RU2012102602A - Легковыделяемые биспецифические антитела с природным иммуноглобулиновым форматом - Google Patents
Легковыделяемые биспецифические антитела с природным иммуноглобулиновым форматом Download PDFInfo
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- 108060003951 Immunoglobulin Proteins 0.000 title 1
- 102000018358 immunoglobulin Human genes 0.000 title 1
- 102000004169 proteins and genes Human genes 0.000 claims abstract 23
- 108090000623 proteins and genes Proteins 0.000 claims abstract 23
- 230000002163 immunogen Effects 0.000 claims abstract 12
- 238000012986 modification Methods 0.000 claims abstract 12
- 230000004048 modification Effects 0.000 claims abstract 12
- 229920001184 polypeptide Polymers 0.000 claims abstract 10
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract 10
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 claims abstract 7
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 claims abstract 7
- 241000282412 Homo Species 0.000 claims abstract 6
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 claims abstract 4
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 claims abstract 4
- 108700024394 Exon Proteins 0.000 claims abstract 2
- 102000025171 antigen binding proteins Human genes 0.000 claims abstract 2
- 108091000831 antigen binding proteins Proteins 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims 11
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 claims 5
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 claims 5
- 150000007523 nucleic acids Chemical group 0.000 claims 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 3
- 210000004027 cell Anatomy 0.000 claims 3
- 239000000178 monomer Substances 0.000 claims 3
- 239000000203 mixture Substances 0.000 claims 2
- 102220142694 rs192332456 Human genes 0.000 claims 2
- 239000007787 solid Substances 0.000 claims 2
- 238000001042 affinity chromatography Methods 0.000 claims 1
- 102220349284 c.287A>T Human genes 0.000 claims 1
- 238000002955 isolation Methods 0.000 claims 1
- 210000004962 mammalian cell Anatomy 0.000 claims 1
- 102220210869 rs1057524586 Human genes 0.000 claims 1
- 102220117530 rs112626848 Human genes 0.000 claims 1
- 102200148758 rs116840795 Human genes 0.000 claims 1
- 102220206698 rs142514490 Human genes 0.000 claims 1
- 102220238658 rs1468529365 Human genes 0.000 claims 1
- 102220268018 rs201210997 Human genes 0.000 claims 1
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- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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Abstract
1. Гетеродимерный биспецифический антигенсвязывающий белок, содержащий:a. первый полипептид, содержащий от N-конца до C-конца первую область связывания эпитопа, которая селективно связывает первый эпитоп, константную область иммуноглобулина, которая содержит первую область CH3 IgG человека, выбранную из IgG1, IgG2 и IgG4; иb. второй полипептид, содержащий, от N-конца до C-конца, вторую область связывания эпитопа, которая селективно связывает второй эпитоп, константную область иммуноглобулина, которая содержит вторую область CH3 IgG человека, выбранного из IgG1, IgG2 и IgG4, где вторая область CH3 содержит модификацию, которая снижает или предотвращает связывание второго домена CH3 с белком A.2. Биспецифический белок по п.1, где первый полипептид и второй полипептид представляют собой тяжелые цепи IgG человека.3. Биспецифический белок по п.1, дополнительно содержащий легкую цепь иммуноглобулина.4. Биспецифический белок по п.3, где легкая цепь иммуноглобулина представляет собой легкую цепь иммуноглобулина человека.5. Биспецифический белок по п.1, где первый и второй полипептиды каждый представляют собой тяжелые цепи IgG1 человека.6. Биспецифический белок по п.1, где модификация выбрана из группы, состоящей из (a) 95R и (b) 95R и 96F в нумерации экзонов IMGT, или (a') 435R и (b') 435R и 436F в нумерации системы EU.7. Биспецифический белок по п.6, дополнительно содержащий от одной до пяти модификаций, выбранных из группы, состоящей из 16E, 18M, 44S, 52N, 57M и 82I в системе нумерации экзонов IMGT или 356E, 358M, 384S, 392N, 397M и 422I в системе нумерации EU.8. Биспецифический белок по п.6, где домен CH3 биспецифического антитела является неиммуногенным или по существу неиммуногенным для человека.9. Биспециф
Claims (20)
1. Гетеродимерный биспецифический антигенсвязывающий белок, содержащий:
a. первый полипептид, содержащий от N-конца до C-конца первую область связывания эпитопа, которая селективно связывает первый эпитоп, константную область иммуноглобулина, которая содержит первую область CH3 IgG человека, выбранную из IgG1, IgG2 и IgG4; и
b. второй полипептид, содержащий, от N-конца до C-конца, вторую область связывания эпитопа, которая селективно связывает второй эпитоп, константную область иммуноглобулина, которая содержит вторую область CH3 IgG человека, выбранного из IgG1, IgG2 и IgG4, где вторая область CH3 содержит модификацию, которая снижает или предотвращает связывание второго домена CH3 с белком A.
2. Биспецифический белок по п.1, где первый полипептид и второй полипептид представляют собой тяжелые цепи IgG человека.
3. Биспецифический белок по п.1, дополнительно содержащий легкую цепь иммуноглобулина.
4. Биспецифический белок по п.3, где легкая цепь иммуноглобулина представляет собой легкую цепь иммуноглобулина человека.
5. Биспецифический белок по п.1, где первый и второй полипептиды каждый представляют собой тяжелые цепи IgG1 человека.
6. Биспецифический белок по п.1, где модификация выбрана из группы, состоящей из (a) 95R и (b) 95R и 96F в нумерации экзонов IMGT, или (a') 435R и (b') 435R и 436F в нумерации системы EU.
7. Биспецифический белок по п.6, дополнительно содержащий от одной до пяти модификаций, выбранных из группы, состоящей из 16E, 18M, 44S, 52N, 57M и 82I в системе нумерации экзонов IMGT или 356E, 358M, 384S, 392N, 397M и 422I в системе нумерации EU.
8. Биспецифический белок по п.6, где домен CH3 биспецифического антитела является неиммуногенным или по существу неиммуногенным для человека.
9. Биспецифический белок по п.7, где домен CH3 биспецифического антитела является неиммуногенным или по существу неиммуногенным для человека.
10. Способ получения биспецифического антитела, включающий:
a. получение последовательности нуклеиновой кислоты, кодирующей первую тяжелую цепь иммуноглобулина, содержащую первый вариабельный домен, который распознает первый эпитоп, где первая тяжелая цепь иммуноглобулина содержит константный домен изотипов IgG1, IgG2 или IgG4;
b. получение второй последовательности нуклеиновой кислоты, кодирующей вторую тяжелую цепь иммуноглобулина, содержащую второй вариабельный домен, который распознает второй эпитоп, где вторая тяжелая цепь иммуноглобулина содержит константный домен изотипов IgG1, IgG2 или IgG4, который содержит модификацию в его домене CH3, которая препятствует или снижает связывание с белком A;
с. получение третьей последовательности нуклеиновой кислоты, кодирующей легкую цепь иммуноглобулина, которая образует пару с первой и второй тяжелой цепью иммуноглобулина;
d. введение первой, второй и третьей последовательности нуклеиновой кислоты в клетку млекопитающего;
e. обеспечение возможности клетке экспрессировать биспецифическое антитело; и
f. выделение биспецифического антитела, основанное на способности биспецифического антитела связываться с белком А.
11. Способ по п.10, где модификация выбрана из группы, состоящей из (a) 95R и (b) 95R и 96F в системе нумерации экзонов IMGT или (a') 435R, и (b') 435R и 436F в системе нумерации EU.
12. Способ по п.11, дополнительно включающий от одной до пяти модификаций, выбранных из группы, состоящей из 16E, 18M, 44S, 52N, 57M, и 82I в системе нумерации экзонов IMGT или 356E, 358M, 384S, 392N, 397M и 422I в системе нумерации EU.
13. Способ по п.11, где домен CH3 биспецифического антитела является неиммуногенным или по существу неиммуногенным для человека.
14. Способ по п.12, где домен CH3 биспецифического антитела является неиммуногенным или по существу неиммуногенным для человека.
15. Способ по п.10, где биспецифическое антитело выделяют на твердом носителе, содержащем белок A.
16. Способ по п.15, где твердый носитель включает колонку для аффинной хроматографии с белком А и биспецифическое антитело выделяют, используя градиент pH.
17. Способ по п.16, где градиент pH представляет собой ступенчатый градиент, включающий один или несколько ступеней pH между значениями pH, равными 3 и 5.
18. Способ выделения биспецифического антитела, включающий выделение из разрушенной клетки или смеси антител биспецифического антитела, обладающего селективно модифицированными доменами CH3 IgG1, IgG2 или IgG4, где селективно модифицированные домены CH3 являются неиммуногенными или по существу неиммуногенными для человека, и где модификации приводят в результате к получению биспецифического антитела с константной областью гетеродимерной тяжелой цепи, мономеры которой обладают различной аффинностью для белка А, и биспецифическое антитело выделяют из разрушенной клетки или смеси, основываясь на его аффинности к белку А.
19. Способ по п.18, где один мономер константной области гетеродимерной тяжелой цепи представляет собой IgG1 человека, а другой мономер константной области гетеродимерной тяжелой цепи представляет собой модифицированный IgG1 человека, содержащий модификацию, выбранную из группы, состоящей из (a) H95R и (b) H95R и Y96F по системе нумерации экзонов IMGT или (a') H435R, и (b') H435R и Y436F в системе нумерации EU.
20. Способ по п.19, где модифицированный IgG1 человека дополнительно содержит модификацию, выбранную из группы, состоящей из D16E, L18M, N44S, K52N, V57M и V82I в системе нумерации экзонов IMGT или D356E, L358M, N384S, K392N, V397M и V422I в системе нумерации EU.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US22068709P | 2009-06-26 | 2009-06-26 | |
| US61/220,687 | 2009-06-26 | ||
| PCT/US2010/040028 WO2010151792A1 (en) | 2009-06-26 | 2010-06-25 | Readily isolated bispecific antibodies with native immunoglobulin format |
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