидом является человеческий гормон роста, интерферон-альфа, гранулоцитарный колониестимулирующий фактор, эритропоэтин или Fab'-фрагмент антитела.
20. Способ получения белкового конъюгата по п.1, предусматривающий
(а) ковалентное связывание одного или нескольких непептидных полимеров, имеющих на своих обоих концах реакционноспособную группу, с одним или несколькими физиологически активными полипептидами и с одним или несколькими Fc-фрагментами иммуноглобулина; и
(b) выделение указанного белкового конъюгата, содержащего, главным образом, физиологически активный полипептид, непептидный полимер и Fc-фрагмент иммуноглобулина, ковалентно связанные друг с другом.
21. Способ по п.20, где стадия (а) включает
(а1) ковалентное связывание Fc-фрагмента иммуноглобулина или физиологически активного полипептида с одним концом активированного непептидного полимера;
(а2) выделение из полученной реакционной смеси комплекса, содержащего Fc-фрагмент иммуноглобулина или физиологически активный полипептид, связанный с непептидным полимером; и
(а3) ковалентное связывание Fc-фрагмента иммуноглобулина или физиологически активного полипептида с другим концом непептидного полимера выделенного комплекса с получением белкового конъюгата, содержащего Fc-фрагмент иммуноглобулина и физиологически активный полипептид, связанные с каждым концом указанного не-пептидного полимера.
22. Способ по п.21, где в указанной стадии (а1), молярное соотношение физиологически активного полипептида и непептидного полимера, используемых в реакционной смеси, составляет от 1:2,5 до 1:5.
23. Способ по п.21, где в указанной стадии (а1), молярное соотношение Fc-фрагмента иммуноглобулина и непептидного полимера, используемых в реакционной смеси, составляет от 1:5 до 1:10.
24. Способ по п.21, где в указанной стадии (а3), молярное соотношение комплекса, полученного в стадии (а2) и Fc-фрагмента иммуноглобулина, или физиологически активного полипептида, используемых в реакционной смеси, составляет от 1:0,5 до 1:20.
25. Способ по п.21, где указанные стадии (а1) и (а3) осуществляют в присутствии восстановителя.
26. Способ по п.25, где указанный восстановитель выбран из группы, включающей цианоборогидрид натрия (NaCNBH3), борогидрид натрия, борат диметиламина и борат пиридина.
27. Фармацевтическая композиция для увеличения продолжительности действия in vivo и стабильности физиологически активного полипептида, где указанная композиция включает белковый конъюгат по п.1 и его фармацевтически приемлемый носитель.
28. Фармацевтическая композиция по п.27, где указанный непептидный полимер, по своим обоим концам, ковалентно связан через реакционноспособную группу с физиологически активным полипептидом и Fc-фрагментом иммуноглобулина.
29. Фармацевтическая композиция по п.28, где один или несколько комплексов физиологически активного полипептида и непептидного полимера ковалентно связаны с одной молекулой Fc-фрагмента иммуноглобулина.
30. Фармацевтическая композиция по п.27, где указанный Fc-фрагмент иммуноглобулина является негликозилированным.
31. Фармацевтическая композиция по п.27, где указанный Fc-фрагмент иммуноглобулина состоит из 1-4 доменов, выбранных из группы, включающей домены СН1, СН2, СН3 и СН4.
32. Фармацевтическая композиция по п.31, где указанный Fc-фрагмент иммуноглобулина, кроме того, содержит шарнирную область.
33. Фармацевтическая композиция по п.27, где указанный Fc-фрагмент иммуноглобулина выбран из группы, включающей Fc-фрагменты IgG, IgA, IgD, IgE, IgM и их комбинации и гибриды.
34. Фармацевтическая композиция по п.33, где указанный Fc-фрагмент иммуноглобулина выбран из группы, включающей Fc-фрагменты IgG1, IgG2, IgG3, IgG4 и их комбинации и гибриды.
35. Фармацевтическая композиция по п.34, где указанным Fc-фрагментом иммуноглобулина является Fc-фрагмент IgG4.
36. Фармацевтическая композиция по п.35, где указанным Fc-фрагментом иммуноглобулина является Fc-фрагмент человеческого агликозилированного IgG4.
37. Фармацевтическая композиция по п.28, где указанная реакционноспособная группа непептидного полимера выбрана из группы, состоящей из альдегидной группы, пропиональдегидной группы, бутилальдегидной группы, малеимидной группы и сукцинимидного производного.
38. Фармацевтическая композиция по п.37, где указанным сукцинимидным производным является сукцинимидилпропионат, сукцинимидилкарбоксиметил, гидроксисукцинимидил, или сукцинимидилкарбонат.
39. Фармацевтическая композиция по п.38, где указанный непептидный полимер имеет на обоих концах реакционноспособую альдегидную группу в качестве реакционноспособной группы.
40. Фармацевтическая композиция по п.27, где указанный непептидный полимер каждым своим концом посредством свободной реакционноспособной группы присоединен у аминоконца к лизиновому остатку, гистидиновому остатку или цистеиновому остатку Fc-фрагмента иммуноглобулина и физиологически активного полипептида.
41. Фармацевтическая композиция по п.27, где указанный непептидный полимер выбран из группы, включающей линейные полимеры полиэтиленгликоля, линейные полимеры полипропиленгликоля, сополимеры этиленгликоля и пропиленгликоля, полиоксиэтилированные полиолы, поливиниловые спирты, полисахариды, декстраны, поливинилэтиловые эфиры, биологически разлагаемые полимеры, липидные полимеры, хитины, гиалуроновые кислоты и их комбинации.
42. Фармацевтическая композиция по п.41, где указанным непептидным полимером является полиэтиленгликоль.
43. Фармацевтическая композиция по п.27, где указанный физиологически активный полипептид выбран из группы, состоящей из гормонов, цитокинов, ферментов, антител, факторов роста, факторов регуляции транскрипции, факторов свертывания крови, вакцин, структурных белков, белков-лигандов и рецепторов.
44. Фармацевтическая композиция по п.43, где указанный физиологически активный полипептид выбран из группы, включающей человеческий гормон роста, релизинг-фактор гормона роста, пептид, высвобождающий гормон роста, интерфероны, рецепторы интерферона, колониестимулирующие факторы, глюкагонподобные пептиды (например, GLP-1 и тому подобное), рецептор, связанный с G-белком, интерлейкины, рецепторы интерлейкинов, ферменты, интерлейкинсвязывающих белков, цитокинсвязывающие белки, фактор активации макрофагов, пептид макрофага, В-клеточный фактор, Т-клеточный фактор, белок А, ингибитор аллергии, гликопротеины некроза клеток, иммунотоксин, лимофтоксин, фактор некроза опухоли, супрессоры опухолей, метастатическыий фактор роста, антитрипсин альфа-1, альбумин, альфа-лактальбумин, аполипопротеин-Е, эритропоэтин, высокогликозилированный эритропоэтин, ангиопоэтины, гемоглобин, тромбин, пептид, активирующий рецептор тромбина, тромбомодулин, фактор VII, фактор VIIa, фактор VIII, фактор IX, фактор XIII, фактор активации плазминогена, фибрин-связывающий пептид, урокиназу, стрептокиназу, гирудин, белок С, С-реактивный белок, ингибитор ренина, ингибитор коллагеназы, супероксиддисмутазу, лептин, тромбоцитарный фактор роста, эпителиальный фактор роста, эпидермальный фактор роста, ангиостатин, ангиотензин, фактор роста костей, белок, стимулирующий рост костей, кальцитонин, инсулин, атриопептин, фактор, индуцирующий образование хряща, элкатонин, фактор активации соединительной ткани, ингибитор пути тканевого фактора, фолликуло-стимулирующий гормон, лютеинизирующий гормон, релизинг-фактор лютеинизирующего гормона, факторы роста нервной ткани, паратиреоидный гормон, релаксин, секретин, соматомедин, инсулинподобный фактор роста, адренокортикоидный гормон, глюкагон, холецистокинин, панкреатический полипептид, пептид, высвобождающий гастрин, релизинг-фактор кортикотропина, тиреоидстимулирующий гормон, аутотаксин, лактоферрин, миостатин, рецепторы, антагонисты рецепторов, антигены клеточной поверхности, антигены вирусных вакцин, моноклональные антитела, поликлональные антитела и фрагменты антител.
45. Фармацевтическая композиция по п.44, где указанным физиологически активным полипептидом является человеческий гормон роста, интерферона-альфа, гранулоцитарный колониестимулирующий фактор, эритропоэтин или Fab'-фрагмент антитела.
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