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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Roberto Burioni
Massimo Clementi
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Pomona Ricerca SRL
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6839—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses
    • A61K47/6841—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses the antibody targeting a RNA virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4216—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-viral Ig
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/686—Anti-idiotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms

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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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Application Number Priority Date Filing Date Title
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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ES2544968T3 true ES2544968T3 (es) 2015-09-07
ES2544968T5 ES2544968T5 (es) 2022-11-22

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US (3) US9200063B2 (es)
EP (1) EP2274335B2 (es)
JP (1) JP5542118B2 (es)
KR (1) KR101605573B1 (es)
CN (1) CN102037013B (es)
AU (1) AU2009227567B2 (es)
BR (1) BRPI0909123B8 (es)
CA (1) CA2718923C (es)
DK (1) DK2274335T3 (es)
EA (1) EA027069B1 (es)
ES (1) ES2544968T5 (es)
HU (1) HUE025329T2 (es)
IL (1) IL208210A0 (es)
IT (1) ITTO20080204A1 (es)
MX (1) MX2010010120A (es)
MY (1) MY157359A (es)
NZ (1) NZ588238A (es)
PL (1) PL2274335T3 (es)
PT (1) PT2274335E (es)
SG (1) SG188890A1 (es)
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