RESUMO
NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Especificidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Proteínas do Sistema Complemento/imunologia , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Cinética , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Ligação Proteica/imunologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
Upon searching for glucocorticoid-regulated cDNA sequences associated with the transformed to normal phenotypic reversion of C6/ST1 rat glioma cells, we identified Nrp/b (nuclear restrict protein in brain) as a novel rat gene. Here we report on the identification and functional characterization of the complete sequence encoding the rat NRP/B protein. The cloned cDNA presented a 1767 nucleotides open-reading frame encoding a 589 amino acids residues sequence containing a BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain in its N-terminal region and kelch motifs in its C-terminal region. Sequence analysis indicates that the rat Nrp/b displays a high level of identity with the equivalent gene orthologs from other organisms. Among rat tissues, Nrp/b expression is more pronounced in brain tissue. We show that overexpression of the Nrp/b cDNA in C6/ST1 cells suppresses anchorage independence in vitro and tumorigenicity in vivo, altering their malignant nature towards a more benign phenotype. Therefore, Nrp/b may be postulated as a novel tumor suppressor gene, with possible relevance for glioblastoma therapy.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Proteínas dos Microfilamentos/genética , Neuropeptídeos/genética , Proteínas Nucleares/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Clonagem Molecular , DNA Complementar , Regulação da Expressão Gênica , Glioma/patologia , Humanos , Imuno-Histoquímica , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Neuropeptídeos/química , Proteínas Nucleares/química , Ratos , Homologia de Sequência de AminoácidosRESUMO
We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.
Assuntos
Software , Transcrição Gênica/genética , Processamento Alternativo/genética , Linhagem Celular , Linhagem Celular Tumoral , Biologia Computacional/métodos , Biologia Computacional/estatística & dados numéricos , Sequência Consenso/genética , DNA de Neoplasias , Bases de Dados Genéticas/classificação , Etiquetas de Sequências Expressas , Genes/genética , Genoma Humano , Células HeLa/patologia , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Design de Software , Validação de Programas de Computador , Células U937/patologiaRESUMO
São poucos os avanços obtidos em intervenções cirúrgicas ou terapias para melhora na qualidade de vida e/ou prognósticos dos pacientes acometidos por gliomas, que são os tumores mais comuns e fatais que acometem o sistema nervoso central. O modelo celular ST1 de glioma de rato responde ao tratamento com hormônio glicocorticóide (GC) com uma reversão fenotípica tumoral-normal in vitro e in vivo e o modelo celular P7 é resistente a este tratamento. Ambas as linhagens são utilizadas, no laboratório, como modelos de estudo de genes associados à origem de neoplasias e controle de proliferação celular, com potenciais aplicações na doença humana. Este trabalho possui dois objetivos gerais: a) clonagem e caracterização funcional do gene NRP/B de rato, previamente descrito no laboratório como sendo induzido durante o tratamento das células ST1 com GC; b) identificação de genes humanos no locus 22q13, frequentemente deletado e associado à progressão tumoral de gliomas. A identificação, clonagem e determinação da seqüência completa de NRP/B de rato, até então desconhecida, foi realizada neste trabalho e depositada no GenBank (número de acesso AY669396). Sua expressão parece ser cérebro-específica, sendo modulada positivamente durante o tratamento com GC nas células ST1. A superexpressão de NRP/B em células ST1 foi realizada para avaliar o papel do gene na reversão fenotípica tumoral-normal induzida por GC. Os resultados obtidos sugerem que NRP/B provoca diminuição da capacidade de crescimento independente de ancoragem em meio semi-sólido e do potencial tumorigênico das células ST1 em ensaios de tumorigênese in vivo. Experimentos preliminares sugerem o envolvimento da expressão de NRP/B no processo de progressão celular e em tumores humanos cerebrais e de mama...