RESUMEN
The availability of mouse monoclonal antibodies has been integral to the classification of human leukocyte cell surface proteins under the "Cluster of Differentiation" or "CD" nomenclature system. The sequencing of the human genome has identified many more proteins that have characteristics similar to the known leukocyte cell surface proteins, but which have not so far been identified using monoclonal antibodies. One factor that may have limited the generation of monoclonal antibodies to some of these proteins is the high level of sequence conservation between the mouse and human proteins, in particular in the extracellular regions that are recognized by most of the widely used antibodies. An alternative approach is to use a more distant species, such as chickens, for the generation of antibody reagents. Here we compare the extent of amino acid differences in the protein CD molecules expressed by human leukocytes and their mouse and chicken homologs. The analysis confirms that the human proteins are more similar to the mouse homologs than the chicken homologs. The results indicate that chicken antibodies have the potential to be used as an alternative to mouse reagents where human-mouse sequence conservation is high.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Leucocitos/química , Proteínas de la Membrana/análisis , Secuencia de Aminoácidos , Animales , Antígenos CD/análisis , Antígenos CD/aislamiento & purificación , Pollos , Secuencia Conservada , Humanos , Proteínas de la Membrana/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
We have prepared single-chain immunoglobulin Fv fragments from the CD20-specific hybridoma HB13d. One scFv clone demonstrated strong binding to a CD20-derived peptide by ELISA and to CD20-positive cells by flow cytometry, a second had reduced binding, and a third clone did not bind the target antigen. Sequence analysis showed that all three constructs contained shared and unique amino acid changes when compared to the nearest germline match. Molecular modelling of the scFv variants revealed that several of the mutations are located in regions predicted to contact antigen, including a mutation in the heavy chain CDR1 of the strongest binding scFv construct. No similar mutation is present in the highly conserved protein sequences of a number of CD20-specific monoclonal antibodies. BIACORE analysis demonstrated that the mutated scFv had approximately three-fold greater antigen-binding activity than another clone. Competition studies showed that the scFv is able to compete with intact CD20 monoclonal antibody for binding to the target antigen. The improved antigen binding of this scFv will permit the construction of novel CD20-specific reagents for the therapy of lymphomas.
Asunto(s)
Reacciones Antígeno-Anticuerpo/genética , Antígenos CD20/inmunología , Regiones Determinantes de Complementariedad/genética , Fragmentos de Inmunoglobulinas/genética , Mutación , Secuencia de Aminoácidos , Humanos , Hibridomas , Fragmentos de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina , Modelos MolecularesRESUMEN
The success of recombinant antibody fragments as diagnostic reagents and therapeutic agents depends on the availability of sufficient functional material. We have produced a bacterial expression vector that combines high-level expression driven by a modified Shine-Dalgarno sequence with the periplasmic chaperonin Skp. Using this vector, we are able to obtain higher yields of soluble antibody fragments from cultures without the need for supplementation of the culture medium during expression. The fragments produced in the presence of the Skp show improved antigen binding activity compared to when the chaperonin is absent.