RU2326127C2 - Варианты иммуноглобулинов и их применение - Google Patents
Варианты иммуноглобулинов и их применение Download PDFInfo
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- RU2326127C2 RU2326127C2 RU2005122448/13A RU2005122448A RU2326127C2 RU 2326127 C2 RU2326127 C2 RU 2326127C2 RU 2005122448/13 A RU2005122448/13 A RU 2005122448/13A RU 2005122448 A RU2005122448 A RU 2005122448A RU 2326127 C2 RU2326127 C2 RU 2326127C2
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- Orthopedic Medicine & Surgery (AREA)
Abstract
Описаны гуманизированные и химерные антитела к CD20, предназначенные для лечения СD20-позитивных злокачественных заболеваний и аутоиммунных заболеваний. Антитело обладает эффективностью в отношении истощения В-клеток приматов in vivo, содержит в вариабельной области Н-цепи CDR3-последовательность из антитела к человеческому CD20 и практически все остатки консенсусного каркасного участка (FR) человеческой Н-цепи подгруппы III. Антитело по изобретению используют в составе композиции и изделия, обладающих способностью связывать CD20. Антитело также используют в способах для индукции апоптоза, лечения СD20-позитивного рака, аутоиммунного заболевания, ревматоидного артрита. Изобретение включает нуклеиновую кислоту (НК), кодирующую антитело, экспрессионный вектор, содержащий указанную НК, и клетку-хозяин, продуцирующую рекомбинантное антитело, а также способ получения указанного антитела. Антитела по изобретению обладают минимальной антигенностью или не обладают ею вообще, что позволяет использовать их для продолжительного лечения, тем самым преодолевая ограничения, связанные с применением существующих терапевтических композиций. 15 н. и 68 з.п. ф-лы, 23 ил., 12 табл.
Description
Claims (83)
1. Гуманизированное антитело, которое связывает человеческий CD20, или его антигенсвязывающий фрагмент, где антитело обладает эффективностью в отношении истощения В-клеток приматов in vivo, причем антитело содержит в вариабельной области Н-цепи (Vн) по меньшей мере СDR3-последовательность, представленную в SEQ ID NO.2, из антитела к человеческому CD20 и практически все остатки человеческого консенсусного каркасного участка (FR) человеческой тяжелой цепи подгруппы III (VнIII).
2. Антитело по п.1, дополнительно содержащее CDR1-последовательность Н-цепи, представленную в SEQ ID NO.10, и CDR2-последовательность Н-цепи, представленную в SEQ ID NO. 11.
3. Антитело по п.2, дополнительно содержащее CDR1-последовательность L-цепи, представленную в SEQ ID NO. 4, CDR2-последовательность L-цепи, представленную в SEQ ID NO. 5, CDR3-последовательность L-цепи, представленную в SEQ ID NO. 6, практически все остатки каркасного участка (FR) человеческой легкой к-цепи подгруппы I (VкI).
4. Антитело по п.1, содержащее последовательность Vн, представленную в SEQ ID NO. 8.
5. Антитело по п.4, дополнительно содержащее последовательность VL, представленную в SEQ ID NO. 2.
6. Антитело по п.3, в котором Vн-область связана с константной областью цепи человеческого IgG.
7. Антитело по п.6, в котором человеческий IgG представляет собой IgG1 или IgG3.
8. Антитело по п.1, где антитело включает аминокислотные последовательности легкой и тяжелой цепи, представленные в SEQ ID NO. 21 и 22, соответственно.
9. Антитело по п.1, где антитело включает аминокислотные последовательности легкой и тяжелой цепи, представленные в SEQ ID NO. 21 и 23, соответственно.
10. Антитело по п.9, в котором N100 в Vн заменен на А и М32 в VL заменен на L.
11. Антитело по п.5, в котором D56 и N100 в Vн и S92 в VL заменены на А.
12. Антитело по п.11, которое дополнительно содержит замену M32L в VL.
13. Антитело по п.12, содержащее Fc-фрагмент IgG1, который несет аминокислотные замены S298A/E333A/K334A.
14. Антитело по п.13, в котором Fc-фрагмент содержит дополнительные аминокислотные замены E356D и M358L.
15. Антитело по п.1, дополнительно содержащее по меньшей мере одну аминокислотную замену в Fc-фрагменте, которая повышает ADCC- и/или CDC-активность.
16. Антитело по п.15, в котором аминокислотные замены представляют собой S298A/ЕЗЗЗА/K334A.
17. Антитело по п.15, дополнительно содержащее по меньшей мере одну аминокислотную замену в Fc-фрагменте, которая повышает CDC-активность.
18. Антитело по п.17, в котором аминокислотная замена представляет собой К326А или K326W.
19. Антитело по п.13, в котором Fc-фрагмент содержит дополнительную аминокислотную замену К326А.
20. Антитело по любому из пп.1-14, дополнительно содержащее по меньшей мере одну аминокислотную замену в Fc-фрагменте, которая понижает CDC-активность.
21. Антитело по п.20, содержащее по меньшей мере замену К322А.
22. Антитело по любому из пп.1, 13 и 14, где антитело обладает по меньшей мере в 10 раз более высокой ADCC- активностью, чем ритуксимаб.
23. Антитело по п.1, где В-клетки приматов представляют собой В-клетки человека или яванских макак-крабоедов.
24. Антитело по п.1, конъюгированное с цитотоксическим агентом.
25. Антитело по п.24, где цитотоксический агент представляет собой радиоактивный изотоп или токсин.
26. Антитело по п.1, где антитело получают в СНО-клетках.
27. Композиция, обладающая способностью связывать CD20 и содержащая антитело по п.1 и носитель.
28. Композиция по п.27, где антитело является антителом по п.13 и носитель является фармацевтически приемлемым носителем.
29. Композиция по п.27, в которой антитело включает аминокислотные последовательности легкой и тяжелой цепи, представленные в SEQ ID NO:21 и SEQ ID NO:22, соответственно, а носитель представляет собой фармацевтически приемлемый носитель.
30. Изделие, обладающее способностью связывать CD20 и представляющее собой контейнер и содержащуюся в нем композицию, где композиция включает антитело по п.1.
31. Изделие по п.30, дополнительно содержащее листовку-вкладыш в упаковке, где указано, что композицию можно применять для лечения не-ходжкинской лимфомы.
32. Способ индукции апоптоза В-клеток in vivo, заключающийся в том, что В-клетки приводят в контакт с антителом по п.1, что приводит к уничтожению В-клеток.
33. Способ лечения СD20-позитивного рака, заключающийся в том, что пациенту, который страдает раком, вводят терапевтически эффективное количество гуманизированного СD20-связывающего антитела по п.1.
34. Способ по п.33, в котором СD20-позитивный рак представляет собой В-клеточную лимфому или лейкоз.
35. Способ по п.34, в котором СD20-позитивный рак представляет собой не-ходжкинскую лимфому (НХЛ) или преимущественно лимфоцитарную болезнь Ходжкина (ПЛБХ).
36. Способ по п.33, в котором рак представляет хронический лимфоцитарный лейкоз (ХЛЛ) или мелкоклеточную лимфоцитарную лимфому (МЛЛ).
37. Способ по любому из пп.35 и 36, где антитело выбирают из группы, включающей антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, и тяжелую цепь с аминокислотной последовательностью, представленной SEQ ID NO:22; антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, и тяжелую цепь с аминокислотной последовательностью, представленной SEQ ID NO:23; антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, имеющей аминокислотную замену M32L в соответствии с системой нумерации, предложенной Кэботом, и тяжелую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:22, имеющей аминокислотные замены N100A, в соответствии с системой нумерации, предложенной Кэботом, и S289A, ЕЗЗЗА и К334А в соответствии с EU-нумерацией; антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, имеющей аминокислотную замену S92A в соответствии с системой нумерации, предложенной Кэботом, и тяжелую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:22, имеющей аминокислотные замены D56A и N100A, в соответствии с системой нумерации, предложенной Кэботом; антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, имеющей аминокислотную замену M32L и S92A в соответствии с системой нумерации, предложенной Кэботом, и тяжелую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:22, имеющей аминокислотные замены D56A и N100A в соответствии с системой нумерации, предложенной Кэботом, и S289A, ЕЗЗЗА и К334А в соответствии с EU-нумерацией; антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, имеющей аминокислотную замену M32L и S92A в соответствии с системой нумерации, предложенной Кэботом, и тяжелую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:22, имеющей аминокислотные замены D56A и N100A в соответствии с системой нумерации, предложенной Кэботом, и S289A, ЕЗЗЗА, К334А, E356D и M358L в соответствии с EU-нумерацией и антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, имеющей аминокислотную замену M32L и S92A в соответствии с системой нумерации, предложенной Кэботом, и тяжелую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:22, имеющей аминокислотные замены D56A и N100A в соответствии с системой нумерации, предложенной Кэботом, и S289A, ЕЗЗЗА, К334А и К326А в соответствии с EU-нумерацией.
38. Способ по п.37, в котором антитело включает аминокислотную последовательность легкой и тяжелой цепи, представленную SEQ ID NO:21 и 22, соответственно, или является антителом по п.13.
39. Способ по любому из пп.34, 38, в котором антитело вводят в дозе, составляющей примерно 275-375 мг/м.
40. Способ по любому из пп.34, 38, в котором антитело вводят в дозе, составляющей от примерно 250 мг/м до примерно 500 мг/м.
41. Способ по п.38, в котором пациенту вводят по меньшей мере две дозы антитела из расчета 375 мг/м на дозу.
42. Способ по п.41, в котором две дозы вводят с интервалом в две недели.
43. Способ по любому из пп.34, 38, заключающийся в том, что дополнительно вводят пациенту по меньшей мере один химиотерапевтический агент.
44. Способ по п.43, в котором рак представляет собой не-ходжкинскую лимфому (НХЛ) и химиотерапевтический агент выбирают из группы, включающей доксорубицин, циклофосфамид, винкристин, преднизолон и CHOP.
45. Способ лечения аутоиммунного заболевания, заключающийся в том, что пациенту, страдающему аутоиммунным заболеванием, вводят терапевтически эффективное количество гуманизированного CD20-связывающего антитела по п.1.
46. Способ по п.45, в котором аутоиммунное заболевание выбирают из группы, включающей ревматоидный артрит, ювенильный ревматоидный артрит (болезнь Стилла), системную красную волчанку (СКВ), волчаночный нефрит, неспецифический язвенный колит, болезнь Вегенера, воспалительное заболевание кишечника, идиопатическую тромпоцитопеническую пурпуру (ИТП), тромботическую тромбогемолитическую пурпуру (ТТЛ), аутоиммунную тромбоцитопению, рассеянный склероз, псориаз, связанную с IgA нефропатию, связанные с IgM полиневропатии, тяжелую псевдопаралитическую миастению, васкулит, ANCA васкулитов, отторжение трансплантата паренхиматозных органов, реакцию «трансплантат против хозяина» (РТПХ), сахарный диабет, синдром Рейно, синдром Шегрена и гломерулонефрит.
47. Способ по п.45, где гуманизированное CD20-связывающее антитело включает аминокислотные последовательности легкой и тяжелой цепи, представленные в SEQ ID NO:21 и SEQ ID NO:22, соответственно.
48. Способ по п.47, где гуманизированное CD20-связывающее антитело является антителом по п.13.
49. Способ по п.46, где аутоиммунным заболеванием является системная красная волчанка (СКВ) или волчаночный нефрит.
50. Способ по п.46, где аутоиммунным заболеванием является язвенный колит.
51. Способ по 46, где аутоиммунным заболеванием является ANCA васкулитов.
52. Способ по п.46, где аутоиммунным заболеванием является отторжение трансплантата паренхиматозных органов или реакция «трансплантат против хозяина».
53. Способ по любому из пп.47, 48, где антитело вводят внутривенной инфузией.
54. Способ по п.46, где аутоиммунное заболевание представляет собой ревматоидный артрит.
55. Способ по п.54, в котором пациент страдает ревматоидным артритом от средней до серьезной степени тяжести и не поддается лечению по меньшей мере одним модифицирующим заболевание противоревматоидным лекарственным средством.
56. Способ по п.54, в котором пациенту вводят дополнительно второй терапевтический агент.
57. Способ по п.56, в котором второй терапевтический агент представляет собой иммуносупрессор.
58. Способ по п.57, в котором иммуносупрессор представляет собой метотрексат.
59. Способ по п.54, в котором гуманизированное СD20-связывающее антитело включает аминокислотные последовательности легкой и тяжелой цепи, представленные в SEQ ID NO. 21 и 23 соответственно.
60. Способ по п.58, в котором антитело вводят в дозе, выбранной из 2×10 мг, 2×50 мг, 2×200 мг и 2×500 мг.
61. Способ по любому из пп.59, 60, в котором антитело вводят путем внутривенной инфузии.
62. Способ лечения аутоиммунного заболевания, выбранного из группы, включающей дерматомиозит, грануломатоз Вегенера, ANCA васкулитов, гипопластическую анемию, аутоиммунную гемолитическую анемию (АИГА), дефицит фактора VIII, гемофилию А, аутоиммунную нейтропению, синдром Кастельмана, синдром Гудпасчера, отторжение трансплантата паренхиматозных органов, реакцию «трансплантат против хозяина» (РТПХ), опосредуемое IgM заболевание, тромботическую тромбогемолитическую пурпуру (ТТП), тиреодит Хашимото, аутоимунный гепатит, лимфоидный интерстициальный пневмонит (ВИЧ), бронхолитические облитерации (не связанные с трансплантацией) против NSIP, синдром Гийена-Барре-Штроя, васкулит крупных сосудов, гигантоклеточный артериит (синдром Такаясу), васкулит средних сосудов, болезнь Кавасаки, нодозный полиартериит, заключающийся в том, что пациенту, страдающему указанным заболеванием, вводят терапевтически эффективное количество СD20-связывающего антитела или его функционального фрагмента.
63. Способ по п.62, в котором СD20-связывающее антитело представляет собой ритуксимаб.
64. Выделенная нуклеиновая кислота, кодирующая антитело по п.1.
65. Экспрессионный вектор, включающий нуклеиновую кислоту по п.64, кодирующую антитело по п.1.
66. Клетка-хозяин, содержащая нуклеиновую кислоту по п.64, продуцирующая рекомбинантное антитело по любому из пп.1-26.
67. Клетка-хозяин по п.66, продуцирующая гуманизированное антитело, выбранное из группы, включающей антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, и тяжелую цепь с аминокислотной последовательностью, представленной SEQ ID NO:22; антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, и тяжелую цепь с аминокислотной последовательностью, представленной SEQ ID NO:23; антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, имеющей аминокислотную замену M32L в соответствии с системой нумерации, предложенной Кэботом, и тяжелую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:22, имеющей аминокислотные замены N100A в соответствии с системой нумерации, предложенной Кэботом, и S289A, ЕЗЗЗА и К334А в соответствии с EU-нумерацией; антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, имеющей аминокислотную замену S92A в соответствии с системой нумерации, предложенной Кэботом, и тяжелую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:22, имеющей аминокислотные замены D56A и N100A в соответствии с системой нумерации, предложенной Кэботом; антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, имеющей аминокислотную замену M32L и S92A в соответствии с системой нумерации, предложенной Кэботом, и тяжелую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:22, имеющей аминокислотные замены D56A и N100A в соответствии с системой нумерации, предложенной Кэботом, и S289A, ЕЗЗЗА и К334А в соответствии с EU-нумерацией; антитело, выключающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, имеющей аминокислотную замену M32L и S92A в соответствии с системой нумерации, предложенной Кэботом, и тяжелую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:22, имеющей аминокислотные замены D56A и N100A в соответствии с системой нумерации, предложенной Кэботом, и S289A, ЕЗЗЗА, К334А, E356D и M358L в соответствии с EU-нумерацией; и антитело, включающее легкую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:21, имеющей аминокислотную замену M32L и S92A в соответствии с системой нумерации, предложенной Кэботом, и тяжелую цепь с аминокислотной последовательностью, представленной в SEQ ID NO:22, имеющей аминокислотные замены D56A и N100A в соответствии с системой нумерации, предложенной Кэботом, и S289A, ЕЗЗЗА, К334А и К326А в соответствии с EU-нумерацией.
68. Клетка-хозяин по п.67, представляющая собой СНО-клетку.
69. Способ получения гуманизированного антитела 2Н7, выбранного из 2H7.v16, v31, v75, v114, v115, v116 и v138, заключающийся в том, что культивируют клетку по п.67, и выделяют антитело из клеточной культуры.
70. Жидкая композиция, предназначенная для лечения ревматоидного артрита, содержащая гуманизированное антитело 2Н7 в концентрации 20 мг/мл, 10 мМ сульфат гистидина, рН 5,8, 60 мг/мл сахарозы, 0,2 мг/мл полисорбата 20.
71. Жидкая композиция по п.70, в которой антитело имеет аминокислотные последовательности легкой и тяжелой цепи, представленные в SEQ ID NO:21 и SEQ ID NO:22, соответственно.
72. Способ лечения ревматоидного артрита (РА) у человека, заключающийся в том, что пациенту вводят антитело к CD20 в дозе, выбранной из 2×50 мг, 2×200 мг и 2×500 мг.
73. Способ по п.72, в котором доза составляет 2×50 мг.
74. Способ по п.72, в котором доза составляет 2×200 мг.
75. Способ по п.72, в котором доза составляет 2×500 мг.
76. Способ по п.72, в котором антитело к CD20 представляет собой гуманизированное антитело.
77. Способ по п.72, в котором гуманизированное CD20-связывающее антитело включает аминокислотные последовательности легкой и тяжелой цепи, представленные в SEQ ID NO. 21 и 22, соответственно.
78. Способ по п.72, в котором антитело к CD20 представляет собой ритуксимаб.
79. Способ по п.72, в котором РА представляет собой РА умеренной или серьезной степени тяжести.
80. Гуманизированное CD20-связывающее антитело, содержащее в VL CDR1, CDR2 и CDR3, которые имеют последовательности, представленные в SEQ ID NO.4, SEQ ID NO.5 и SEQ ID NO.6, соответственно.
81. Антитело по п.80, дополнительно содержащее аминокислотные замены M32L и S92A в VL.
82. Антитело по п.81, содержащее Fc-фрагмент IgG1, который несет аминокислотные замены S298A/ЕЗЗЗА/K334A.
83. Гуманизированное CD20-связывающее антитело, содержащее Fc-фрагмент IgG1, который несет аминокислотные замены S298A, ЕЗЗЗА, К334А и К326А.
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