Human Antibodies with Sub-nanomolar Affinities Isolated from a Large Non-immunized Phage Display Library

Abstract

To generate a stable resource from which high affinity human antibodies to any given antigen can be rapidly isolated, functional V-gene segments from 43 non-immunized human donors were used to construct a repertoire of 1.4 x 10(10) single-chain Fv (scFv) fragments displayed on the surface of phage. Fragments were cloned in a phagemid vector, enabling both phage displayed and soluble scFv to be produced without subcloning. A hexahistidine tag has been incorporated to allow rapid purification of scFv by nickel chelate chromatography. This library format reduces the time needed to isolate monoclonal antibody fragments to under two weeks. All of the measured binding affinities show a Kd < 10 nM and off-rates of 10(-3) to 10(-4) s-1, properties usually associated with antibodies from a secondary immune response. The best of these scFvs, an anti-fluorescein antibody (0.3 nM) and an antibody directed against the hapten DTPA (0.8 nM), are the first antibodies with subnanomolar binding affinities to be isolated from a naive library. Antibodies to doxorubicin, which is both immunosuppressive and toxic, as well as a high affinity and high specificity antibody to the steroid hormone oestradiol have been isolated. This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.

Design and testing of PCR primers for the construction of scFv libraries representing the immunoglobulin repertoire of rats

“…The results (not shown) demonstrated that at least 60% percent, and probably more, of the randomly picked clones were efficiently displayed using the phage production methods described in the Materials and Methods. This value is similar or better to those reported for human scFv libraries (Vaughan et al, 1996;O'Connell et al, 2002). The failure to observe scFv/pIII fusion proteins in some clones is likely caused by the poor expression levels of these scFv fusions, or by aberrant or introduced stop codons.…”

Controlling complement resistance in cancer by using human monoclonal antibodies that neutralize complement-regulatory proteins CD55 and CD59

“…To neutralize the inhibitory activity of CRP and to potentiate complement-mediated killing of B lymphoma cells, we have isolated scFv to CD55 and CD59 from a human phage-display library [18] to be used in combination with complement-fixing Rituximab and other complement-activating mAb. The human phage Ab libraries offer the advantage over conventional mAb of a large Ab repertoire that is not shaped by the constraints of the immune system, with a dramatic increase in the chances of isolating Ab to selfantigens [20]. On the other hand, the Ab currently used in cancer therapy, unlike human scFv, are of murine origin and, although engineered to become chimeric or humanized, they still contain murine variable portions of the original Ig that may elicit an anti-idiotypic response [21,22] resulting in their rapid clearance.…”

HGS-ETR1, a fully human TRAIL-receptor 1 monoclonal antibody, induces cell death in multiple tumour types in vitro and in vivo

“…Human soluble extracellular domain (ECD) TRAIL-R1-flag fusion protein at 10 mg ml À1 in PBS was immobilised onto immunotubes (Nunc, Rochester, NY, USA) overnight at 41C. Three or four rounds of panning selections were performed using the scFv human antibody phage library with approximately 1.4 Â 10 10 individual recombinants (Vaughan et al, 1996). A deselection round was performed on an irrelevant fusion protein to remove any nonspecific binders.…”