RESUMEN
OBJECTIVE: The guided selection strategy for isolation of human antibody (Ab) fragments specific for human interferon gamma receptor 1 (IFNGR-1) from a cloned Ab VH and VL repertoire has been investigated. In order to identify recombinant Abs binding to soluble antigen, a novel method termed affinity sedimentation was introduced here. RESULTS AND CONCLUSIONS: The VH region of murine monoclonal Ab (IR gamma-1) against human IFNGR-1 was combined with human VL repertoire and used for selection of human VL regions. One of these human VL regions (kappa 2) possesses high homology to the murine template VL region, also in CDR3 (77%). A chimeric Fab consisting of kappa 2 and the murine IR gamma-1 VH region was highly IFNGR-1 specific and exerted the same epitope specificity and a comparable binding affinity as the parental murine Fab. In a further step, the selected human VL region kappa 2 was combined with a human VH repertoire and led by guided selection to the generation of a completely human Fab (1b5) specific for human IFNGR-1. The overall VH region homology of 1b5 compared to the parental antibody IR gamma-1 was 81%, with a rather low homology in CDR3. Binding competition studies revealed that the epitope recognized by 1b5 differs from the parental Ab IR gamma-1.
Asunto(s)
Anticuerpos/inmunología , Especificidad de Anticuerpos/genética , Clonación Molecular/métodos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Receptores de Interferón/inmunología , Secuencia de Aminoácidos , Anticuerpos/genética , Anticuerpos/aislamiento & purificación , Afinidad de Anticuerpos/genética , Genes de Inmunoglobulinas , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Receptor de Interferón gammaRESUMEN
Tumour necrosis factor (TNF) is an important mediator of immune and inflammatory responses and has been recognized as a major pathogenic factor in several autoimmune and inflammatory diseases. TNF receptor TR60 plays a critical role in signalling the pathogenic activities of TNF. We here describe molecular cloning and bacterial production of a single-chain antibody (scFv H398) directed against TR60 which possesses antagonistic activity. VH and VL encoding sequences were isolated by PCR from the murine hybridoma cell line H398, cloned into a scFv expression vector and expressed in Escherichia coli. The recombinant antibody (Ab) fragment was found as an active soluble protein in the periplasm but also formed inclusion bodies. Re-folded scFv H398 purified from inclusion bodies was shown to be functional and stable at 37 degrees C with a half-life of 50 h. Comparison of the antigen binding characteristics of scFv with the parental enzymatically produced Fab H398 revealed that both Ab fragments have the same epitope specificity and an identical antigen binding affinity of 1.5 nM. In an in vitro assay it was demonstrated that scFv H398 is an efficient inhibitor of TNF mediated cytotoxicity with an IC50 of 22 nM, which is comparable to the antagonistic activity of natural Fab H398 with an IC50 of 12 nM. As scFv H398 possesses the high affinity TR60 binding and receptor antagonistic activity of the parental Ab H398 but is expected to be less antigenic in man, it provides a valuable tool for the development of novel therapeutic reagents against TNF mediated diseases.