ES2665317T3 - Anticuerpos humanizados dirigidos contra el dominio EC1 de la cadherina-11 y composiciones y procedimientos relacionados - Google Patents

Anticuerpos humanizados dirigidos contra el dominio EC1 de la cadherina-11 y composiciones y procedimientos relacionados Download PDF

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ES2665317T3
ES2665317T3 ES11741717.0T ES11741717T ES2665317T3 ES 2665317 T3 ES2665317 T3 ES 2665317T3 ES 11741717 T ES11741717 T ES 11741717T ES 2665317 T3 ES2665317 T3 ES 2665317T3
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James G. Mcarthur
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Abstract

Un anticuerpo humanizado que se une especificamente a un dominio EC1 de una proteina cadherina-11 de mamifero y antagoniza dicha proteina cadherina-11 de mamifero, que comprende una region variable de la cadena pesada de anticuerpo que comprende la SEQ ID NO: 69 y una region variable de la cadena ligera de anticuerpo que comprende la SEQ ID NO: 71.

Description

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La CI50 relativa se calculó dividiendo el valor para el anticuerpo de ensayo por el de SYN0012. Los niveles de expresión de anticuerpos dados son para cultivos estáticos en matraces T. Se calculan los PI.
5 En la selección inicial de los anticuerpos anti-cad-11 humanizados contra el antígeno cad-11 y los antígenos cad-7, 8, -9, -11, -18, -20, -24 a un revestimiento de 500 ng/ml ( Figura 41A), SDP051 demostró una unión mucho mayor a cad-11 que cualquier otra cadherina. El mayor grado de reactividad cruzada se observó con el antígeno de cad-8. Este ensayo se repitió con los antígenos de cad-11, cad-8 y cad-18 con una concentración de revestimiento de antígeno inferior de 50 ng/ml y una serie de dilución más amplia del anticuerpo SDP051 (Figura 41B). En este
10 ensayo, SDP051 tenía una CE50 de 6,8 ng/ml para cad-11 y una CE50 de 1421 ng/ml de cad-8, un diferencial de unión de más de 200 veces. No se pudo establecer una CE50 para ad-18 debido al bajo nivel de unión observado. Estos resultados confirmaron que SDP051 era altamente específico para cad-11. Una búsqueda BLAST con la secuencia del dominio EC1 de cad-11 indicó que las coincidencias más cercanas estaban entre los antígenos cad-7, -8, -9, -11, -18, -20 y -24. Una búsqueda BLAST con el epítopo de unión a hexapéptido SDP051 dilucidado en el
15 dominio EC1 de cad-11 indicó que ninguna otra proteína humana conocida poseía esta secuencia.
SDP051 tenía afinidad subnanomolar por la proteína rhCad-11 con una constante de disociación en el equilibrio (KD) de 0,37 nM (Tabla 4).
20 Tabla 4: Propiedades físicas de SDP051. mAb SDP051
ka1 (1/Ms) 6,13x105 kd1 (1/s) 0,004301 ka2 (1/s) 0,01160 kd2 (1/s) 6,618 x104 KD (M) 3,787 x1010 Chi2 0,194
Los experimentos de caracterización y control para la superficie del chip sugirieron que la rhCad-11 inmovilizada en la densidad única era adecuada para determinar los valores cinéticos de las interacciones del anticuerpo anti-cad-11.
25 Para el análisis cinético, se usó un modelo de reacción de 2 estados (Ecuación 1, Figura 42) basándose en los resultados del control de reacción de unión, que indicaba que se producía otro suceso dependiente del tiempo una vez que el antígeno se había unido al anticuerpo. Dado que rhCad-11 es homodimérica y, por lo tanto, contiene dos epítopos de unión idénticos, es probable que las moléculas de mAb individuales se unan secuencialmente a través de un primer sitio de unión y luego a ambos sitios de unión.
30
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Ecuación 1: Reacción de dos estados
En los ensayos de unión a células, SDP051 y SDP071 demostraron una unión dependiente de la dosis a las células que expresan cad-11 y no se unen a la línea celular parental negativa para cad-11 (Figura 43).
35 Una forma alternativa de determinar la especificidad de SDP051 para cad-11 es medir la capacidad de diferentes cadherinas para bloquear la unión de SDP051 a células que expresan cad-11. Esto se realizó para cada una de las cadherinas-7, -8, -9, -18, -20, -24 o -MN. Solo el péptido cad-11 bloqueó significativamente la unión de SDP051 a las células cad-11+. Los resultados del análisis se muestran en las figuras 44A-C.
40
Ejemplo 15: SDP051, un anticuerpo humanizado Anti EC1 de cad11, exhibe baja inmunogenicidad en ensayos que usan células humanas
Materiales y procedimientos 45 Purificación de anticuerpos
SDP051 se purificó a partir de cultivos de 5 l de células que expresaban anticuerpos que crecían hasta la saturación. Los sobrenadantes se separaron de las células y los desechos, se ajustaron a pH 7,4, se esterilizaron por filtración y 50 se procesaron a través de columnas de afinidad de proteína A Hi-Trap Mab Select Sure de 5 ml (GE Healthcare, Amersham, Reino Unido), previamente desinfectadas con NaOH 0,5 M, a un caudal de 5 ml/min. La columna se lavó con 100 ml de PBS a pH 7,4. El anticuerpo se eluyó en fracciones de 5 ml con citrato de sodio 0,1M a pH 3,0 y cada fracción se neutralizó inmediatamente con 0,25 ml de Tris-HCl 1M. El contenido de proteína de cada fracción se controló por absorción de UV a 280 nm y las fracciones que contenían proteína se juntaron, se cambió el tampón en
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Claims (1)

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ES11741717.0T 2010-07-15 2011-07-15 Anticuerpos humanizados dirigidos contra el dominio EC1 de la cadherina-11 y composiciones y procedimientos relacionados Active ES2665317T3 (es)

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