RU2013110875A - БИСПЕЦИФИЧЕСКИЕ АНТИТЕЛА, СОДЕРЖАЩИЕ СТАБИЛИЗИРОВАННЫЙ ДИСУЛЬФИДОМ ФРАГМЕНТ Fv - Google Patents
БИСПЕЦИФИЧЕСКИЕ АНТИТЕЛА, СОДЕРЖАЩИЕ СТАБИЛИЗИРОВАННЫЙ ДИСУЛЬФИДОМ ФРАГМЕНТ Fv Download PDFInfo
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- 102000017727 Immunoglobulin Variable Region Human genes 0.000 title claims abstract 7
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 title claims abstract 7
- 239000000427 antigen Substances 0.000 claims abstract 23
- 102000036639 antigens Human genes 0.000 claims abstract 23
- 108091007433 antigens Proteins 0.000 claims abstract 23
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract 16
- 210000004027 cell Anatomy 0.000 claims 7
- 108091005804 Peptidases Proteins 0.000 claims 6
- 239000004365 Protease Substances 0.000 claims 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 6
- 125000000539 amino acid group Chemical group 0.000 claims 6
- 238000003776 cleavage reaction Methods 0.000 claims 6
- 230000007017 scission Effects 0.000 claims 6
- 238000000034 method Methods 0.000 claims 5
- 210000004962 mammalian cell Anatomy 0.000 claims 3
- 102000004961 Furin Human genes 0.000 claims 2
- 108090001126 Furin Proteins 0.000 claims 2
- 108700011066 PreScission Protease Proteins 0.000 claims 2
- 238000004113 cell culture Methods 0.000 claims 2
- 239000012228 culture supernatant Substances 0.000 claims 2
- 108020004707 nucleic acids Proteins 0.000 claims 2
- 150000007523 nucleic acids Chemical class 0.000 claims 2
- 102000039446 nucleic acids Human genes 0.000 claims 2
- 239000004475 Arginine Substances 0.000 claims 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims 1
- 239000004473 Threonine Substances 0.000 claims 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims 1
- 235000004279 alanine Nutrition 0.000 claims 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims 1
- 235000018417 cysteine Nutrition 0.000 claims 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000008194 pharmaceutical composition Substances 0.000 claims 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims 1
- 239000004474 valine Substances 0.000 claims 1
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- C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
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Abstract
1. Биспецифическое антитело, содержащее:a) полноразмерное антитело, специфически связывающееся с первым антигеном и состоящее из двух тяжелых цепей антитела и двух легких цепей антитела;b) фрагмент Fv, специфически связывающийся со вторым антигеном и содержащий домен VHи домен VL, при этом оба домена соединены посредством дисульфидного мостика; ипри этом только один из доменов, либо домен VH, либо домен VL, соединен посредством пептидного линкера с тяжелой или легкой цепью полноразмерного антитела, специфически связывающегося с первым антигеном.2. Биспецифическое антитело по п.1, которое отличается тем, что биспецифическое антитело является тривалентным и:либо домен VH, либо домен VLсоединен посредством пептидного линкера с тяжелой цепью полноразмерного антитела, специфически связывающегося с первым антигеном.3. Биспецифическое антитело по п.2, которое отличается тем, чтодомен VHили домен VLсоединен своим N-концом посредством пептидного линкера с С-концом полноразмерного антитела, специфически связывающегося с первым антигеном.4. Биспецифическое антитело по п.2, которое отличается тем, чтодомен VHили домен VLсоединен своим С-концом посредством пептидного линкера с N-концом полноразмерного антитела, специфически связывающегося с первым антигеном.5. Биспецифическое антитело по п.1, которое отличается тем, что домен VHили домен VLсоединен своим N-концом посредством пептидного линкера с С-концом тяжелой или легкой цепи полноразмерного антитела, специфически связывающегося с первым антигеном.6. Биспецифическое антитело по п.1, которое отличается тем, чтодомен VHили домен VLсоединен своим С-концом посредством пептидного линкер
Claims (18)
1. Биспецифическое антитело, содержащее:
a) полноразмерное антитело, специфически связывающееся с первым антигеном и состоящее из двух тяжелых цепей антитела и двух легких цепей антитела;
b) фрагмент Fv, специфически связывающийся со вторым антигеном и содержащий домен VH2 и домен VL2, при этом оба домена соединены посредством дисульфидного мостика; и
при этом только один из доменов, либо домен VH2, либо домен VL2, соединен посредством пептидного линкера с тяжелой или легкой цепью полноразмерного антитела, специфически связывающегося с первым антигеном.
2. Биспецифическое антитело по п.1, которое отличается тем, что биспецифическое антитело является тривалентным и:
либо домен VH2, либо домен VL2 соединен посредством пептидного линкера с тяжелой цепью полноразмерного антитела, специфически связывающегося с первым антигеном.
3. Биспецифическое антитело по п.2, которое отличается тем, что
домен VH2 или домен VL2 соединен своим N-концом посредством пептидного линкера с С-концом полноразмерного антитела, специфически связывающегося с первым антигеном.
4. Биспецифическое антитело по п.2, которое отличается тем, что
домен VH2 или домен VL2 соединен своим С-концом посредством пептидного линкера с N-концом полноразмерного антитела, специфически связывающегося с первым антигеном.
5. Биспецифическое антитело по п.1, которое отличается тем, что домен VH2 или домен VL2 соединен своим N-концом посредством пептидного линкера с С-концом тяжелой или легкой цепи полноразмерного антитела, специфически связывающегося с первым антигеном.
6. Биспецифическое антитело по п.1, которое отличается тем, что
домен VH2 или домен VL2 соединен своим С-концом посредством пептидного линкера с N-концом тяжелой или легкой цепи полноразмерного антитела, специфически связывающегося с первым антигеном.
7. Биспецифическое антитело по любому из пп.1-6, которое отличается тем, что:
домен VH2 и домен VL2 соединены
посредством дисульфидного мостика, который внесен между следующими положениями:
i) положением 44 в домене VH2 и положением 100 в домене VL2;
ii) положением 105 в домене VH2 и положением 43 в домене VL2; или
iii) положением 101 в домене VH2 и положением 100 в домене VL2
8. Биспецифическое антитело по любому из пп.1-6, которое отличается тем, что:
домен VH2 и домен VL2 соединены
посредством дисульфидного мостика, который внесен между положением 44 в домене VH2 и положением 100 в домене VL2.
9. Биспецифическое антитело по любому из пп.1-6, которое отличается тем, что:
первый домен СН3 тяжелой цепи целого антитела и второй домен СН3 целого антитела соединяются друг с другом, образуя поверхность контакта, которая содержит изменения относительно исходной поверхности контакта между доменами СН3 антитела;
при этом i) в домене СН3 одной тяжелой цепи:
аминокислотный остаток заменен аминокислотным остатком, имеющим более объемную боковую цепочку, в результате чего образуется выступ на поверхности контакта домена СН3 одной тяжелой цепи, который способен помещаться в полости на поверхности контакта домена СН3 другой тяжелой цепи,
и
ii) в домене СН3 другой тяжелой цепи:
аминокислотный остаток заменен аминокислотным остатком, имеющим меньшую по объему боковую цепочку, в результате чего на поверхности контакта второго домена СН3 образуется полость, в которой способен помещаться выступ, имеющийся на поверхности контакта первого домена СН3.
10. Биспецифическое антитело по п.9, которое отличается тем, что:
указанный аминокислотный остаток, имеющий более объемную боковую цепочку, выбран из группы, состоящей из аргинина (R), фенилаланина (F), тирозина (Y) и триптофана (W), а указанный аминокислотный остаток, имеющий меньшую по объему боковую цепочку, выбран из группы, состоящей из аланина (А), серина (S), треонина (Т) и валина (V).
11. Биспецифическое антитело по п.9, которое отличается тем, что:
оба домена СН3 дополнительно изменены путем внесения остатка цистеина (С) в такие положения в составе каждого домена СН3, чтобы мог образоваться дисульфидный мостик между доменами СН3.
12. Способ получения биспецифического антитела по любому из пп.1-11, включающий следующие этапы:
A) экспрессию в клетке млекопитающего нуклеиновой кислоты, кодирующей биспецифическое антитело, содержащее:
а) полноразмерное антитело, специфически связывающееся с первым антигеном и состоящее из двух тяжелых цепей антитела и двух легких цепей антитела;
b) фрагмент Fv, специфически связывающийся со вторым антигеном и содержащий домен VH2 и домен VL2, при этом оба домена соединены посредством дисульфидного мостика; и
при этом фрагмент Fv соединен:
через N-концы домена VH2 и домена VL2 с обоими С-концами тяжелой цепи полноразмерного антитела, специфически связывающегося с первым антигеном, посредством первого и второго пептидных линкеров; или
через С-концы домена VH2 и домена VL2 с обоими N-концами одной тяжелой и одной легкой цепи полноразмерного антитела, специфически связывающегося с первым антигеном, посредством первого и второго пептидных линкеров,
которые отличаются тем, что:
один из линкеров содержит сайт расщепления протеазой, который может расщеплять фурин, а другой линкер не содержит сайт расщепления протеазой;
B) извлечение указанного антитела из указанной клетки или супернатанта клеточной культуры.
13. Способ получения тривалентного, биспецифического антитела по любому из пп.2-4 и 7-11, включающий следующие этапы:
A) экспрессию в клетке млекопитающего нуклеиновой кислоты, кодирующей биспецифическое антитело, содержащее:
a) полноразмерное антитело, специфически связывающееся с первым антигеном и состоящее из двух тяжелых цепей антитела и двух легких цепей антитела;
b) фрагмент Fv, специфически связывающийся со вторым антигеном и содержащий домен VH2 и домен VL2, при этом оба домена соединены посредством дисульфидного мостика; и
при этом фрагмент Fv соединен:
через N-концы домена VH2 и домена VL2 с обоими С-концами тяжелых цепей полноразмерного антитела, специфически связывающегося с первым антигеном, посредством первого и второго пептидных линкеров; или
через С-концы домена VH2 и домена VL2 с обоими N-концами тяжелых цепей полноразмерного антитела, специфически связывающегося с первым антигеном, посредством первого и второго пептидных линкеров,
которые отличаются тем, что:
один из линкеров содержит сайт расщепления протеазой, который может расщеплять протеаза PreScission, а другой линкер не содержит сайт расщепления протеазой;
B) извлечение указанного антитела из указанной клетки или супернатанта клеточной культуры.
14. Способ по любому из пп.12 или 13, который отличается тем, что:
сайт расщепления протеазой, который может расщеплять фурин, имеет последовательность SEQ ID NO: 13 или SEQ ID NO: 14.
15. Способ по любому из пп.12 или 13, который отличается тем, что:
сайт расщепления протеазой, который может расщеплять протеаза PreScission, имеет последовательность SEQ ID NO: 15.
16. Способ по любому из пп.12-13, который отличается тем, что:
клетка млекопитающего является клеткой СНО, клеткой NS0, клеткой SP2/0, клеткой HEK293, клеткой COS или клеткой PER.С6.
17. Антитело, полученное способом по любому из пп.12-16.
18. Фармацевтическая композиция, содержащая биспецифическое антитело по любому из пп.1-11.
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| EP10173914 | 2010-08-24 | ||
| PCT/EP2011/064476 WO2012025530A1 (en) | 2010-08-24 | 2011-08-23 | Bispecific antibodies comprising a disulfide stabilized - fv fragment |
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| RU2013110875A true RU2013110875A (ru) | 2014-09-27 |
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| US (1) | US9879095B2 (ru) |
| EP (1) | EP2609111B1 (ru) |
| JP (1) | JP5758004B2 (ru) |
| KR (1) | KR101586128B1 (ru) |
| CN (1) | CN103068846B9 (ru) |
| BR (1) | BR112013001847A2 (ru) |
| CA (1) | CA2807278A1 (ru) |
| MX (1) | MX340558B (ru) |
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-
2011
- 2011-08-23 CA CA2807278A patent/CA2807278A1/en not_active Abandoned
- 2011-08-23 RU RU2013110875/10A patent/RU2013110875A/ru not_active Application Discontinuation
- 2011-08-23 JP JP2013525290A patent/JP5758004B2/ja not_active Expired - Fee Related
- 2011-08-23 BR BR112013001847A patent/BR112013001847A2/pt not_active IP Right Cessation
- 2011-08-23 MX MX2013001982A patent/MX340558B/es active IP Right Grant
- 2011-08-23 EP EP11746259.8A patent/EP2609111B1/en not_active Not-in-force
- 2011-08-23 WO PCT/EP2011/064476 patent/WO2012025530A1/en not_active Ceased
- 2011-08-23 KR KR1020137004582A patent/KR101586128B1/ko not_active Expired - Fee Related
- 2011-08-23 CN CN201180040675.3A patent/CN103068846B9/zh not_active Expired - Fee Related
-
2013
- 2013-02-21 US US13/773,167 patent/US9879095B2/en active Active
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| Publication number | Publication date |
|---|---|
| US9879095B2 (en) | 2018-01-30 |
| WO2012025530A1 (en) | 2012-03-01 |
| CN103068846B9 (zh) | 2016-09-28 |
| KR101586128B1 (ko) | 2016-01-15 |
| CA2807278A1 (en) | 2012-03-01 |
| MX340558B (es) | 2016-07-14 |
| CN103068846A (zh) | 2013-04-24 |
| KR20130041968A (ko) | 2013-04-25 |
| EP2609111B1 (en) | 2017-11-01 |
| US20130267686A1 (en) | 2013-10-10 |
| CN103068846B (zh) | 2016-06-22 |
| JP5758004B2 (ja) | 2015-08-05 |
| EP2609111A1 (en) | 2013-07-03 |
| JP2013539463A (ja) | 2013-10-24 |
| HK1184471A1 (zh) | 2014-01-24 |
| BR112013001847A2 (pt) | 2016-05-31 |
| MX2013001982A (es) | 2013-05-09 |
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