ES2596855T3 - Inducción de tolerancia mucosa a antígenos - Google Patents

Inducción de tolerancia mucosa a antígenos Download PDF

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ES2596855T3
ES2596855T3 ES06830192.8T ES06830192T ES2596855T3 ES 2596855 T3 ES2596855 T3 ES 2596855T3 ES 06830192 T ES06830192 T ES 06830192T ES 2596855 T3 ES2596855 T3 ES 2596855T3
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ova
microorganism
autoimmune
disease
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Pieter Rottiers
Veerle Snoeck
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Intrexon Actobiotics NV
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Abstract

Una combinación que comprende un microorganismo secretor de IL-10 y un alérgeno, aloantígeno, antígeno propio o autoantígeno para su uso en la inducción de tolerancia inmunitaria o para su uso en el tratamiento de una enfermedad elegida del grupo que consiste en una reacción alérgica que incluye alergia alimentaria, enfermedad celíaca, asma alérgica, uveítis autoinmunitaria, tiroiditis autoinmunitaria, miastenia gravis autoinmunitaria, artritis reumatoide, diabetes tipo I, esclerosis múltiple, enfermedad de injerto contra huésped, inmunoactivación de producto terapéutico, y producción de anticuerpos contra el Factor VIII no endógeno, en un mamífero.

Description

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vida restante del mamífero); por ejemplo desde aproximadamente (2 o 3 o 5 días, 1 o 2 semanas, o 1 mes) hacia arriba y/o por ejemplo hasta aproximadamente (5 años, 1 año, 6 meses, 1 mes, 1 semana, o 3 o 5 días). Es típica la administración de la dosis de mantenimiento diaria durante de aproximadamente 3 a aproximadamente 5 días o durante de aproximadamente 1 semana a aproximadamente 1 año. Otros constituyentes de las formulaciones
5 líquidas pueden incluir conservantes, sales inorgánicas, ácidos, bases, tampones, nutrientes, vitaminas u otros productos farmacéuticos.
El microorganismo que secreta el compuesto inmunomodulador y/o el antígeno puede administrarse en una dosis de al menos 104 unidades formadoras de colonias (ufc) a 1012 ufc al día, preferiblemente entre 106 ufc y 1012 ufc al día,
10 lo más preferiblemente entre 109 ufc y 1012 ufc al día. Según el método descrito en Steidler y col. (Science 2000), el compuesto inmunomodulador de por ejemplo de 109 ufc se secreta hasta de al menos 1 ng a 100 ng. A través de ELISA, conocido por un experto en la técnica, el antígeno de, por ejemplo, de 109 ufc se secreta hasta de al menos 1 ng a 100 ng; el experto en la técnica puede calcular el intervalo de secreción de compuesto inmunomodulador y/o antígeno en relación con cualquier otra dosis de ufc.
15 El antígeno puede administrarse en una dosis que induce una respuesta a baja dosis. Preferiblemente, dicho antígeno se administra en una dosis de al menos 10 fg a 100 µg al día, preferiblemente entre 1 pg y 100 µg al día, lo más preferiblemente entre 1 ng y 100 µg al día.
20 El compuesto inmunomodulador de la invención puede administrarse en una dosis de al menos 10 fg a 100 µg al día, preferiblemente entre 1 pg y 100 µg al día, mucho más preferiblemente entre 1 ng y 100 µg al día.
Preferiblemente, los compuestos o la composición se proporcionan en una forma farmacéutica unitaria, por ejemplo un comprimido, una solución, una cápsula o una dosis de aerosol medida, de modo que se administra una dosis 25 única al sujeto, por ejemplo un paciente.
Dependiendo del modo de administración, por ejemplo oral, o cualquiera de los descritos anteriormente, el experto en la técnica conoce cómo definir o calcular la dosis real que va a administrarse a un paciente. El experto en la técnica tendrá conocimientos sobre cómo ajustar las dosis dependiendo del paciente, el microorganismo, el vector,
30 etc.
Los compuestos de la presente invención también pueden tomar la forma de una sal, hidrato, solvato o metabolito farmacológicamente aceptable. Las sales farmacológicamente aceptables incluyen sales básicas de ácidos orgánicos e inorgánicos, incluyendo, pero sin limitación, ácido clorhídrico, ácido bromhídrico, ácido sulfúrico, ácido 35 fosfórico, ácido nítrico, ácido metanosulfónico, ácido etanosulfónico, ácido p-toluenosulfónico, ácido naftalenosulfónico, ácido málico, ácido acético, ácido oxálico, ácido tartárico, ácido cítrico, ácido láctico, ácido fumárico, ácido succínico, ácido maleico, ácido salicílico, ácido benzoico, ácido fenilacético, ácido mandélico y similares. Cuando los compuestos de la invención incluyen una función ácida, tal como un grupo carboxilo, entonces los expertos en la técnica conocen bien pares catiónicos farmacéuticamente aceptables para el grupo carboxilo e
40 incluyen cationes alcalinos, alcalinotérreos, de amonio, de amonio cuaternario y similares.
El microorganismo puede ser cualquier microorganismo, incluyendo bacterias, levaduras u hongos, adecuados para administración mucosa. Preferiblemente, dicho microorganismo es un microorganismo no patógeno, incluso más preferiblemente dicho microorganismo es un microorganismo probiótico. El experto en la técnica conoce organismos 45 probióticos. Los organismos probióticos incluyen, pero sin limitación, bacterias tales como Lactobacillus sp., Lactococcus sp., y levaduras tales como Saccharomyces cerevisiae subspecies boulardii. Preferiblemente, dicha bacteria es una bacteria de ácido láctico; incluso más preferiblemente, dicha bacteria de ácido láctico se elige entre el grupo que consiste en Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Teragenococcus, Vagococcus y Weisella. En una realización preferida
50 adicional, dicho microorganismo es Lactococcus lactis. En otras realización preferida, dicho microorganismo es Saccharomyces cerevisiae.
En una realización preferida, la citocina inmunosupresora se combina con anticuerpos antagonizantes contra citocinas inmunoinductoras, tales como anti-IL-2, anti-IL-12 y/o anti-IFNγ; y moléculas coestimuladoras, tales como 55 anti-CD40L y anti-CD3. Como alternativa, pueden administrarse compuestos que estimulan la producción de las citocinas inmunosupresoras, tales como subunidad B de la toxina del cólera; y moléculas que estimulan la función de linfocitos T reguladores, tales como agonistas de CTLA-4 e ICOS. Como se ha descrito anteriormente, preferiblemente, dicho microorganismo es un microorganismo no patógeno, incluso más preferiblemente es un microorganismo probiótico. El experto en la técnica conoce organismos probióticos, e incluyen, pero sin limitación,
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Dos modelos de ratón de asma alérgica que imita a la enfermedad humana son el modelo de alérgeno Ova y el modelo SCID humanizado.
El modelo de alérgeno Ova
5 Se exponen mediante inhalación ratones sensibilizados a OVA con aerosol de OVA que conduce a inflamación eosinofílica de las vías respiratorias dependiente de citocinas Th2, hiperreactividad bronquial y producción de IgE, hallazgos ampliamente característicos del asma alérgica humana (Brusselle, 1994, Clin Exp Allergy 24: 73; Kips y col. 1996, Am J Respir Crit Care Med 153: 535; Brusselle y col. 1995, Am J Respir Cell Mol Biol 12: 254).
10
Bacterias
Se usa la cepa MG1363 de L. lactis a lo largo de todo este estudio. Se cultivan las bacterias en medio GM17, es decir, M17 (Difco Laboratories, Detroit, MI) complementado con glucosa al 0,5 %. Se almacenan las suspensiones
15 madre de todas las cepas a -20 ºC en glicerol al 50 % en GM17. Para inoculaciones intragástricas, se diluyen las suspensiones madre 200 veces en GM17 nuevo y se incuban a 30 ºC. Alcanzaron una densidad de saturación de 2 x 109 unidades formadoras de colonias (UFC) por ml en el plazo de 16 horas. Se recogen las bacterias mediante centrifugación y se concentran 10 veces en medio BM9. Para el tratamiento, cada ratón recibe 100 µl de esta suspensión diariamente mediante catéter intragástrico.
20
Plásmidos
Se recupera la secuencia de ARNm que codifica para ovoalbúmina de Gallus gallus de Genbank (número de registro AY223553). Se aísla ARN total de útero de pollo y se sintetiza ADNc usando 2 µg de ARN total, cebadores de oligo
25 dT2 µM (Promega Corporation Benelux, Leiden, Países Bajos), DTT 0,01 mM (Sigma-Aldrich, Zwijndrecht, Países Bajos), dNTP 0,5 mM (Invitrogen, Merelbeke, Bélgica), 20 U de Rnasin (Promega Incorporation Benelux) y 100 U de transcriptasa inversa Superscript II (Invitrogen) en un volumen de 25 µl. Se amplifica el fragmento de ADNc de OVA mediante reacción en cadena de la polimerasa (PCR) usando las siguientes condiciones: 94 ºC durante 2 min seguido de 30 ciclos a 94 ºC durante 45 segundos, 62 ºC durante 30 segundos y 72 ºC durante 90 segundos, con los
30 siguientes cebadores directo e inverso 5'-GGCTCCATCGGTGCAGCAAGCATGGAATT-3' y 5'-ACTAGTTAAGGGGAAAC-ACATCTGCCAAAGAAGAGAA-3'.
Se fusiona el fragmento amplificado a la señal de secreción Usp45 del vector pT1NX resistente a eritromicina, en el 35 sentido de 3' del promotor P1 de lactococos.
Las cepas MG1363 transformadas con plásmidos que portan IL-10 murina y ADNc de OVA se designan LL-IL10 y LL-OVA. LL-pT1NX, que es MG1363 que contiene el vector vacío pT1NX, sirven como control.
40 Cuantificación de OVA
Se determina OVA a partir de LL-OVA usando un ensayo inmunoabsorbente ligado a enzimas (ELISA) específico de OVA desarrollado en el laboratorio. También se evalúa la producción de las proteínas recombinantes mediante análisis de transferencia de tipo Western.
45
El modelo de alérgeno OVA
Ratones
50 Se adquieren ratones BALB/c (de 6 a 8 semanas de edad) de Charles River Laboratories (Calco, Italia). Se mantienen los ratones en condiciones libres de patógenos específicos.
Inmunización de los ratones
55 Se inmunizan los ratones por vía i.p. con 2 µg de OVA (calidad V; Sigma-Aldrich) en 2 mg de hidróxido de aluminio (alumbre). Se repite esta inmunización tras un intervalo de 10 días (en los días 0 y 10). Los ratones control reciben una inyección de solución salina en lugar de la disolución de OVA/alumbre. Siete días tras la inmunización, los ratones sensibilizados inhalan una disolución aerosolizada de OVA al 3 % disuelta en PBS durante 10 min. Se realiza la inhalación de OVA durante 3 días en una fila (días 18, 19, y 20). Los ratones control inhalan PBS solo en
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