ES2659229T3 - Anticuerpos anti-FcRH5 - Google Patents
Anticuerpos anti-FcRH5 Download PDFInfo
- Publication number
- ES2659229T3 ES2659229T3 ES14742047.5T ES14742047T ES2659229T3 ES 2659229 T3 ES2659229 T3 ES 2659229T3 ES 14742047 T ES14742047 T ES 14742047T ES 2659229 T3 ES2659229 T3 ES 2659229T3
- Authority
- ES
- Spain
- Prior art keywords
- antibody
- fcrh5
- human
- antibodies
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
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- A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
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- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- 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
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
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Abstract
Un anticuerpo anti-FcRH5 aislado que se une a una región específica de isoforma c del dominio extracelular de FcRH5c mostrada como los aminoácidos 743-850 de SEQ ID NO: 1, en donde el anticuerpo no se une significativamente a otro dominio de tipo Ig de FcRH5.
Description
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actividad de unión al antígeno deseada.
Un "fragmento de anticuerpo" se refiere a una molécula distinta de un anticuerpo intacto que comprende una porción de un anticuerpo intacto y que se une al antígeno al que se une el anticuerpo intacto. Ejemplos de fragmentos de anticuerpos incluyen, pero no se limitan a, Fv, Fab, Fab', Fab'-SH, F(ab')2; diacuerpos; anticuerpos lineales; moléculas de anticuerpo monocatenarias (por ejemplo, scFv); y anticuerpos multiespecíficos formados de fragmentos de anticuerpos.
El término "epítope" se refiere al sitio particular en una molécula de antígeno a la que se une un anticuerpo.
Un "anticuerpo que se une al mismo epítope" que un anticuerpo de referencia se refiere a un anticuerpo que bloquea la unión del anticuerpo de referencia a su antígeno en un ensayo de competición el 50 % o más, y en cambio, el anticuerpo de referencia bloquea la unión del anticuerpo a su antígeno en un ensayo de competición el 50 % o más. Un ensayo de competición a modo de ejemplo se proporciona en el presente documento.
El término "anticuerpo monoclonal", como se usa en el presente documento, se refiere a un anticuerpo obtenido a partir de una población de anticuerpos sustancialmente homogéneos, es decir, los anticuerpos individuales que comprenden la población son idénticos y/o se unen al mismo epítope, excepto por posibles anticuerpos de variante, por ejemplo, que contienen mutaciones que existen de forma natural o que surgen durante la producción de una preparación de anticuerpos monoclonales, estando tales variantes generalmente presentes en cantidades menores. A diferencia de las preparaciones de anticuerpos policlonales, que normalmente incluyen diferentes anticuerpos dirigidos contra diferentes determinantes (epítopes), cada anticuerpo monoclonal de una preparación de anticuerpos monoclonales se dirige contra un único determinante en un antígeno. Así, el adjetivo "monoclonal" indica el carácter del anticuerpo como que se obtiene de una población sustancialmente homogénea de anticuerpos, y no debe interpretarse como que requiere la producción del anticuerpo por cualquier método particular. Por ejemplo, los anticuerpos monoclonales que van a usarse según la presente invención pueden prepararse mediante varias técnicas, que incluyen, pero no se limitan a, el método de hibridoma, métodos de ADN recombinante, métodos de presentación en fago y métodos que utilizan animales transgénicos que contienen todos o parte de los loci de inmunoglobulina humana, siendo tales métodos y otros métodos a modo de ejemplo de preparación de anticuerpos monoclonales descritos en el presente documento.
Los términos "anticuerpo de longitud completa", "anticuerpo intacto" y "anticuerpo completo" se usan en el presente documento indistintamente para referirse a un anticuerpo que tiene una estructura sustancialmente similar a una estructura de anticuerpo nativa o que tiene cadenas pesadas que contienen una región Fc como se define en el presente documento.
Un "anticuerpo desnudo" se refiere a un anticuerpo que no está conjugado con un resto heterólogo (por ejemplo, un resto citotóxico) o radiomarca. El anticuerpo desnudo puede estar presente en una formulación farmacéutica.
"Anticuerpos nativos" se refiere a moléculas de inmunoglobulina que existen de forma natural con estructuras variables. Por ejemplo, los anticuerpos IgG nativos son glucoproteínas heterotetraméricas de aproximadamente
150.000 dalton, compuestas de dos cadenas ligeras idénticas y dos cadenas pesadas idénticas que están unidas por disulfuro. De extremo N a C, cada cadena pesada tiene una región variable (VH), también llamado un dominio pesado variable o un dominio variable de la cadena pesada, seguido de tres dominios constantes (CH1, CH2 y CH3). Similarmente, de extremo N a C, cada cadena ligera tiene una región variable (VL), también llamado un dominio ligero variable o un dominio variable de la cadena ligera, seguido de un dominio ligero constante (CL). La cadena ligera de un anticuerpo puede asignarse a uno de dos tipos, llamados kappa (κ) y lambda (λ), basándose en la secuencia de aminoácidos de su dominio constante.
El término anticuerpo "quimérico" se refiere a un anticuerpo en el que una porción de la cadena pesada y/o ligera deriva de una fuente o especie particular, mientras que el resto de la cadena pesada y/o ligera deriva de una fuente
o especie diferente.
Un "anticuerpo humano" es uno que posee una secuencia de aminoácidos que se corresponde con la de un anticuerpo producido por un ser humano o una célula humana o derivada de una fuente no humana que utiliza repertorios de anticuerpos humanos u otras secuencias codificantes de anticuerpos humanos. Esta definición de un anticuerpo humano excluye específicamente un anticuerpo humanizado que comprende restos de unión al antígeno no humanos.
Un anticuerpo "humanizado" se refiere a un anticuerpo quimérico que comprende restos de aminoácidos de HVR no humanas y restos de aminoácidos de FR humanas. En ciertas realizaciones, un anticuerpo humanizado comprenderá sustancialmente todos de al menos uno, y normalmente dos, dominios variables, en los que todas o sustancialmente todas las HVR (por ejemplo, CDR) se corresponden con aquellas de un anticuerpo no humano, y todas o sustancialmente todas las FR se corresponden con aquellas de un anticuerpo humano. Un anticuerpo humanizado puede comprender opcionalmente al menos una porción de una región constante de anticuerpo derivada de un anticuerpo humano. Una "forma humanizada" de un anticuerpo, por ejemplo, un anticuerpo no
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Los trastornos de células plasmáticas resultan de la división o multiplicación no controlada de un clon de células plasmáticas. Las células plasmáticas surgen de los linfocitos B activados (es decir, linfocitos B). Cada linfocito B produce un receptor único, conocido como el receptor de linfocitos B, presentado sobre su superficie celular que es específico para una sustancia extraña, es decir, antígeno. Cuando un receptor de linfocitos B se une a su antígeno relacionado, la célula que expresa el receptor se activa para volver a entrar en el ciclo celular, produciendo muchas copias clonales de él mismo. Los clones maduran en células plasmáticas que residen principalmente en la médula ósea y que están especializados para producir copias del receptor de linfocitos B que son liberadas en la corriente sanguínea como anticuerpos. En un trastorno de células plasmáticas, la célula plasmática o el linfocito B parental sufre daño genético, que produce la supresión de o la insensibilidad a las limitaciones normales en la división celular y/o actividad. Células plasmáticas hijas derivadas de tales células son malignas porque pueden dividirse libremente y/o generar cantidad en exceso de la misma inmunoglobulina (anticuerpo). Frecuentemente, la inmunoglobulina producida es incompleta o tiene una conformación incorrecta que puede producir la acumulación de la proteína (también conocida como proteína monoclonal, proteína M, paraproteína o proteína de amiloide, dependiente del trastorno específico) en el suero, tejidos u órganos (especialmente los riñones), que conduce a disfunción y/o insuficiencia orgánica. Los trastornos de células plasmáticas incluyen gammapatías monoclonales de significancia indeterminada (MGUS), mieloma múltiple (MM), macroglobulinemia, enfermedades de la cadena pesada y amiloidosis sistémica de la cadena ligera (AL), que se diferencian basándose en la naturaleza proliferativa del clon, el grado de participación de la médula ósea y el tipo de proteína M expresada. Trastornos de células plasmáticas adicionales son plasmacitoma solitario, plasmacitoma extramedular, plasmacitomas solitarios múltiples, leucemia de células plasmáticas, macroglobulinemia de Waldenström, linfoma no Hodgkin de linfocitos B, leucemia linfocítica crónica de linfocitos B.
El término "cáncer FcRH5-positiva" se refiere a un cáncer que comprende células que expresan FcRH5 sobre su superficie. Para los fines de determinar si una célula expresa FcRH5 sobre la superficie, se considera que la expresión de ARNm de FcRH5 se correlaciona con la expresión de FcRH5 en la superficie celular. En algunas realizaciones, la expresión de ARNm de FcRH5 se determina por un método seleccionado de hibridación in situ y RT-PCR (incluyendo RT-PCR cuantitativa). Alternativamente, la expresión de FcRH5 sobre la superficie celular puede determinarse, por ejemplo, usando anticuerpos contra FcRH5 en un método tal como inmunohistoquímica, FACS, etc. En algunas realizaciones, FcRH5 es uno o más de FcRH5a, FcRH5b, FcRH5c, identificador de UniProt Q96RD9-2 y/o FcRH5d. En algunas realizaciones, FcRH5 es FcRH5c.
El término "célula FcRH5-positiva" se refiere a una célula que expresa FcRH5 en su superficie. En algunas realizaciones, FcRH5 es uno o más de FcRH5a, FcRR5b, FcRH5c, identificador de UniProt Q96RD9-2, y/o FcRH5d. En algunas realizaciones, FcRH5 es FcRH5c.
Una "cantidad eficaz" de un agente, por ejemplo, una formulación farmacéutica, se refiere a una cantidad eficaz, a dosificaciones y durante periodos de tiempo necesarios, para lograr el resultado terapéutico o profiláctico deseado.
Un "individuo" o "sujeto" es un mamífero. Mamíferos incluyen: pero no se limitan a, animales domesticados (por ejemplo, vacas, ovejas, gatos, perros y caballos), primates (por ejemplo, seres humanos y primates no humanos tales como monos), conejos y roedores (por ejemplo, ratones y ratas). En ciertas realizaciones, el individuo o sujeto es un ser humano.
El término "prospecto" se usa para referirse a instrucciones habitualmente incluidas en envases comerciales de productos terapéuticos, que contienen información sobre las indicaciones, uso, dosificación, administración, terapia de combinación, contraindicaciones y/o advertencias referentes al uso de tales productos terapéuticos.
El término "formulación farmacéutica" se refiere a una preparación que está en una forma tal como para permitir que la actividad biológica de un principio activo contenido en ella sea eficaz, y que no contiene componentes adicionales que son inaceptablemente tóxicos para un sujeto al que se administraría la formulación.
Un "vehículo farmacéuticamente aceptable" se refiere a un componente en una formulación farmacéutica, distinto de un principio activo, que es no tóxico para un sujeto. Un vehículo farmacéuticamente aceptable incluye, pero no se limita a, un tampón, excipiente, estabilizador o conservante.
"Alquilo" es hidrocarburo C1-C18 que contiene átomos de carbono normales, secundarios, terciarios o cíclicos. Ejemplos son metilo (Me, -CH3), etilo (Et, -CH2CH3), 1-propilo (n-Pr, n-propilo, -CH2CH2CH3), 2-propilo (i-Pr, i-propilo, -CH(CH3)2), 1-butilo (n-Bu, n-butilo, -CH2CH2CH2CH3), 2-metil-1-propilo (i-Bu, i-butilo, -CH2CH(CH3)2), 2-butilo (s-Bu, s-butilo, -CH(CH3)CH2CH3), 2-metil-2-propilo (t-Bu, t-butilo, -C(CH3)3), 1-pentilo (n-pentilo, -CH2CH2CH2CH2CH3), 2pentilo (-CH(CH3)CH2CH2CH3), 3-pentilo (-CH(CH2CH3)2), 2-metil-2-butilo (-C(CH3)2CH2CH3), 3-metil-2-butilo (CH(CH3)CH(CH3)2), 3-metil-1-butilo (-CH2CH2CH(CH3)2), 2-metil-1-butilo (-CH2CH(CH3)CH2CH3), 1-hexilo (-CH2CH2CH2CH2CH2CH3), 2-hexil(-CH(CH3)CH2CH2CH2CH3), 3-hexilo (-CH(CH2CH3)(CH2CH2CH3)), 2-metil-2-pentil(C(CH3)2CH2CH2CH3), 3-metil-2-pentilo (-CH(CH3)CH(CH3)CH2CH3), 4-metil-2-pentilo (-CH(CH3)CH2CH(CH3)2), 3metil-3-pentilo (-C(CH3)(CH2CH3)2), 2-metil-3-pentil(-CH(CH2CH3)CH(CH3)2), 2,3-dimetil-2-butilo (-C(CH3)2CH(CH3)2), 3,3-dimetil-2-butilo (-CH(CH3)C(CH3)3. El término "alquilo C1-C8", como se usa en el presente documento, se refiere a un hidrocarburo de cadena lineal o ramificada, saturado o insaturado, que tiene de 1 a 8 átomos de carbono.
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para la unión de FcR5 a anticuerpos usando FACS. Se detectó reactividad significativa de células SVT2 que expresan FcRH5 de longitud completa humana, FcRH5 de longitud completa de cino, o el dominio transmembranario del dominio de E11 humano y dominios citoplásmicos, pero no las células SVT2 transfectadas con vector, que indica que los anticuerpos reactivos con FcRH5 estaban presentes en los sueros de los 5 ratones inmunizados.
5 Después de 9 dosis, se electrofusionaron linfocitos de los ratones inmunizados con células de mieloma de ratón X63-Ag8.653. Se seleccionaron 323 subclones de hibridomas IgG-positiva para cribado adicional. Los clones se probaron para unirse a proteína E11 recombinante (restos de aminoácidos 745-850 de SEQ ID NO: 1) por ELISA (no mostrado) y la unión a células SVT2 que expresan FcRH5 de longitud completa humana, FcRH5 de longitud
10 completa de cino o dominio transmembranario del dominio de E11 humano y dominios citoplásmicos de FcRH5 por FACS. Se identificaron un total de 26 clones que se unieron a células que expresan FcRH5 humana y células que expresan FcRH5 de cino, indicativo de reactividad de especies cruzada (Tabla 2). Se caracterizaron adicionalmente sobrenadantes de subclones para unirse a A) células de mieloma múltiple transfectadas con FcRH5 humana, B) células que expresan FcRH5 humana endógenamente (células de mieloma MOLP-2, linfocitos B CD19+ humanos
15 periféricos de donantes sanos), C) células SVT2 transfectadas para expresar FcRH1, FcRH2, FcRH3 o FcRH4 humana, D) células 293 que expresan la versión truncada de FcRH5 humana (que carece de dominios 4 de Ig que incluyen E11; aminoácidos 464-850 de SEQ ID NO: 1) y E) linfocitos NK. Además, la unión de sobrenadantes a FcRH5a soluble se analizó por ELISA. Basándose en estos análisis, se seleccionaron anticuerpos monoclonales para la purificación.
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Tabla 2.
- Muestra
- SVT2-huFcRH5 SVT2-cyFcRH5 SVT2-huE11
- 1B8
- +++ ++ ++
- 4H8
- +++ ++ ++
- 1H11
- +++ ++ +
- 4G8
- ++ + +
- 4D4
- + + +
- 1C8
- +++ ++ +
- 3C10
- +++ ++ ++
- 3A4
- +++ ++ ++
- 6D2
- +++ +++ +
- 3G3
- ++ + +++
- 1F4
- ++ + +
- 3F10
- ++ + +++
- 1G7
- +++ ++ ++
- 3B12
- ++ + +++
- 3G7
- +++ ++ +
- 5A10
- +++ ++ ++
- 1C12
- ++ + +
- 3D12
- ++ + ++
- 5H4
- ++ ++ +
- 5H9
- ++ ++ +++
- 3C5
- ++ ++ +
- 2D10
- + + +++
- 5B12
- ++ + ++
- 1H2
- + + +
- 5F1
- ++ ++ ++
- 2H7
- ++ +++ ++
La Figura 2 muestra el intervalo de dosis de la unión de cinco anticuerpos E11 purificados, anticuerpo anti-FcRH5 selectivo no de isoforma 10A8 (que se une a los dominios 4-5 de tipo Ig de FcRH5c) y un anticuerpo de control 25 específico para la marca gD del extremo N a las células SVT2 que expresan ya sea FcR5 humana (Figura 2A) o FcR5 de cino (Figura 2B). Los anticuerpos en este ensayo se marcaron directamente con APC-fluoróforo según el protocolo del fabricante (Invitrogen N.º z25051, z25151, z25251). Se encontró que la unión del anticuerpo E11 representativo 5A10 a múltiples líneas de células de mieloma EJM (Figura 3A) y OPM2 (Figura 3B) transfectadas con FcRH5 humana era similar o mejor en comparación con los anticuerpos contra FcRH5 selectivos no de isoforma 30 previamente descritos 10A8 y 7D11 (ambos se unen a los dominios 4-5 de tipo Ig de FcRH5c) (Elkins et al., 2012;
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Claims (1)
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imagen1 imagen2 imagen3 imagen4
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