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PT2352763E - Anticorpos biespecíficos de cadeia única com especificidade para antigénios-alvo com elevado peso molecular - Google Patents

Anticorpos biespecíficos de cadeia única com especificidade para antigénios-alvo com elevado peso molecular Download PDF

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PT2352763E
PT2352763E PT09736398T PT09736398T PT2352763E PT 2352763 E PT2352763 E PT 2352763E PT 09736398 T PT09736398 T PT 09736398T PT 09736398 T PT09736398 T PT 09736398T PT 2352763 E PT2352763 E PT 2352763E
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Kufer Peter
Blümel Claudia
Kischel Roman
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Amgen Res Munich Gmbh
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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Claims (13)

  1. REIVINDICAÇÕES
    1. Processo para a seleção de anticorpos biespecíficos de cadeia única contendo um primeiro domínio de ligação capaz de se ligar a um determinante antigénico de CD3 e a um segundo domínio de ligação capaz de se ligar ao domínio extracelular do antigénio da membrana específica da próstata (AMEP), caracterizado pelo facto de compreender as etapas de: (a) providenciar pelo menos três tipos de células hospedeiras gue expressam (i.) o domínio extracelular humano de tipo selvagem de AMEP tendo a seg ID: 447 na superfície celular; (ii) uma forma mutada do AMEP humano de tipo selvagem na superfície celular, em que os resíduos de aminoácidos, nas posições 140, 169, 191, 308, 334, 339, 344, 624, 626, 716, 717 e 721 estão mutados nos correspondentes resíduos de aminoácidos do AMEP de tipo selvagem de roedor; e (iii) o domínio extracelular de tipo selvagem de AMEP de roedor na superfície celular; (b) fazer contatar cada tipo das células hospedeiras de (i) , (ii) e (iii) da etapa (a) com os anticorpos biespecíficos de cadeia única e células T efetoras; e (c) identificar e isolar anticorpos biespecíficos de cadeia única que medeiam a lise das células hospedeiras que expressam o domínio extracelular humano de tipo selvagem de AMEP na superfície celular, de acordo com (b) (i) e das células hospedeiras que expressam a forma mutada do AMEP humano de tipo selvagem, na superfície celular, de acordo com (b) (ii) , mas não das células hospedeiras que expressam o domínio extracelular AMEP de tipo selvagem de roedor, na superfície celular, de acordo com b (iii) .
  2. 2. Processo para a seleção de anticorpos biespecíficos de cadeia única contendo um primeiro domínio capaz de se ligar a um determinante antigénico de CD3 e um segundo domínio de ligação capaz de se ligar ao domínio extracelular da proteína de ativação de fibroblastos α (PAFa), caracterizado pelo facto de compreender as etapas de: (a) providenciar pelo menos três tipos de células hospedeiras que expressam (i) o domínio extracelular humano de tipo selvagem de PAFa tendo a seq ID: 448 na superfície celular; (ii) uma forma mutada do PAFa humana de tipo selvagem, na superfície celular, em que os resíduos de aminoácidos, nas posições 144, 185, 186, 229, 267, 273, 274, 278, 284, 301, 328, 329, 331, 335 e 362 estão mutados nos correspondentes resíduos de aminoácidos do PAFa de tipo selvagem de roedor; e (iii) o domínio extracelular de tipo selvagem de roedor de PAFa na superfície celular; (b) fazer contatar cada tipo de células hospedeiras (i) , (ii) e (iii) , da etapa (a) , com os anticorpos biespecíficos de cadeia única e células T efetoras; e (c) identificar e isolar os anticorpos biespecíficos de cadeia única que medeiam a lise das células hospedeiras que expressam o domínio extracelular humano de tipo selvagem de PAFa, na superfície celular, de acordo com (b) (i) e das células hospedeiras que expressam a forma mutada do PAFa humano de tipo selvagem, na superfície celular, de acordo com (b) (ii) , mas não das células hospedeiras que expressam o domínio extracelular de PAFa, de tipo selvagem, de roedor, PAFa na superfície celular, de acordo com b(iii).
  3. 3. Processo para a seleção de anticorpos biespecificos de cadeia única, caracterizado pelo facto de compreender um primeiro domínio de ligação capaz de se ligar a um determinante antigénico de CD3 e um segundo domínio de ligação capaz de se ligar ao domínio extracelular do recetor do fator de crescimento de hepatócitos (c-MET), endosialina (TEMI) e do recetor do tipo 1 do fator de crescimento semelhante à insulina (Rl-FCI), compreendendo as etapas de: (a) identificação dos 640 aminoácidos da membrana proximal do homólogo humano e do homólogo de roedor do domínio de c-MET, TEMI ou Rl-FCI; (b) fornecimento de células hospedeiras que expressam (i )o tipo selvagem humano do domínio extracelular dos domínios extracelulares c-MET, TEMI ou Rl-FCI, na superfície celular; (ii) uma proteína de fusão que contém 640 resíduos de aminoácidos da membrana proximal humana identificados na etapa (a) e > 640 resíduos de aminoácidos de roedor de c-MET, TEMI ou Rl-FCI; e (iii) o domínio extracelular de tipo selvagem de roedor de c-MET, TEMI ou Rl-FCI; (c) fazer contatar as células hospedeiras, de acordo com a etapa (b) , com os anticorpo biespecífico de cadeia única e células T efetoras; e (d) identificação e isolamento dos anticorpos biespecíficos de cadeia única que medeiam a lise das células hospedeiras, de acordo com (b)(i) e (b)(ii), mas não as células hospedeiras de acordo com b(iii).
  4. 4. Processo de acordo com uma qualquer das reivindicações 1 a 3, caracterizado pelo facto de o primeiro domínio de ligação se ligar a CD3 épsilon (CD3s) de seres humanos e de Callithrix jacchus, Saguinus oedipus ou Saimiri sciureus.
  5. 5. Processo, de acordo com a reivindicação 4, caracterizado pelo facto de o primeiro domínio de ligação compreender uma região VL que contém RDC-Ll, RDC-L2 e RDC-L3 selecionadas entre: (a) RDC-Ll tal como descrito na SEQ ID N2. 27, RDC-L2 tal como descrito na SEQ ID N2. 28, RDC-L3 tal como descrito na SEQ ID N2. 29; (b) RDC-Ll tal como descrito na SEQ ID N2. 117, RDC-L2 tal como descrito na SEQ ID N2. 118, RDC-L3 tal como descrito na SEQ ID N2. 119; e (c) RDC-Ll tal como descrito na SEQ ID N2. 153, RDC-L2 tal como descrito na SEQ ID N2. 154, RDC-L3 tal como descrito na SEQ ID N2. 155.
  6. 6. Processo, de acordo com a reivindicação 4, caracterizado pelo facto de o primeiro domínio de ligação compreender uma região VP que contém as RDC-P1, RDC-P2 e RDC-P3 selecionadas entre: (a) RDC-P1 tal como descrito na SEQ ID N2. 12, RDC-P2 tal como descrito na SEQ ID N2. 13 e RDC-P3 tal como descrito na SEQ ID N2. 14; (b) RDC-P1 tal como descrito na SEQ ID N2. 30, RDC-P2 tal como descrito na SEQ ID N2. 31 e RDC-P3 tal como descrito na SEQ ID N2. 32; (c) RDC-P1 tal como descrito na SEQ ID N2. 48, RDC-P2 tal como descrito na SEQ ID N2. 49 e RDC-P3 tal como descrito na SEQ ID N2. 50; (d) RDC-P1 tal como descrito na SEQ ID N2. 66, RDC-P2 tal como descrito na SEQ ID N2. 67 e RDC-P3 tal como descrito na SEQ ID N2. 68; (e) RDC-P1 tal como descrito na SEQ ID N2. 84, RDC-P2 tal como descrito na SEQ ID N2. 85 e RDC-P3 tal como descrito na SEQ ID N2. 86; (f) RDC-P1 tal como descrito na SEQ ID N2. 102, RDC-P2 tal como descrito na SEQ ID N2. 103 e RDC-P3 tal como descrito na SEQ ID N2. 104; (g) RDC-P1 tal como descrito na SEQ ID N2. 120, RDC-P2 tal como descrito na SEQ ID N2. 121 e RDC-P3 tal como descrito na SEQ ID N2. 122; (h) RDC-P1 tal como descrito na SEQ ID N2. 138, RDC-P2 tal como descrito na SEQ ID N2. 139 e RDC-P3 tal como descrito na SEQ ID N2. 140; (i) RDC-P1 tal como descrito na SEQ ID N2. 156, RDC-P2 tal como descrito na SEQ ID N2. 157 e RDC-P3 tal como descrito na SEQ ID N2. 158; e (j) RDC-P1 tal como descrito na SEQ ID N2. 174, RDC-P2 tal como descrito na SEQ ID N2. 175 e RDC-P3 tal como descrito na SEQ ID N2. 176.
  7. 7. Processo, de acordo com uma qualquer das reivindicações 3 a 6, caracterizado pelo facto de o segundo domínio de ligação se ligar aos determinantes antigénicos/sítios de ligação selecionados no grupo que consiste em: determinantes antigénicos/sítios de ligação nos quatro domínios de Ig com a seq ID: 436, um domínio rico em cisteína com a seq ID:437 ou a cadeia beta de um domínio sema com a seq ID: 438 do domínio extracelular de c-MET, determinantes antigénicos/sítios de ligação no domínio de mucino com a seq ID: 440, os três domínios semelhantes a FCE com a seq ID: 441 ou o domínio Sushi/SCR/CCP com a seq ID: 442 do domínio extracelular de TEMI e determinantes antigénicos/sítios de ligação nos três domínios de fibronectin do tipo III com a seq ID: 444 e o domínio L2 com a seq ID: 445 do domínio extracelular de Rl-FCI.
  8. 8. Anticorpo biespecífico de cadeia única compreendendo um primeiro domínio de ligação capaz de se ligar a um determinante antigénico de CD3 épsilon (CD3s) de seres humano e primatas não chimpanzés, em que o determinante antigénico faz parte de uma sequência de aminoácidos compreendida no grupo que consiste nas SEQ ID N2s. 2, 4, 6 e 8 e um segundo domínio de ligação selecionado no grupo que consiste em: (a) um domínio de ligação capaz de se ligar ao domínio extracelular de AMEP humano mutado com uma sequência de aminoácidos tal como a representada na SEQ ID N2 . 447, em que os resíduos de aminoácidos, nas posições 140, 169, 191, 308, 334, 339, 344, 624, 626, 716, 717 e 721 estão mutados nos correspondentes resíduos de aminoácidos de AMEP de tipo selvagem de roedor, mas não com o domínio extracelular de AMEP de roedor; (b) um domínio de ligação capaz de se ligar ao domínio extracelular da PAFa quimérica humana, mutada, com uma sequência de aminoácidos, tal como descrito na SEQ ID N2: 448, em que os resíduos de aminoácidos nas posições 144, 185, 186, 229, 267, 273, 274, 278, 284, 301, 328, 329, 331, 335 e 362 estão mutados nos correspondentes resíduos de aminoácidos de PAFa de tipo selvagem de roedor, mas não com o domínio extracelular de PAFa de roedor; (c) um domínio de ligação capaz de se ligar aos quatro domínios de Ig com a SEQ ID N2: 436, um domínio rico de cisteína com a seq ID: 437 ou a cadeia beta de um somínio sema com a seq ID: 438 do domínio extracelular de c-MET; (d) um domínio de ligação capaz de se ligar ao domínio de mucina com a seq ID: 440, aos três domínios semelhantes a FCE com a seq ID: 441 ou o domínio de Sushi/SCR/CCP com a seq ID: 442 do domínio extracelular de TEMI; e (e) um domínio de ligação capaz de se ligar aos três domínios de fibronectina do tipo III, domínio que tem a seq ID: 444 e o domínio L2 com a seq ID: 445 do domínio extracelular de Rl-FCI.
  9. 9. Sequência de ácidos nucleicos, caracterizada pelo facto de codificar para uma molécula de anticorpo bi-específico de cadeia única, tal como definido na reivindicação 8.
  10. 10. Vetor, caracterizado pelo facto de compreender uma sequência de ácidos nucleicos, tal como definido na reivindicação 9.
  11. 11. Célula hospedeira caracterizada pelo facto de estar transformada ou transfectada com um vetor definido na reivindicação 10.
  12. 12. Processo para a produção de uma molécula de anticorpo biespecifico de cadeia única, de acordo com a reivindicação 8, caracterizado pelo facto de o referido processo compreender a cultura de uma célula hospedeira, definida na reivindicação 11, em condições que permitem a expressão do polipéptido, tal como definido na reivindicação 8 e a recuperação do polipéptido produzido a partir da cultura.
  13. 13. Composição farmacêutica, caracterizada pelo facto de compreender uma molécula de anticorpo biespecifico de cadeia única, de acordo com a reivindicação 8 ou por ser produzida de acordo com o processo da reivindicação 12 . Lisboa, 25 de Maio de 2016.
PT09736398T 2008-10-01 2009-10-01 Anticorpos biespecíficos de cadeia única com especificidade para antigénios-alvo com elevado peso molecular PT2352763E (pt)

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