RESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD28/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Linfoma não Hodgkin/imunologia , Biblioteca de Peptídeos , Receptores de Antígenos de Linfócitos T/imunologia , Ribossomos/imunologia , Antígenos CD28/isolamento & purificação , Humanos , Imunoensaio/métodos , Linfoma não Hodgkin/patologiaRESUMO
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.
Assuntos
Cisteína Endopeptidases/metabolismo , Hemaglutininas/metabolismo , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Receptores de Antígenos/química , Receptores de Antígenos/metabolismo , Adesinas Bacterianas , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , DNA/genética , Mapeamento de Epitopos , Cisteína Endopeptidases Gingipaínas , Hemaglutininas/genética , Hemaglutininas/imunologia , Imunoglobulinas/genética , Técnicas In Vitro , Dados de Sequência Molecular , Biblioteca de Peptídeos , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/genética , Estrutura Terciária de Proteína , Receptores de Antígenos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Tubarões/imunologiaRESUMO
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.