RESUMEN
Novel in vitro methods for the display of antibody libraries against disease-related antigens have led to the development of powerful protein-based biotherapeutics. Eukaryotic ternary ribosome complexes can be used to display human single chain antibodies (scFvs) to isolate specific binding reagents to these antigens. Here, we present the isolation of human scFv against the immunotherapeutic target antigen CD22 from a patient-derived human scFv library using ribosome display technology. The ribosome complexes were enriched against the extra-cellular domain of human CD22 conjugated to magnetic beads. Isolated constructs were further affinity-matured and specific binding activity was demonstrated by surface plasmon resonance and validated using in vitro cell assays. The isolated human anti-CD22 scFvs can provide a basis for the development of new immunotherapeutic strategies in CD22-expressing malignant diseases.
Asunto(s)
Anticuerpos/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Ribosomas/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Línea Celular , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
Engineered antibodies have become an invaluable source of biopharmaceuticals against a wide range of diseases. About 200 antibody-based biologicals have been tested in clinical trials. Single chain variable fragments of antibodies (scFvs) provide binding specificity and offer an increased ease of in vitro display selection. Here, we present the generation of a human scFv library from peripheral blood lymphocyte RNA of a patient with relapsed T-cell non-Hodgkin lymphoma (T-NHL) who experienced a rare case of "spontaneous" remission. Antibodies against human T-cell antigen CD28, a co-stimulatory protein that influences the immune response by amplification of the transcriptional effects of T-cell receptors, might have contributed to the patient's remission. The scFv library was panned against CD28 using ribosome display and further subjected to affinity maturation. Isolated scFv were assessed for binding specificity and affinity and may provide the basis for the development of novel immunotherapeutic strategies. This work demonstrates the selection of a fully human antibody fragment from a patient-derived gene pool by in vitro ribosome display technology.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD28/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Linfoma no Hodgkin/inmunología , Biblioteca de Péptidos , Receptores de Antígenos de Linfocitos T/inmunología , Ribosomas/inmunología , Antígenos CD28/aislamiento & purificación , Humanos , Inmunoensayo/métodos , Linfoma no Hodgkin/patologíaRESUMEN
The new antigen receptor (NAR) from sharks consists of a single immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antibody. Two closely related NARs with affinity for the Kgp (lysine-specific) gingipain protease from Porphyromonas gingivalis were selected by panning an NAR variable domain library. When produced in Escherichia coli, these recombinant NARs were stable, correctly folded, and specifically bound Kgp (K(d)=1.31+/-0.26x10(-7) M). Binding localised to the Kgp adhesin domains, however without inhibiting adhesin activity. These naturally occurring proteins indicate an immune response to pathogenic bacteria and suggest that the NAR is a true antibody-like molecule.