RESUMEN
The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential, but broad application has been hindered by the difficulty of identifying bispecific antibody formats that exhibit favorable pharmacokinetic properties and ease of large-scale manufacturing. Previously, the development of an antibody technology utilizing heavy chain knobs-into-holes mutations and a single common light chain enabled the small-scale generation of human full-length bispecific antibodies. Here we have extended the technology by developing a two-part bispecific antibody discovery strategy that facilitates proof-of-concept studies and clinical candidate antibody generation. Our scheme consists of the efficient small-scale generation of bispecific antibodies lacking a common light chain and the hinge disulfides for proof-of-concept studies coupled with the identification of a common light chain bispecific antibody for large-scale production with high purity and yield. We have applied this technology to generate a bispecific antibody suitable for development as a human therapeutic. This antibody directly inhibits the activation of the high affinity IgE receptor FcepsilonRI on mast cells and basophils by cross-linking FcepsilonRI with the inhibitory receptor FcgammaRIIb, an approach that has strong therapeutic potential for asthma and other allergic diseases. Our approach for producing human bispecific full-length antibodies enables the clinical application of bispecific antibodies to a validated therapeutic pathway in asthma.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Receptores de IgE/fisiología , Sustitución de Aminoácidos , Animales , Anticuerpos Biespecíficos/genética , Especificidad de Anticuerpos , Basófilos/inmunología , Línea Celular Tumoral , Codón/genética , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Genes , Glutatión Transferasa/genética , Humanos , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Ratones , Ratones SCID , Anafilaxis Cutánea Pasiva/inmunología , Receptores de IgE/antagonistas & inhibidores , Receptores de IgE/efectos de los fármacos , Receptores de IgE/inmunología , Receptores de IgG/inmunología , Proteínas Recombinantes/uso terapéutico , Neoplasias de la Retina/inmunología , Retinoblastoma/inmunología , Sensibilidad y EspecificidadRESUMEN
Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a favorable ratio leads to the high-level expression and assembly of full-length IgGs in the Escherichia coli periplasm. The technology described offers a rapid, generally applicable and potentially inexpensive method for the production of full-length therapeutic antibodies, as verified by the expression of several humanized IgGs. One E. coli-derived antibody in particular, anti-tissue factor IgG1, has been thoroughly evaluated and has all of the expected properties of an aglycosylated antibody, including tight binding to antigen and the neonatal receptor. As predicted, the protein lacks binding to C1q and the FcgammaRI receptor, making it an ideal candidate for research purposes and therapeutic indications where effector functions are either not required or are actually detrimental. In addition, a limited chimpanzee study suggests that the E. coli-derived IgG1 retains the long circulating half-life of mammalian cell-derived antibodies.
Asunto(s)
Escherichia coli , Expresión Génica , Vectores Genéticos , Inmunoglobulinas/genética , Tromboplastina/inmunología , Animales , Genes , Glicosilación , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Pan troglodytes , Factores de TiempoRESUMEN
Lec13 cells, a variant Chinese hamster ovary cell line, were used to produce human IgG1 that were deficient in fucose attached to the Asn(297)-linked carbohydrate but were otherwise similar to that found in IgG1 produced in normal Chinese hamster ovary cell lines and from human serum. Lack of fucose on the IgG1 had no effect on binding to human FcgammaRI, C1q, or the neonatal Fc receptor. Although no change in affinity was found for the His(131) polymorphic form of human FcgammaRIIA, a slight improvement in binding was evident for FcgammaRIIB and the Arg(131) FcgammaRIIA polymorphic form. In contrast, binding of the fucose-deficient IgG1 to human FcgammaRIIIA was improved up to 50-fold. Antibody-dependent cellular cytotoxicity assays using purified peripheral blood monocytes or natural killer cells from several donors showed enhanced cytotoxicity, especially evident at lower antibody concentrations. When combined with an IgG1 Fc protein variant that exhibited enhanced antibody-dependent cellular cytotoxicity, the lack of fucose was synergistic.