ES2544968T3 - Anticuerpos monoclonales capaces de reaccionar con una pluralidad de subtipos del virus de la influenza A - Google Patents

Anticuerpos monoclonales capaces de reaccionar con una pluralidad de subtipos del virus de la influenza A Download PDF

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ES2544968T3
ES2544968T3 ES09722394.5T ES09722394T ES2544968T3 ES 2544968 T3 ES2544968 T3 ES 2544968T3 ES 09722394 T ES09722394 T ES 09722394T ES 2544968 T3 ES2544968 T3 ES 2544968T3
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influenza
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monoclonal antibodies
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Roberto Burioni
Massimo Clementi
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Pomona Ricerca SRL
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    • C07K16/10—RNA viruses
    • C07K16/108—Orthomyxoviridae (F), e.g. influenza virus
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
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Abstract

Un anticuerpo monoclonal humano dirigido contra el antígeno hemaglutinina del virus de la influenza A, caracterizado porque es capaz de unirse a una pluralidad de subtipos del virus de la influenza A y neutralizarlos, en donde dicha pluralidad de subtipos comprende, al menos, un subtipo del virus de la influenza A que contiene hemaglutinina H1 y, al menos, un subtipo del virus de la influenza A que contiene hemaglutinina H3.

Description

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E09722394
10-08-2015
de diluciones de albúmina de suero bovina (BSA) de concentración conocida. La absorbancia de cada muestra se midió en un espectrofotómetro a una longitud de onda de 540 nm. Los lisados así obtenidos se usaron entonces (300 ng por pocillo) para cubrir una placa de ELISA (COSTAR) que se incubó a 4 °C durante la noche. Al día siguiente, la placa se lavó con agua destilada y se bloqueó con PBS /BSA 1% (Sigma) durante 45 minutos a 37 °C. 5 Después, 40 µl de sobrenadante de cada clon se agregaron a cada pocillo, que se incubó 1 hora a 37 °C. Después de 5 lavados (WASHER ETI-SYSTEM, DiaSorin) con PBS /0.5% de Tween 20 (Sigma), se agregaron a cada pocillo 40 µl de anti-Fc humano conjugado con peroxidasa (1:4000 en PBS /1% de BSA, Sigma), y la placa se incubó 1 hora a 37 °C. Después de 5 lavados más con PBS /0.5% de Tween 20, se agregaron a cada pocillo 40 µl de substrato de peroxidasa TMB (Pierce). Aproximadamente 15 minutos después se bloqueó la actividad enzimática agregando 40
10 µl de H2SO4, y la señal se midió en un espectrofotómetro a 450 nm. Se puso atención especial al sobrenadante de seis clones putativos capaces de producir anticuerpos de reactividad cruzada (denominados cINF4, cINF16, cINF28, cINF39, cINF43 y cINF47, respectivamente), esto es, capaces de reconocer lisados celulares tanto infectados con la cepa que pertenece al subtipo H1N1 como los infectados con la cepa que pertenece al subtipo H3N2.
15 4. Preparación de los fragmentos Fab de clones de reactividad cruzada
Los genes que codifican las cadenas de Fab monovalentes capaces de reaccionar con los virus de la influenza se clonaron en un vector de expresión eucariótico. Esto permite evitar problemas debido a la inestabilidad de los clones de células productoras de anticuerpo, para caracterizar mejor los genes codificadores desde un punto de vista
20 molecular, para tener disponibles moléculas que son indudablemente monoclonales, así como también mayores cantidades de cada anticuerpo individual.
El ARN mensajero (ARNm) se extrajo de los clones cultivados y se transcribió inversamente usando oligo-dT de acuerdo con los métodos conocidos per se. Los ADNc’s que codifican la cadena ligera y el fragmento Fd (esto es, la 25 porción de cadena pesada presente dentro del fragmento Fab), fueron entonces amplificados mediante los métodos descritos (CSH Press, “Phage display manual”, ed. D.R.Burton, p. A1.6). Luego, los ADNc’s así obtenidos se clonaron en un vector de expresión conocido per se denominado pCb3/CAF (Burioni et al, J. Imm. Meth, 1988). Brevemente, el gen (ADN amplificado) que codifica la porción Fd de cadena pesada de cada Fab, se digirió con las enzimas de restricción XhoI y SpeI (Roche) durante 1.5 horas a 37 °C, y subsiguientemente se insertó en el sitio de
30 clonación del vector para cadenas pesadas, a su vez digeridas con las mismas enzimas. En cambio, las cadenas ligeras (ADN amplificado) se digirieron con las enzimas SacI y XbaI (Roche) y se clonaron en el vector digerido similarmente.
Las construcciones recombinantes así obtenidas de cada clon se usaron para electrotransformación en la cepa
35 XL1Blue de E. coli (hecha competente mediante lavados fríos en glicerol), de acuerdo con los protocolos estandarizados para el uso de cubetas de 0.2 cm (Voltaje: 2500 V; Capacitancia: 25 µF; Resistencia: 200 �). En paralelo, se analizaron las secuencias de ADN de la parte variable de cadena ligera y la parte variable de cadena pesada de los clones seleccionados. Las secuencias son las provistas en el listado de secuencias. El análisis molecular del patrón mutacional mostró una imagen atribuible a procesos de mutación somática inducidos por
40 antígeno para cada uno de los clones.
5. Análisis de ELISA de los Fab’s monoclonales obtenidos por clonación en PCb3/CAF
Al terminarse la clonación, se analizaron 40 clones bacterianos recombinantes para cada anticuerpo monoclonal por
45 medio de ELISA, usando lisados crudos de cultivos bacterianos obtenidos por choque de calor. En particular, clones de bacterias transformadas con la construcción PCb3/CAF se inocularon en 10 ml de medio SB que contenía ampicilina y tetraciclina a 50 µg/ml y 10 µg/ml, respectivamente, y se desarrollaron bajo agitación a 37 °C hasta alcanzar una DO 600 = 1. Subsiguientemente se les agregó un inductor específico (IPTG – isopropil-β-Dtiogalactopiranósido) a una concentración final de 1 mM, y el cultivo se dejó agitando a 30 °C durante la noche. Las
50 células se lisaron por choque de calor (3 rondas de congelación/descongelación, a -80 °C y 37 °C, respectivamente), y después se centrifugaron para separar los desechos celulares del sobrenadante que contiene el Fab. Los Fab’s solubles obtenidos se analizaron por medio de ELISA. Se cubrieron placas de microtitulación de 96 pocillos (Nunc) con lisados de células infectadas con los aislados de virus de referencia anteriormente mencionados. Los lisados obtenidos de células no infectadas se usaron como control negativo. Las placas de ELISA cubiertas con 300 ng de
55 los lisados obtenidos como se describe se dejaron entonces a 4 °C durante la noche. El siguiente día, después de quitar el antígeno no unido, las placas se lavaron 5 veces con PBS y los sitios de unión inespecífica se bloquearon con albúmina al 3% en PBS durante 1 hora a 37 °C. Después de quitar la solución bloqueadora, se les agregaron los sobrenadantes de los cultivos celulares tratados como se describe arriba y que contienen los Fab’s solubles, seguido por un paso de incubación a 37 °C durante 2 horas. Después de 10 ciclos de lavado con PBS /0.05% de Tween 20,
60 se agregaron 40 µl de una dilución 1:700 de un preparado policlonal de inmunoglobulinas anti-Fab humano de cabra conjugadas con peroxidasa de rábano (Sigma) en PBS /1% de BSA. Después de 1 hora de incubación a 37 °C y una serie adicional de 10 lavados, se agregó el substrato (OPD-o-fenilendiamina) a los pocillos. Después, las placas se incubaron 30 minutos a temperatura ambiente en la oscuridad. La reacción se desactivó con ácido sulfúrico 1N y la densidad óptica se determinó por lectura espectrofotométrica a 450 nm. Todos los clones probados mostraron
65 reactividad contra los lisados obtenidos de las células infectadas. Así, para cada uno de los monoclonales de
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Claims (1)

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ES09722394T 2008-03-17 2009-03-16 Anticuerpos monoclonales capaces de reaccionar con una pluralidad de subtipos del virus de la influenza A Active ES2544968T5 (es)

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ITTO20080204 2008-03-17
IT000204A ITTO20080204A1 (it) 2008-03-17 2008-03-17 Anticorpi monoclonali atti a reagire con una pluralita di sottotipi del virus influenzale a
PCT/IB2009/051068 WO2009115972A1 (en) 2008-03-17 2009-03-16 Monoclonal antibodies capable of reacting with a plurality of influenza virus a subtypes

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ITTO20070066A1 (it) 2007-01-30 2008-07-31 Pomona Biotechnologies Llc Anticorpi monoclonali anti-idiotipo mimotopi dell'antigene gp 120 di hiv
EP3524619A1 (en) 2007-12-06 2019-08-14 Dana-Farber Cancer Institute, Inc. Antibodies against influenza virus and methods of use thereof
ITTO20080204A1 (it) * 2008-03-17 2009-09-18 Pomona Biotechnologies Llc Anticorpi monoclonali atti a reagire con una pluralita di sottotipi del virus influenzale a
ITTO20080398A1 (it) 2008-05-27 2009-11-28 Pomona Biotechnologies Llc Anticorpi monoclonali aventi proprieta' di cross-neutralizzazione omosubtipica per virus influenzali di tipo a sottotipo h1
CA2731686C (en) 2008-07-25 2020-04-07 Institute For Research In Biomedicine Neutralizing anti-influenza a virus antibodies and uses thereof
ITTO20080964A1 (it) 2008-12-22 2010-06-23 Natimab Therapeutics S R L Anticorpo monoclonale anti-hcv come medicamento per il trattamento terapeutico e la prevenzione di infezioni da hcv
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