ES2618526T3 - Anticuerpos para M-CSF - Google Patents

Anticuerpos para M-CSF Download PDF

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Publication number
ES2618526T3
ES2618526T3 ES04809712.5T ES04809712T ES2618526T3 ES 2618526 T3 ES2618526 T3 ES 2618526T3 ES 04809712 T ES04809712 T ES 04809712T ES 2618526 T3 ES2618526 T3 ES 2618526T3
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csf
amino acid
seq
acid sequence
antibodies
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ES04809712.5T
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Vahe Bedian
Madhav Narasimha Devalaraja
Joseph Edwin Low
James Leslie Mobley
Sirid-Aimee Kellermann
Ian Foltz
Mary Haak-Frendscho
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Amgen Fremont Inc
Warner Lambert Co LLC
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Amgen Fremont Inc
Warner Lambert Co LLC
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Application filed by Amgen Fremont Inc, Warner Lambert Co LLC filed Critical Amgen Fremont Inc
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Publication of ES2618526T3 publication Critical patent/ES2618526T3/es
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Abstract

Un anticuerpo monoclonal humano o una porción de unión a antígeno del mismo que se une de forma específica a M-CSF, en donde el anticuerpo comprende: (a) una secuencia de aminoácidos de cadena pesada que es al menos un 90 % idéntica a la secuencia de aminoácidos de cadena pesada de la SEQ ID NO: 30 sin los diecinueve restos de aminoácidos de la secuencia señal (restos 1-19 de la SEQ ID NO: 30), y (b) una secuencia de aminoácidos de cadena ligera que es al menos un 90 % idéntica a la secuencia de aminoácidos de cadena ligera de la SEQ ID NO: 32 sin los veinte restos de aminoácidos de la secuencia señal (restos 1-20 de la SEQ ID NO: 32); y en donde el anticuerpo tiene al menos una de las propiedades seleccionadas del grupo que consiste en: (i) inhibe la proliferación celular dependiente del M-CSF con una CI50 de 8 x 10-8 M o menor; (ii) inhibe el cambio de forma de los monocitos humanos dependiente del M-CSF con una CI50 de 9 x 10-8 M o menor; y (iii) inhibe la unión del receptor de M-CSF con una CI50 de 7 x 10-8 M o menor.

Description

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presente (Lewis D.A., et al., Anal. Chem, 66(5): 585-95 (1994)). En varias realizaciones de la invención, las cadenas pesada y ligera delos anticuerpos anti-M-CSF pueden incluir demanera opcional una secuencia señal.
En un aspecto, la invención se refiere a la inhibición de anticuerpos anti-M-CSF monoclonales y a las líneas celulares modificadas por ingeniería genética para producirlos. La tabla 1 enumera los identificadores de secuencia 5 (SEQ ID NOS) de los ácidos nucleicos que codifican la región variable de las cadenas pesada y ligera y las correspondientes secuencias de aminoácidos predichas para los anticuerpos monoclonales: 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3 y 9.7.2. Las variantes adicionales de los anticuerpos 9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4 8.10.3FG1 o 9.14.4G1 se pueden producir mediantemétodos conocidos por el experto en la 10 materia.
Tabla 1
ANTICUERPOS ANTI-M-CSF HUMANOS
MAb
IDENTIFICADOR DE SECUENCIA (SEQ ID NO:)
Longitud completa
Pesada
Ligera
ARN
Proteína ADN Proteína
252
1 2 3 4
88
5 6 7 8
100
9 10 11 12
3.8.3
14 16
2.7.3
18 20
1.120.1
22 24
9.14.41
25 26 27 28
9.14.4
37 38 27 28
9.14.4C-Ser
54 56
9.14.4-CG2
74 56
9.14.4-CG4
78 56
9.14.4-Ser
82 27 28
9.14.4-G1
101 102 27 28
8.10.3F
29 30 31 32
8.10.3
29 30 43 44
8.10.3C-Ser
58 60
8.10.3-CG2
62 60
8.10.3-Ser
90 43 44
8.10.3-CG4
94 60
8.10.3FG1
97 98 31 32
9.7.2IF
33 34 35 36
9.7.2
45 46 47 48
9.7.2C-Ser
50 52
18
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Hibridoma9.14.4(LN15895) PTA-5394 Hibridoma8.10.3(LN15896) PTA-5395 Hibridoma88-gamma(UC25489) PTA-5396 Hibridoma 88-kappa (UC 25490) PTA-5397 Hibridoma 100-gamma (UC 25491) PTA-5398 Hibridoma 100-kappa (UC 25492) PTA-5399 Hibridoma 252-gamma (UC 25493) PTA-5400 Hibridoma 252-kappa (UC 25494) PTA-5401
EJEMPLO II Análisis de utilización de genes El ADN que codifica las cadenas pesada y ligera de los anticuerpos monoclonales 252, 88, 100, 3.8.3, 2.7.3,
1,120.1, 9.14.4, 8.10.3 y 9.7.2 se clonó a partir de las respectivas líneas celulares de hibridoma y se determinaron
5 las secuencias de ADN mediante métodos conocidos por el experto en la materia. De forma adicional, se mutó el ADN de las líneas celulares de hibridoma 9.14.4, 8.10.3 y 9.7.2 en regiones marco conservadas específicas en el dominio variable y/o se les cambió el isotipo para obtener, por ejemplo 9.14.4I, 8.10.3F y 9.7.2IF, respectivamente. Se determinó la identidad del uso de genes para cada cadena de anticuerpos ("VBASE") a partir de la secuencia de ácidos nucleicos y la secuencia de aminoácidos predicha de los anticuerpos. La tabla 2 expone la utilización de
10 genes de los anticuerpos seleccionados según la invención: Tabla 2
Utilización de genes de cadena pesada y ligera
Clon
Cadena pesada Cadena ligera kappa
SEQ ID NO:
VH DH JH SEQ ID NO: Vκ Jκ
252
1, 2 3-11 7-27 6 3, 4 O12 3
88
5, 6 3-7 6-13 4 7, 8 O12 3
100
9, 10 3-23 1-26 4 11, 12 L2 3
3.8.3
14 3-11 7-27 4 16 L5 3
2.7.3
18 3-33 1-26 4 20 L5 4
1.120.1
22 1-18 4-23 4 24 B3 1
9.14.4I
25, 26 3-11 7-27 4b 27, 28 O12 3
8.10.3F
29, 30 3-48 1-26 4b 31, 32 A27 4
9.7.2IF
33, 34 3-11 6-13 6b 35, 36 O12 3
9.14.4
37, 38 3-11 7-27 4b 27, 28 O12 3
8.10.3
29, 30 3-48 1-26 4b 43, 44 A27 4
9.7.2
45, 46 3-11 6-13 6b 47, 48 O12 3
8.10.3FG1
97, 98 3-48 1-26 4b 31, 32 A27 4
9.14.4G1
101, 102 3-11 7-27 4b 27, 28 O12 3
9.14.4C-Ser
54 3-11 7-27 4b 56 O12 3
41 5
10
15
20
25
30
Utilización de genes de cadena pesada y ligera
Clon
Cadena pesada Cadena ligera kappa
SEQ ID NO:
VH DH JH SEQ ID NO: Vκ Jκ
9.14.4-CG2
74 3-11 7-27 4b 56 O12 3
9.14.4-CG4
78 3-11 7-27 4b 56 O12 3
8.10.3C-Ser
58 3-48 1-26 4b 60 A27 4
8.10.3-CG2
62 3-48 1-26 4b 60 A27 4
8.10.3-CG4
94 3-48 1-26 4b 60 A27 4
8.10.3-Ser
90 3-48 1-26 4b 43, 44 A27 4
9.7.2C-Ser
50 3-11 6-13 6b 52 O12 3
9.7.2-CG2
66 3-11 6-13 6b 52 O12 3
9.7.2-CG4
70 3-11 6-13 6b 52 O12 3
9.7.2-Ser
86 3-11 6-13 6b 47, 48 O12 3
9.14.4-Ser
82 3-11 7-27 4b 27, 28 O12 3
La mutagénesis de restos específicos de las cadenas pesada y ligera se llevó a cabo mediante el diseño de cebadores y el uso del kit para mutagénesis de sitio dirigido QuickChange de Stratagene, según las instrucciones del fabricante. Las mutaciones se confirmaron mediante secuenciación automática y los insertos mutagenizados se subclonaron en vectores de expresión. Los vectores de expresión se transfectaron en células HEK293 para producir suficientes anticuerpos para su caracterización.
EJEMPLO III
Ensayo de proliferación de células monocíticas de ratón M-CSF
Se llevaron a cabo ensayos in vitro para medir la proliferación celular demonocitos de ratón dependientes de M-CSF en presencia de anticuerpos anti-M-CSF para determinar el grado de inhibiciónmediante anticuerpos anti-M-CSF.
Se obtuvieron células monocíticas de ratón, células M-NFS-60, de la American Type Culture Collection (ATCC) (Manassas, VA) y se mantuvieron en medio RPMI-1640 que contenía L-glutamina 2 mM (ATCC), suero fetal bovino inactivado por calor al 10 % (FBS) (Invitrogen, Carlsbad, CA), 2-mercaptoetanol 0,05 mM (Sigma, St. Louis MO) (medio de ensayo), con 15 ng/ml de M-CSF humano. Las células M-NSF-60 se separaron en 5 x 104 para su uso al día siguiente o en 2,5 x 104 para su uso en 2 días. Antes de usarlas en el ensayo, las células se lavaron tres veces con RPMI-1640, se contaron y se ajustó el volumen conmedio de ensayo para producir 2 x 105 células/ml. Todas las condiciones se llevaron a cabo por triplicado en placas de 96 pocillos tratadas para cultivo tisular (Corning, Corning, NY). A cada pocillo se añadieron 50 μl de las células lavadas, o bien 100 pM o 1000 pM de M-CSF en un volumen de 25 μl y un anticuerpo de ensayo o control a varias concentraciones en un volumen de 25 μl en tampón acetato (cloruro de sodio 140 mM, acetato de sodio 20 mM y 0,2 mg/ml de polisorbato 80, pH 5,5) hasta un volumen final de 100 μl. Los anticuerpos de la invención se ensayaron solos y con M-CSF humano. Las placas se incubaron durante 24 horas (h) a 37 °C con de CO2 al 5%.
Tras 24 h, se añadieron 10 µl/pocillo de 0,5 µCi 3H-timidina (Amersham Biosciences, Piscataway, NJ) y se pulsó con las células durante 3 h. Para detectar la cantidad de timidina incorporada, las células se recolectaron en placas de filtro unifilter GF/C prehumedecidas (Packard, Meriden, CT) y se lavaron 10 veces con agua. Se dejaron secar las placas durante la noche. Se añadieron sellos inferiores a las placas de filtro. A continuación se añadieron 45 μl de Microscint 20 (Packard, Meriden, CT) por pocillo. Después de añadir un sello superior, se contaron las placas en un contador Triluxmicrobeta (Wallac, Norton, OH).
Estos experimentos demuestran que los anticuerpos anti-M-CSF de la invención inhiben la proliferación celular de monocitos de ratón en respuesta al M-CSF. Además, usando varias concentraciones de anticuerpos, se determinó la CI50 para la inhibición de la proliferación celular de monocitos de ratón para los anticuerpos 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, y 9.7.2 (ensayo de proliferación celular, tabla 3a y tabla 3b).
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