RESUMEN
Two internalizing monovalent single chain antibody fragments (scFv), C6.5 and F5, that recognize distinct ErbB2 extracellular domain (ECD) epitopes, and their bivalent forms dbC6.5 and F5(scFv')(2), were compared to the growth-inhibiting anti-ErbB2 antibody Herceptin/trastuzumab, in either its bivalent (Her) or monovalent (4D5Fab') form, for their abilities to induce biological responses in the ErbB2-overexpressing breast cancer cells, SkBr-3. Assays compared internalization by receptor-mediated endocytosis, effects on cell cycling and culture growth, and interference with intracellular MAPK and PI3K signaling pathways. We found no correlation between ErbB2 epitope affinity or valency on degree of antibody-induced endocytosis, since all the scFv were able to internalize better than Her. Unlike Her, neither the monovalent or bivalent forms of the internalizing scFv had any sustained effect on cell growth. Basal levels of MAPK and PI3K signaling in SkBr-3 cells were not inhibited by up to 8 h scFv treatment, while decreased MAPK and PI3K signals were noted within 8 h of Her treatment. In summary, antibody-induced ErbB2-mediated endocytosis is not a surrogate marker for resultant biological response, as it shows no correlation with cell cycle, culture proliferation, or intracellular kinase signal induction by internalizing antibodies. Thus, the enhanced endocytotic property of scFv like C6.6 and F5 in conjunction with their absence of any growth or signaling impact on ErbB2-overexpressing cells favors their choice as ErbB2 targeting moieties for intracellular delivery of novel cancer therapeutics.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Fragmentos de Inmunoglobulinas/farmacología , Receptor ErbB-2/inmunología , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Afinidad de Anticuerpos , Neoplasias de la Mama , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , División Celular/efectos de los fármacos , Línea Celular , Endocitosis/efectos de los fármacos , Epítopos/inmunología , Femenino , Humanos , Fragmentos de Inmunoglobulinas/metabolismo , Radioisótopos de Yodo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Trastuzumab , Células Tumorales CultivadasRESUMEN
Antibody internalization into the cell is required for many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes or viral DNA into cells. To generate directly tumor-specific internalizing antibodies, a non-immune single chain Fv (scFv) phage antibody library was selected on the breast tumor cell line SKBR3. Internalized phage were recovered from within the cell and used for the next round of selection. After three rounds of selection, 40 % of clones analyzed bound SKBR3 and other tumor cells but did not bind normal human cells. Of the internalizing scFv identified, two (F5 and C1) were identified as binding to ErbB2, and one (H7) to the transferrin receptor. Both F5 and H7 scFv were efficiently endocytosed into SKBR3 cells, both as phage antibodies and as native monomeric scFv. Both antibodies were able to induce additional functional effects besides triggering endocytosis: F5 scFv induces downstream signaling through the ErbB2 receptor and H7 prevents transferrin binding to the transferrin receptor and inhibits cell growth. The results demonstrate the feasibility of selecting internalizing receptor-specific antibodies directly from phage libraries by panning on cells. Such antibodies can be used to target a variety of molecules into the cell to achieve a therapeutic effect. Furthermore, in some instances endocytosis serves as a surrogate marker for other therapeutic biologic effects, such as growth inhibition. Thus, a subset of selected antibodies will have a direct therapeutic effect.
Asunto(s)
Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/inmunología , Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/inmunología , Endocitosis , Biblioteca de Péptidos , Animales , Anticuerpos Antineoplásicos/farmacología , Afinidad de Anticuerpos , Especificidad de Anticuerpos/inmunología , Bacteriófagos/genética , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Clonación Molecular , Cricetinae , Endocitosis/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Fibroblastos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Receptores de Transferrina/antagonistas & inhibidores , Receptores de Transferrina/inmunología , Receptores de Transferrina/metabolismo , Transducción de Señal/efectos de los fármacos , Transferrina/antagonistas & inhibidores , Transferrina/metabolismo , Células Tumorales CultivadasRESUMEN
We report that prokaryotic viruses can be re-engineered to infect eukaryotic cells resulting in expression of a reporter gene inserted into the bacteriophage genome. Phage capable of binding mammalian cells expressing the growth factor receptor ErbB2 and undergoing receptor-mediated endocytosis were isolated by selection of a phage antibody library on breast tumor cells and recovery of infectious phage from within the cell. As determined by immunofluorescence, F5 phage were efficiently endocytosed into 100 % of ErbB2 expressing SKBR3 cells. To achieve reporter gene expression, F5 phage were engineered to package the green fluorescent protein (GFP) reporter gene driven by the CMV promoter. These phage when applied to cells underwent ErbB2-mediated endocytosis leading to GFP expression. GFP expression occurred only in cells overexpressing ErbB2, was dose-dependent reaching, 4 % of cells after 60 hours and was detected with phage titers as low as 2.0 x 10(7) cfu/ml (500 phage/cell). The results demonstrate that bacterial viruses displaying the appropriate antibody can bind to mammalian receptors and utilize the endocytic pathway to infect eukaryotic cells, resulting in expression of a reporter gene inserted into the viral genome. This represents a novel method to discover targeting molecules capable of delivering a gene intracellularly into the correct trafficking pathway for gene expression by directly screening phage antibodies. This should significantly facilitate the identification of appropriate targets and targeting molecules for gene therapy or other applications where delivery into the cytosol is required. This approach can be adapted to directly select, rather than screen, phage antibodies for targeted gene expression. The results also demonstrate the potential of phage antibodies as an in vitro or in vivo targeted gene delivery vehicle.
Asunto(s)
Bacteriófagos/genética , Genes erbB-2 , Vectores Genéticos/genética , Receptor ErbB-2/genética , Transfección/métodos , Neoplasias de la Mama/patología , Línea Celular , Neoplasias del Colon/patología , Endocitosis , Femenino , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Masculino , Receptor ErbB-2/biosíntesis , Receptores Inmunológicos/fisiología , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
Antibodies which bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. For this work, we studied a human scFv (C6.5) which binds ErbB2 to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage, we define the role of affinity, valency, and display format on phage endocytosis and identify the factors that lead to the greatest enrichment for internalization. Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine. Measurement of phage recovery from within the cytosol as a function of applied phage titer indicates that it is possible to select for endocytosable antibodies, even at the low concentrations that would exist for a single phage antibody member in a library of 10(9).
Asunto(s)
Fragmentos de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Inovirus/inmunología , Inovirus/metabolismo , Biblioteca de Péptidos , Anticuerpos Biespecíficos/metabolismo , Afinidad de Anticuerpos/genética , Neoplasias de la Mama , Endocitosis/genética , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/fisiología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/fisiología , Inovirus/genética , Inovirus/aislamiento & purificación , Microscopía Fluorescente , Modelos Biológicos , Receptor ErbB-2/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas , Ensayo de Placa ViralRESUMEN
We have examined the expression, activity and localization of cyclin dependent kinase 5 (cdk5), during myogenesis. Cdk5 protein was found expressed in adult mouse muscle. In murine C2 cells, both the protein level and kinase activity of cdk5 showed a marked increase during early myogenesis with a peak between 36 and 48 hours of differentiation, decreasing as myotubes fuse after 60 to 72 hours. This increase in cdk5 protein level was specific for differentiation and not simply related to cell cycle arrest since it was not observed in fibroblasts grown for 48 hours in low serum medium. Indirect immunofluorescence using monospecific purified anti-cdk5 antibodies showed a low level cytoplasmic staining in proliferative myoblasts, a rapid increase in nuclear staining during the initial 12 hours of differentiation and a predominant nuclear staining in myotubes. Microinjection of plasmids encoding wild-type cdk5 into C2 myoblasts enhanced differentiation as assessed by both myogenin and troponin T expression after 48 hours of differentiation. In contrast, microinjection of plasmids encoding a dominant negative mutant of cdk5 inhibited the onset of differentiation. These data imply a previously unsuspected role for cdk5 protein kinase as a positive modulator of early myogenesis.
Asunto(s)
Quinasas Ciclina-Dependientes , Desarrollo de Músculos , Músculo Esquelético/enzimología , Músculo Esquelético/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Núcleo Celular/enzimología , Quinasa 5 Dependiente de la Ciclina , Citoplasma/enzimología , Cartilla de ADN/genética , Expresión Génica , Inmunohistoquímica , Ratones , Músculo Esquelético/metabolismo , Mutagénesis Sitio-Dirigida , Miogenina/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Ratas , Fracciones Subcelulares/enzimología , Troponina/metabolismo , Troponina TRESUMEN
BACKGROUND: Various systems have been described for the expression of recombinant monoclonal antibodies for therapeutical applications. Insect cells offer great advantages with respect to post-translational modifications, stability, yields and applications. OBJECTIVES: To construct plasmid cassette transfer vectors in order to express chimeric, humanized or human antibodies in insect cells using baculovirus expression system. STUDY DESIGN: Two transfer vectors, pBHuC kappa and pBHuC gamma 1, were designed. They contain a viral promoter (polyhedrin or p10 promoters, respectively), a signal peptide sequence and a human immunoglobulin light chain C kappa gene or heavy chain C gamma 1 sequence, respectively. Restriction sites have been introduced to allow insertion of rearranged variable genes, after amplification by polymerase chain reaction. RESULTS: Recombinant baculoviruses expressing complete immunoglobulins have been generated by a double-recombination event between baculovirus DNA and the loaded cassette transfer vectors. CONCLUSION: Our genetic cassette approach makes this system a very flexible and convenient one for the rapid production of therapeutic monoclonal antibodies with heavy and light chains of any human isotype. Specific variable regions selected by the antibody phage display technology can be easily transferred in these vectors to obtain a complete antibody.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Baculoviridae , Vectores Genéticos , Mutagénesis Insercional/métodos , Animales , Secuencia de Bases , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Células Jurkat , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes/biosíntesis , SpodopteraAsunto(s)
Anticuerpos Monoclonales/genética , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones/genética , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Ingeniería Genética , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Leucemia-Linfoma de Células T del Adulto , Ratones/inmunología , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Células Tumorales CultivadasRESUMEN
Plasmid cassette-transfer vectors pBHuC chi and pBHuC gamma l have been designed which enable the construction of recombinant baculoviruses directing the co-expression of complete immunoglobulin in insect cells. We describe the application of these vectors for the expression of a human/mouse chimeric monoclonal antibody of potential immunosuppressive clinical value derived from a mouse anti-human CD29 monoclonal antibody (Mu-K20). The chimeric K20 light and heavy chains produced in sf9 insect cells were correctly processed and assembled into a normal immunoglobulin which is secreted into the culture medium of infected cells. The chimeric mAb Ch-K20-sf9 reproduces in vitro the functional properties of the parental mouse K20, including affinity and inhibition of lymphocyte proliferation. These results demonstrate that the baculovirus/insect cell expression system is suitable for the expression of fully active monoclonal antibodies of therapeutic value. Our generic cassette approach makes this system a very flexible and convenient one for the rapid production of either chimeric, humanized or human mAb with heavy and light chains of any isotype.
Asunto(s)
Anticuerpos/genética , Vectores Genéticos , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Especificidad de Anticuerpos , Antígenos CD/metabolismo , Bioensayo , Humanos , Técnicas In Vitro , Integrina beta1 , Integrinas/metabolismo , Ratones , Células PC12 , Ratas , SpodopteraRESUMEN
The murine anti-CD29 mAb K20 (Mu-K20) is known to bind to the beta 1 chain of the human integrins and to inhibit activation and proliferation of T cells, implying an important potential for in vivo immunosuppression. However, use of K20 as an immunosuppressant drug would be impaired by the immunogenicity of mouse mAbs in man. We have therefore engineered K20 into (1) a mouse/human chimeric mAb (Ch-K20) that comprises the human kappa/gamma 1C regions and the K20 V regions; and (2) a humanized mAb (Hu-K20) combining the complementarity-determining regions (CDRs) of the K20 mAb with human framework (FR) and kappa/gamma 1 C regions. Both chimeric and humanized Abs were able to reproduce a range of functional properties of the original mouse mAb K20 (Mu-K20), namely, specific binding of CD29, inhibition of T cell proliferation and elevation of second messenger phosphatidic acid (PA) induced via CD3 in a soluble form, and activation of T cell proliferation in a cross-linked form. When compared to Ch-K20, the avidity of Hu-K20 was only slightly reduced. This demonstrates the feasibility of a successful humanization performed on the sole basis of the primary amino acid sequence analysis of the original mouse antibody V regions.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Integrinas/inmunología , Activación de Linfocitos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Antígenos CD/inmunología , Secuencia de Bases , Unión Competitiva , Clonación Molecular , Complemento C1q/inmunología , Pruebas Inmunológicas de Citotoxicidad , Reordenamiento Génico de Linfocito B/genética , Humanos , Hibridomas/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Integrina beta1 , Ratones , Datos de Secuencia Molecular , Ácidos Fosfatidicos/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
Signal transduction GTP-binding proteins are tightly associated with plasma membrane. In the resting state, the anchorage of the alpha subunit could be indirect by means of the other beta gamma subunits or polydisperse multimers. In the activated state, although the alpha subunit is dissociated from other subunits, it is not released from the membrane and therefore is likely to contain information necessary to remain associated with the plasma membrane. Previous proteolytic experiments suggested that, in contrast to other G proteins alpha subunits, the C-terminal domain of Gs alpha (the G protein involved in adenylate cyclase stimulation) is essential for membrane association of the activated form. To better define the crucial residues involved in membrane attachment, we constructed chimeras between a soluble core and various parts of the Gs alpha C-terminal domain. We first deleted codons 2-6 of Gi1 alpha (the inhibitory G protein of the i1 subtype) to generate a soluble GTP-binding protein, delta N-Gi1 alpha. We then replaced the last 14 C-terminal codons of delta N-Gi1 alpha by different domains of the Gs alpha C terminus and looked for the membrane association of chimeric proteins after in vitro transcription, in vitro translation, and interaction with S49 cyc- membranes (obtained from a mutant cell line that does not express Gs alpha). Our results showed that addition of amino acids 367-376 of Gs alpha is sufficient to promote membrane association of the soluble N-terminal deleted Gi1 alpha.