RESUMEN
Enhanced expression of epidermal growth factor receptor (EGFR) occurs on a variety of malignant tissues thus making anti-EGFR antibodies possible agents for the diagnosis and therapy of human tumors. Standard hybridoma technology has been used successfully to isolate anti-EGFR antibodies from immunized mice and rats. This report demonstrates that phage-antibody libraries are an alternative, and more versatile, method for isolating antibodies from immunized mice. Anti-EGFR antibodies were isolated from phage-antibody libraries constructed not only from the spleen of an immunized mouse but also from the draining lymph node of an immunized mouse and from in vitro immunized mouse cells. Two of the single-chain Fv isolated from the phage-antibody libraries were engineered to create partially humanized whole antibody molecules.
Asunto(s)
Bacteriófagos/genética , Receptores ErbB/inmunología , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Neoplasias/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Bacteriófagos/inmunología , Secuencia de Bases , Biblioteca de Genes , Humanos , Inmunización , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , Ratones , Ratones Endogámicos CBA , Datos de Secuencia MolecularAsunto(s)
Clonación Molecular/métodos , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Historia del Siglo XX , Región Variable de Inmunoglobulina/biosíntesis , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction. The primers incorporate restriction sites that allow the cDNA of the variable domains to be force-cloned for sequencing and expression. Here we have applied the technique to clone and sequence the variable domains of five hybridoma antibodies and to express a mouse-human chimeric antibody that binds to the human mammary carcinoma line MCF-7. The technique should also lead to the cloning of antigen-binding specificities directly from immunoglobulin genes.