ES2593583T3 - Anticuerpos anti-BCMA - Google Patents

Anticuerpos anti-BCMA Download PDF

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ES2593583T3
ES2593583T3 ES10708865.0T ES10708865T ES2593583T3 ES 2593583 T3 ES2593583 T3 ES 2593583T3 ES 10708865 T ES10708865 T ES 10708865T ES 2593583 T3 ES2593583 T3 ES 2593583T3
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amino acids
variable domain
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ES2593583T9 (es
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Susan L. Kalled
Yen-Ming Hsu
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Biogen MA Inc
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Abstract

Un anticuerpo aislado o fragmento de unión a antígeno del mismo que se une al polipéptido de la SEQ ID NO: 9, en el que el anticuerpo o fragmento de unión a antígeno comprende: a) un dominio variable de cadena pesada que comprende una región CDR1 que comprende los aminoácidos 31-35 de la SEQ ID NO: 3, una región CDR2 que comprende los aminoácidos 50-66 de la SEQ ID NO: 3, y una región CDR3 que comprende los aminoácidos 99-106 de la SEQ ID NO: 3; y un dominio variable de cadena ligera que comprende una región CDR1 que comprende los aminoácidos 24-38 de una cualquiera de las SEQ ID NO: 4, 11 o 12, una región CDR2 que comprende los aminoácidos 54-60 de una cualquiera de las SEQ ID NO: 4, 11 o 12, y una región CDR3 que comprende los aminoácidos 93- 101 de una cualquiera de las SEQ ID NO: 4, 11 o 12; b) un dominio variable de cadena pesada que comprende una región CDR1 que comprende los aminoácidos 31-35 de la SEQ ID NO: 5, una región CDR2 que comprende los aminoácidos 50-66 de la SEQ ID NO: 5, y una región CDR3 que comprende los aminoácidos 99-106 de la SEQ ID NO: 5; y un dominio variable de cadena ligera que comprende una región CDR1 que comprende los aminoácidos 24-38 de la SEQ ID NO: 6, una región CDR2 que comprende los aminoácidos 54-60 de la SEQ ID NO: 6, y una región CDR3 que comprende los aminoácidos 93-101 de la SEQ ID NO: 6; o c) un dominio variable de cadena pesada que comprende una región CDR1 que comprende los aminoácidos 31-35 de la SEQ ID NO: 7, una región CDR2 que comprende los aminoácidos 50-66 de la SEQ ID NO: 7, y una región CDR3 que comprende los aminoácidos 99-106 de la SEQ ID NO: 7; y un dominio variable de cadena ligera que comprende una región CDR1 que comprende los aminoácidos 24-38 de la SEQ ID NO: 8, una región CDR2 que comprende los aminoácidos 54-60 de la SEQ ID NO: 8, y una región CDR3 que comprende los aminoácidos 93-101 de la SEQ ID NO: 8.

Description

imagen1
imagen2
La figura 5 representa la tinción por citometría de flujo para las células plasmáticas (PC) en el compartimiento CD45+ humano del esplenocito aisladas de ratones HSC/NSG tratados con anticuerpo anti-BCMA (chC12A3.2 o chC13F12.1) o control de IgG1 humana. Los ratones recibieron una inyección i.p. de Ab anti-BCMA o control de
5 HIgG1 dos veces por semana durante 2 semanas. ** p<0,0001; * p = 0,0066.
La figura 6 representa la tinción por citometría de flujo para las células plasmáticas (PC) en el compartimiento CD45+ humano del esplenocito aisladas de ratones HSC/NSG tratados con anticuerpo anti-BCMA (chCHD5.1 o chA7D12.2) o control de IgG1 humana. Los ratones recibieron una inyección i.p. de anticuerpo anti-BCMA o control
10 de HIgG1 dos veces por semana durante 2 semanas.
Tabla 1. Breve descripción de las secuencias
SEQ ID NO
Descripción de secuencia
1
secuencia de proteína de dominio variable de cadena pesada madura A7D12.2
2
secuencia de proteína de dominio variable de cadena ligera madura A7D12.2
3
secuencia de proteína de dominio variable de cadena pesada madura C11D5.3
4
secuencia de proteína de dominio variable de cadena ligera madura A C11D5.3
5
secuencia de proteína de dominio variable de cadena pesada madura C12A3.2
6
secuencia de proteína de dominio variable de cadena ligera madura C12A3.2
7
secuencia de proteína de dominio variable de cadena pesada madura C13F12.1
8
secuencia de proteína de dominio variable de cadena ligera madura C13F12.1
9
secuencia de proteína de BCMA
10
huBCMA-huFc (como se define por el análisis de secuencia N-terminal)
11
secuencia B de proteína de dominio variable de cadena ligera madura C11D5.3
12
secuencia C de proteína de dominio variable de cadena ligera madura C11D5.3
13
secuencia de proteína de cadena pesada madura quimérica chA7D12.2
14
secuencia de proteína de cadena ligera madura quimérica chA7D12.2
15
secuencia de proteína de cadena pesada madura quimérica chC11 D5.3
16
secuencia A de proteína de cadena ligera madura quimérica chC11 D5.3
17
secuencia C de proteína de cadena ligera madura quimérica chC11 D5.3
18
secuencia de proteína de cadena pesada madura quimérica chC12A3.2
19
secuencia de proteína de cadena ligera madura quimérica chC12A3.2
20
secuencia de proteína de cadena pesada madura quimérica chC13F12.1
21
secuencia de proteína de cadena ligera madura quimérica chC13F12.1
22
secuencia de dominio variable de cadena ligera madura humanizada huC11D5.3L1
23
secuencia de dominio variable de cadena ligera madura humanizada huC11D5.3L2
24
secuencia de dominio variable de cadena ligera madura humanizada huC11D5.3L3
25
secuencia de dominio variable de cadena pesada madura humanizada huC11D5.3H0
26
secuencia de dominio variable de cadena pesada madura humanizada huC11D5.3H1
27
secuencia de dominio variable de cadena pesada madura humanizada huC11D5.3H2
28
secuencia de dominio variable de cadena pesada madura humanizada huC11D5.3H3
29
secuencia de dominio variable de cadena pesada madura humanizada huC11D5.3H4
30
secuencia de dominio variable de cadena ligera madura humanizada huC12A3.2L0
31
secuencia de dominio variable de cadena ligera madura humanizada huC12A3.2L1
32
secuencia de dominio variable de cadena ligera madura humanizada huC12A3.2L2
33
secuencia de dominio variable de cadena ligera madura humanizada huC12A3.2L3
34
secuencia de dominio variable de cadena pesada madura humanizada huC12A3.2H0
35
secuencia de dominio variable de cadena pesada madura humanizada huC12A3.2H1
36
secuencia de dominio variable de cadena pesada madura humanizada huC12A3.2H2
37
secuencia de dominio variable de cadena pesada madura humanizada huC12A3.2H3
38
secuencia de dominio variable de cadena pesada madura humanizada huC12A3.2H4
39
secuencia de dominio variable de cadena ligera madura humanizada huC13F12.1L0
40
secuencia de dominio variable de cadena ligera madura humanizada huC13F12.1L1
41
secuencia de dominio variable de cadena ligera madura humanizada huC13F12.1L2
42
secuencia de dominio variable de cadena ligera madura humanizada huC13F12.1 L3
43
secuencia de dominio variable de cadena pesada madura humanizada huC13F12.1H0
44
secuencia de dominio variable de cadena pesada madura humanizada huC13F12.1H1
4
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que mediante sí mismo, estos valores ligeramente negativos también podrían ser resultado de la variación experimental. Después, los resultados se expresaron como un porcentaje del valor de control positivo, es decir, la lectura de absorbancia ajustada al fondo para el anticuerpo conjugado con biotina en ausencia de un anticuerpo no conjugado de competencia.
5 Se consideró que los anticuerpos unían el mismo epítopo o epítopos en superposición muy cercana si cada uno de ellos reducía la unión del otro (de acuerdo con la fracción calculada como anteriormente) a menos del 20 % del valor del control positivo. En caso de no satisfacer esta condición, se consideró que tenían epítopos al menos parcialmente distintos. La Tabla 3 muestra qué anticuerpos tienen epítopos al menos parcialmente distintos.
10
Ejemplo 5. Análisis por citometría de flujo de la unión de anticuerpos a células de sangre periférica humana
La sangre se obtuvo de voluntarios sanos que dieron su consentimiento y las células mononucleares de sangre periférica (PBMC) se enriquecieron mediante centrifugación a través de Ficoll-Paque (GE Healthcare, Reino Unido), 15 de acuerdo con las recomendaciones del fabricante. Las PBMC se lavaron de forma extensa en PBS antes de su uso. Las células se trataron previamente con tampón FACS que contenía suero de ratón normal al 5 % para bloquear los sitios de unión no específicos. Se usaron los siguientes anticuerpos monoclonales conjugados con fluoróforo dirigidos contra marcadores de células plasmáticas y linfocitos B específicos: anti-CD19-PE-Cy5, anti-IgD-FITC, anti-CD27-APC, anti-CD38-PE-Cy7 (BD Biosciences, San Jose, CA). Se usó estreptavidina-PE (Molecular
20 Probes, Eugene, OR) para visualizar los mAb anti-BCMA conjugados con biotina (10 µg/ml). También se midió la unión de un mAb de control de isotipos como en el Ejemplo 3.
Ninguno de los mAb anti-BCMA tiñó los linfocitos B sin tratar de los voluntarios sanos (figura 2E), mientras que todos tiñeron las células plasmáticas, aunque con intensidades variables (figura 2A). Solamente el clon A7D12.2 tiñó una 25 proporción de los tres subconjuntos de linfocitos B de memoria (figura 2B-D).
Ejemplo 6. Comparación de la unión de anticuerpos a linfocitos B de individuos sanos y con SLE
La sangre se obtuvo de voluntarios sanos que dieron su consentimiento y pacientes con SLE y se procesó como en
30 el Ejemplo 5. La figura 3 muestra una comparación entre las muestras de un voluntario sano y un paciente con SLE representativo. El anticuerpo A7D12.2 se unió a las células plasmáticas tanto de los voluntarios sanos como de los pacientes con SLE (figura 3A). En las muestras con SLE, pero no en las muestras sanas, el anticuerpo A7D12.2 se unió a los linfocitos B sin tratar (figura 3E). La unión del anticuerpo A7D12.2 a los linfocitos B de memoria (figura 3B-D), particularmente a linfocitos B de memoria doble negativos (figura 3D), aumentó en las muestras con SLE.
35
Ejemplo 7. Generación de líneas celulares que producen mAb anti-BCMA quiméricos
Se usó CHO-DG44-I, una línea celular de ovario de hámster chino independiente de insulina y dhfr-deficiente, para construir líneas celulares de tipo silvestre anti-BCMA. Las células huésped se cultivaron en medio CHO-S-SFM II 40 con nucleósidos antes de la transfección.
Los anticuerpos quiméricos se produjeron transfectando las células con plásmidos de expresión que codificaban las secuencias de cadena ligera y pesada maduras enumeradas en la Tabla 4.
45 Tabla 4. Secuencias de cadena pesada y ligera maduras de anticuerpos anti-BCMA quiméricos
Anticuerpo quimérico
Secuencia de cadena pesada madura Secuencia de cadena ligera madura
chA7D12.2
SEQ ID NO: 13 SEQ ID NO: 14
chC11D5.3
SEQ ID NO: 15 SEQ ID NO: 16 (para el Ejemplo 8) SEQ ID NO: 17 (para el Ejemplo 9)
chC12A3.2
SEQ ID NO: 18 SEQ ID NO: 19
chC13F12.1
SEQ ID NO: 20 SEQ ID NO: 20
Los plásmidos de expresión anti-BCMA quiméricos se transfectaron en la línea celular huésped CHO DG44-I usando un método de lípido catiónico (Fugene HD). En resumen, se sembraron 1 x 106 células DG44-I en cada uno de dos pocillos de una placa de 6 pocillos que contenía 3 ml de medio CHO-S-SFMII con nucleósidos por pocillo. Se 50 diluyeron cuatro µg de ADN de plásmido (2 µg de cadena pesada, 2 µg de cadena ligera) en 200 µl de medio CHO-S-SFM II (Invitrogen) a temperatura ambiente. Se dejó que dieciséis µl de reactivo Fugene HD (Roche) formaran un complejo con el ADN durante aproximadamente 15 minutos. Se añadieron 100 µl de la mezcla de ADN en complejo
14
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Claims (1)

  1. imagen1
    imagen2
    imagen3
ES10708865.0T 2009-03-10 2010-03-10 Anticuerpos anti-BCMA Active ES2593583T3 (es)

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US15894209P 2009-03-10 2009-03-10
US158942P 2009-03-10
US16292409P 2009-03-24 2009-03-24
US162924P 2009-03-24
PCT/US2010/026825 WO2010104949A2 (en) 2009-03-10 2010-03-10 Anti-bcma antibodies

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