ES2637801T3 - Anticuerpos multivalentes y usos de los mismos - Google Patents

Anticuerpos multivalentes y usos de los mismos Download PDF

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ES2637801T3
ES2637801T3 ES14189442.8T ES14189442T ES2637801T3 ES 2637801 T3 ES2637801 T3 ES 2637801T3 ES 14189442 T ES14189442 T ES 14189442T ES 2637801 T3 ES2637801 T3 ES 2637801T3
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chain polypeptides
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Kathy L. Miller
Leonard G. Presta
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Abstract

Un anticuerpo aislado que comprende un primer y un segundo polipéptidos de cadena pesada, al menos dos polipéptidos de cadena ligera y cuatro sitios de unión a antígeno, en donde dichos primer y segundo polipéptidos de cadena pesada comprenden VD1-(X1)n-VD2-(X2)n-Fc, en donde VD1 es un primer domino variable de cadena pesada (VH), VD2 es un segundo dominio VH, Fc es una región Fc, X1 y X2 representan un aminoácido o un polipéptido y n es 0 o 1, en donde cada uno de los al menos dos polipéptidos de cadena ligera comprende un dominio variable de cadena ligera (VL), en donde cada uno de dichos cuatro sitios de unión a antígeno está formado por un dominio VH del primer o del segundo polipéptidos de cadena pesada y un dominio VL de los al menos dos polipéptidos de cadena ligera.

Description

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respectivamente), IgG2hum (SEQ ID NO: 3), IgG3hum (SEQ ID NO: 4) e IgG4hum (SEQ ID NO: 5). La secuencia IgG1 humana es el alotipo no A, y las diferencias entre esta secuencia y las del alotipo A (en las posiciones 356 y 358; sistema de numeración EU) se muestran debajo de la secuencia IgG1 humana. También se muestran las secuencias de la región Fc de la IgG murina de secuencia nativa, IgG1mur (SEQ ID NO: 6), IgG2Amur (SEQ ID NO: 7), IgG2Bmur (SEQ ID NO: 8) e IgG3mur (SEQ ID NO: 9). Las Figuras 4A-B representan esquemáticamente anticuerpos tetravalentes de acuerdo con la presente invención. En la Figura 4A, se enumeran los cuatro Fabs de unión a antígeno (1 y 2 para cada brazo del anticuerpo tetravalente) y X representa un dominio de dimerización. En la Figura 4B, el dominio de dimerización del anticuerpo tetravalente es una región Fc. La Figura 5 muestra la construcción usada para la expresión de un anticuerpo anti-HER2 tetravalente (OctHER2) en el Ejemplo 1. Las Figuras 6A-C ilustran la unión de OctHER2 (Fig. 6A); de rhuMAb de IgG1 bivalente 4D5-8 expresado por células 293 (Fig. 6B); y de HERCEPTIN® en viales (expresado por células de ovario de hámster Chino (CHO)) (Fig. 6C) con el dominio extracelular (DEC) de HER2 tal y como se determina usando un ensayo inmunoabsorbente ligado a enzimas (ELISA). La Figura 7 representa un análisis de ultracentrifugación de la unión de OctHER2 al DEC de HER2. Se muestran los pesos moleculares promedio (teóricos o determinados experimentalmente) frente a la proporción molar de OctHER2 contra el DEC de HER2. Los pesos moleculares promedio teóricos calculados suponiendo que el anticuerpo tetravalente tiene cuatro sitios de unión completamente funcionales se muestra con círculos; los pesos moleculares promedio calculados teóricos suponiendo que el anticuerpo tetravalente tiene tres sitios de unión completamente funcionales se muestran con cuadrados; y los triángulos representan pesos moleculares determinados experimentalmente. Las Figuras 8A-D representan la actividad inhibidora del crecimiento de HERCEPTIN® en comparación con OctHER2 usando las líneas celulares SKBR3 (que sobreexpresa HER2 3+) (Fig. 8A), MDA 361 (que sobreexpresa HER2 2+) (Fig. 8B), BT474 (que sobreexpresa HER2 3+) (Fig. 8C) y MCF7 (que expresa HER2 0+) (Fig. 8D). La Figura 9 representa el efecto de enlazadores flexibles sobre la actividad inhibidora del crecimiento de los anticuerpos anti-HER2 tetravalentes con respecto a células MDA 231 (que sobreexpresan HER2 1+) o células SKBR3 (que sobreexpresan HER2 3+). Las Figuras 10A-B comparan la tasa de internalización/catabolismo de OctHER2 (Fig. 10A) con la de la HERCEPTIN® (Fig. 10B) en relación con las líneas celulares tanto de MDA 453 (que sobreexpresan HER2 2+) como de SKBR3 (que sobreexpresan HER2 3+). Las Figuras 11A-I son fotografías de microscopía electrónica que muestran la internalización de OctHER2. Las Figuras 11A-F muestran la localización subcelular de 125I-OctHER2 en células SKBR3. Se observaron en granos de plata autorradiográficos asociados con las vellosidades de la membrana celular apical (Fig. 11A), en estrecha proximidad con una formación de fosa recubierta (Fig. 11B, flecha), con vesículas citosólicas moderadas (Figs. 11C y D) y endosomas (Figs. 11 E y F). Barras = 0,25 µM. Las Figs. 11G-I muestran la internalización a las 0 horas (Fig. 11 G) y 5 horas (Figs. 11H y 11I). Las Figuras 12A-E representan la apoptosis inducida por un anticuerpo tetravalente anti-DR5 (16E2 Octopus), un anticuerpo IgG bivalente anti-DR5 (16E2 IgG) y Apo2L/TRAIL (Apo2L) en líneas de células de cáncer: COLO 205 (Fig. 12A), SK-MES-1 (Fig. 12B), HCT116 (Fig. 12C) y HOP 92 (Fig. 12D), en comparación con una línea celular de control no cancerosa, HUMEC (Fig. 12E). Las Figuras 13A-D son portaobjetos histológicos teñidos para detectar células apoptóticas. Los tejidos tumorales de ratones tratados con 16E2 Octopus o Apo2L/TRAIL se fijaron en formalina al 10 % y después se embebieron en parafilm y se seccionaron en los portaobjetos que después se tiñeron con hematoxilina y eosina y se visualizaron con un aumento de 400X. El efecto de 16E2 Octopus a las 6 y 24 horas se muestra en las Figs. 13A y B, respectivamente; en la Figura 13C se muestran las células tratadas con control; y en la Figura 13D se muestran las células tratadas con Apo2L/TRAIL. La Figura 14 representa la actividad in vivo de Apo2L/TRAIL (60 mg/kg, 5 x/semana), IgG 3H3 bivalente (5 mg/kg administrada los días 0, 3, 5 y 9), IgG bivalente 16E2 (16E2) (5 mg/kg administrada los días 0, 3, 5 y 9) y 16E2 Octopus (5 mg/kg administrado los días 0, 3, 5 y 9) con respecto a tumores COLO 205 en ratones desnudos atímicos. La Figura 15 representa un ensayo in vitro con alamar Azul confirmando la actividad apoptótica del material usado en los estudios con ratones (Apo2L/TRAIL y 16E2 Octopus) en comparación con un control positivo convencional con Apo2L. Se confirmó que el anticuerpo anti-IgE (E25) usado como un control negativo en los estudios con ratones tenía actividad no apoptótica. La Figura 16 representa los resultados de un ensayo de apoptosis con cristal violeta comparando el anti-DR5 3H3 Octopus con diversos lotes de anti-DR5 16E2 Octopus. Las Figuras 17A-B revelan los resultados del ensayo de apoptosis con alamar Azul con respecto a Apo2L/TRAIL (documento WO97/25428), anticuerpo anti-DR5 3H3 Octopus, anticuerpo anti-DR5 16E2 Octopus y Apo2L/TRAIL con una etiqueta epitópica FLAG entrecruzada mediante un anticuerpo anti-FLAG (documento WO97/25428) con respecto a células SK-MES-1 (Fig. 17A) y Jurkat (Fig. 17B) en presencia de suero bovino fetal (FBS) al 5 %. Las Figuras 18A-C representan curvas de respuesta a la dosis que muestran el efecto del anti-DR5 16E2 Octopus (gráficos superiores) en comparación con Apo2L/TRAIL (gráficos inferiores) sobre el crecimiento de líneas celulares tumorales humanas de leucemia, de cáncer pulmonar no microcítico, cáncer de colon, cáncer del
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(véase a continuación) siempre que estos exhiban la actividad biológica deseada.
A menos que se indique de otra manera la expresión “anticuerpo multivalente” se usa a lo largo de esta memoria descriptiva para indicar un anticuerpo que comprende tres o más sitios de unión a antígeno. Preferentemente, el 5 anticuerpo multivalente se modifica genéticamente para que tenga tres o más sitios de unión a antígeno y generalmente no es un anticuerpo de IgM o IgA de secuencia nativa.
Los “fragmentos de anticuerpo” comprenden solo una parte de un anticuerpo intacto, incluyendo generalmente un sitio de unión a antígeno del anticuerpo intacto y por lo tanto conservando la capacidad de unirse al antígeno. Los ejemplos de fragmentos de anticuerpos incluidos en la presente definición incluyen: (i) el fragmento Fab, que tiene los dominios VL, CL, VH y CH1; (ii) el fragmento Fab’, que es un fragmento Fab que tiene uno o más restos de cisteína en el extremo C del dominio CH1; (iii) el fragmento Fd que tiene los dominios VH y CH1; (iv) el fragmento Fd’ que tiene los dominios VH y CH1 y uno o más restos de cisteína en el extremo C del dominio CH1; (v) el fragmento Fv que tiene los dominios VL y VH de un brazo sencillo de un anticuerpo; (vi) el fragmento dAb (Ward et 15 al., Nature 341, 544-546 (1989)) que consiste en un dominio VH; (vii) las regiones CDR aisladas; (viii) los fragmentos F(ab’)2, un fragmento bivalente que incluye dos fragmentos Fab’ unidos por un puente disulfuro en la región bisagra;
(ix) moléculas de anticuerpo monocatenario (por ejemplo, Fv monocatenario; scFv) (Bird et al., Science 242: 423-426 (1988); y Huston et al., PNAS (EE.UU.) 85: 5879-5883 (1988)); (x) “diacuerpos” con dos sitios de unión a antígeno, que comprenden un dominio variable de la cadena pesada (VH) conectado a un dominio variable de la cadena ligera (VL) en la misma cadena polipeptídica (véanse, por ejemplo, los documentos EP 404,097; WO 93/11161; y Hollinger et al., Proc. Natl. Acad. Sci. EE.UU., 90: 6444-6448 (1993)); (xi) “anticuerpos lineales” que comprenden un par de segmentos Fd en tándem (VH-CH1-VH-CH1) que, junto con los polipéptidos complementarios de cadena ligera, forman un par de regiones de unión a antígeno (Zapata et al. Protein Eng. 8(10): 1057-1062 (1995); y Patente de Estados Unidos Nº 5.641.870).
25 La expresión “anticuerpo monoclonal”, tal como se usa en el presente documento, se refiere a un anticuerpo obtenido de una población de anticuerpos sustancialmente homogéneos, es decir, los anticuerpos individuales que comprenden la población son idénticos excepto por posibles mutaciones de origen natural que pueden estar presentes en menores cantidades. Los anticuerpos monoclonales que se dirigen contra un solo antígeno son muy específicos. Además, a diferencia de las preparaciones de anticuerpos policlonales, que normalmente incluyen distintos anticuerpos que se dirigen contra distintos determinantes (epítopos), cada anticuerpo monoclonal se dirige contra un solo determinante en el antígeno. El modificador “monoclonal” no debe interpretarse como que requiere la producción del anticuerpo por cualquier método en particular. Por ejemplo, los anticuerpos monoclonales a usar de acuerdo con la presente invención deben estar constituidos por el método del hibridoma descrito por primera vez por
35 Kohler et al., Nature 256: 495 (1975), o también deben constituirse por métodos de ADN recombinante (véase, por ejemplo, la Patente de Estados Unidos Nº 4.816.567). Los “anticuerpos monoclonales” también pueden aislarse de fagotecas de anticuerpos usando las técnicas descritas en Clackson et al., Nature 352: 624-628 (1991) o en Marks et al., J. Mol. Biol. 222: 581-597 (1991), por ejemplo.
Los anticuerpos monoclonales del presente documento incluyen específicamente anticuerpos “quiméricos” en los que una parte de la cadena pesada y/o ligera es idéntica u homóloga a las secuencias correspondientes en anticuerpos que derivan de una especie particular o que pertenece a una clase o subclase particular de anticuerpos, mientras que el resto de la cadena o de las cadenas es idéntico u homólogo a secuencias correspondientes en anticuerpos derivados de otras especies o que pertenecen a otra clase o subclase de anticuerpos, así como
45 fragmentos de dichos anticuerpos, siempre que estos exhiban la actividad biológica deseada (Patente de Estados Unidos Nº 4.816.567; y Morrison et al., Proc. Natl. Acad. Sci. EE.UU. 81: 6851-6855 (1984)).
Las formas “humanizadas” de anticuerpos no humanos (por ejemplo, murinos) son anticuerpos quiméricos que contienen secuencias mínimas derivadas de inmunoglobulinas no humanas. Para la mayor parte, los anticuerpos humanizados son inmunoglobulinas humanas (anticuerpo receptor) en las que los restos de una región hipervariable del receptor se reemplazan por restos de una región hipervariable de una especie no humana (anticuerpo donante) tal como un ratón, rata, conejo o primate no humano que tenga la especificidad, afinidad, y capacidad deseada. En algunos casos, los restos de la región marco conservada (FR) de la inmunoglobulina humana se reemplazan por restos no humanos correspondientes. Además, los anticuerpos no humanizados pueden comprender restos que no
55 se encuentran en el anticuerpo receptor o en el anticuerpo donante. Estas modificaciones se realizan para refinar adicionalmente el rendimiento del anticuerpo. En general, el anticuerpo humanizado comprenderá sustancialmente todos de al menos uno, y normalmente dos, dominios variables, en los cuales todos o sustancialmente todos los bucles hipervariables corresponden a los de una inmunoglobulina no humana y todas o sustancialmente todas las FR son las de una secuencia de inmunoglobulina humana. El anticuerpo humanizado opcionalmente también comprenderá al menos una parte de una región constante de inmunoglobulina (Fc), normalmente la de una inmunoglobulina humana. Para más detalles, véase Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); y Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).
Un “anticuerpo humano” es uno que posee una secuencia de aminoácidos que se corresponde con la de un
65 anticuerpo producido por un ser humano y/o se ha realizado usando cualquiera de las técnicas para preparar anticuerpos humanos, como se divulga en el presente documento. Esta definición de un anticuerpo humano excluye
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Tabla 1
Resto Original
Sustituciones a modo de ejemplo Sustituciones Preferidas
Ala (A)
val; leu; ile val
Arg (R)
lys; gln; asn lys
Asn (N)
gln; his; asp, lys; arg gln
Asp (D)
glu; asn glu
Cys (C)
ser; ala ser
Gln (Q)
asn; glu asn
Glu (E)
asp; gln asp
Gly (G)
ala ala
His (H)
asn; gln; lys; arg arg
Ile (I)
leu; val; met; ala; phe; norleucina leu
Leu (L)
norleucina; ile; val; met; ala; phe ile
Lys (K)
arg; gln; asn arg
Met (M)
leu; phe; ile leu
Phe (F)
leu; val; ile; ala; tyr tyr
Pro (P)
ala ala
Ser (S)
thr thr
Thr (T)
ser ser
Trp (W)
tyr; phe tyr
Tyr (Y)
trp; phe; thr; ser phe
Val (V)
ile; leu; met; phe; ala; norleucina leu
Las modificaciones sustanciales en las propiedades biológicas del anticuerpo se realizan seleccionando sustituciones que difieren significativamente en su efecto sobre el mantenimiento de (a) la estructura del esqueleto
5 polipeptídico en la zona de la sustitución, por ejemplo, como una conformación en lámina o en hélice, (b) la carga o hidrofobicidad de la molécula en el sitio diana, o (c) el volumen de la cadena lateral. Los restos de origen natural se dividen en grupos basándose en propiedades comunes de las cadenas laterales:
(1) hidrófobos: norleucina, met, ala, val, leu, ile; 10 (2) neutros hidrófilos: cys, ser, thr;
(3)
ácidos: asp, glu;
(4)
básicos: asn, gln, his, lys, arg;
(5)
restos que influyen en la orientación de la cadena: gly, pro; y
(6)
aromáticos: trp, tyr, phe.
15 Las sustituciones no conservativas implicarán el intercambio de un miembro de una de estas clases por otra clase.
Cualquier resto de cisteína que no esté implicado en el mantenimiento de la conformación correcta del anticuerpo también puede sustituirse, generalmente con serina, para aumentar la estabilidad oxidativa de la molécula e impedir
20 el entrecruzamiento aberrante. Al contrario, puede añadirse uno o más enlaces de cisteína al anticuerpo para mejorar su estabilidad.
Un tipo particularmente preferido de variantes sustitucionales implica sustituir uno o más restos de regiones hipervariables de un anticuerpo parental (por ejemplo un anticuerpo humanizado o humano). Generalmente, la 25 variante (o variantes) resultante seleccionada para el desarrollo posterior tendrá propiedades biológicas mejoradas en relación al anticuerpo parental a partir del que se generan. Un modo conveniente para generar dichas variantes sustitucionales implica la maduración por afinidad usando presentación de fagos. Brevemente, varios sitios de la región hipervariable (por ejemplo 6-7 sitios) se mutan para generar todas las sustituciones de aminoácidos posibles en cada sitio. Los anticuerpos multivalentes generados de este modo se presentan de un modo monovalente a partir
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