Beyond natural antibodies: the power of in vitro display technologies

Abstract

In vitro display technologies, best exemplified by phage and yeast display, were first described for the selection of antibodies some twenty years ago. Since that time a large number of antibodies, some with remarkable properties, have been selected and improved upon using these methods. The first antibodies derived using in vitro display methods are now in the clinic, with many more waiting in the wings. Here we discuss the scope of the technology, some of the powerful antibodies selected, and the future potential in a post-genomic world. Unique advantages offered by in vitro display technologies include the ability to carefully define selection conditions, allowing the derivation of antibodies recognizing predefined epitopes or conformations, the further improvement of selected antibodies, the potential for high throughput applications and the immediate availability of genes encoding the selected antibody. We anticipate that the high throughput potential of these technologies will soon lead to their use to select antibodies against all human proteins.

RETRACTED: Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation

“…However, in the case of antibody 93F3, a surprising number of the peripheral mutations are stabilizing relative to what one might expect on the basis of directed evolution experiments with other proteins (9,33,34). Indeed, most in vitro antibody-engineering techniques rely upon display systems that select for affinity alone (35,36) and often produce high-affinity binders with poor biophysical characteristics (13).…”

“…Antigen binding fragments (Fabs) of antibodies generated from hybridomas exhibit a relatively small range of melting temperatures despite significant sequence variation (12). In contrast, phage display and other in vitro selection systems often afford high-affinity antibodies that are poorly expressed, aggregate, and/or have low stability (13). Thus, a subset of naturally occurring somatic mutations, especially those distal to the combining site, may compensate for destabilizing mutations that are selected on the basis of affinity alone.…”

Degradation of C-terminal tag sequences on domain antibodies purified fromE. colisupernatant

“…Today, recombinant antibodies can be generated in vitro by applying various display systems that allow for the selection of high affinity binders to virtually any type of antigen. 1 One of the most common display formats for recombinant antibodies is phage display using filamentous phage. In phage display, a foreign protein is expressed in fusion with one of the phage capsid proteins by cloning the foreign DNA sequence into the phage genome.…”

Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes

“…When the protein is an enzyme, such as that encoded by BRAF, the resulting structural change can provide a pocket for the binding of specific enzymatic inhibitors (3)(4)(5). Antibodies are one of the most successful types of modern pharmaceutical agents and have been shown to be able to specifically recognize proteins that differ only by a single amino acid or by the modification of a single amino acid (5)(6)(7)(8)(9)(10)(11). However, all antibodies used in the clinic are directed against cell-surface or secreted proteins rather than intracellular proteins.…”