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 20 years ago. Since then, many antibodies have been selected and improved upon using these methods. Although it is not widely recognized, many of the antibodies derived using in vitro display methods have properties that would be extremely difficult, if not impossible, to obtain by immunizing animals. The first antibodies derived using in vitro display methods are now in the clinic, with many more waiting in the wings. Unlike immunization, in vitro display permits the use of defined selection conditions and provides immediate availability of the sequence encoding the antibody. The amenability of in vitro display to high-throughput applications broadens the prospects for their wider use in basic and applied research.

Figure 1

The additional capabilities of in vitro selection offer a new approach to antibody generation, allowing the selection of antibodies with particular properties by predefining panning conditions. Variations in salt and pH conditions, the conformational form of the target and the presence of closely related proteins help determine the biochemical properties, fine specificity, cross-reactivities and affinity of resulting binders. Further, the immediate availability of the antibody gene provides significant additional value. Complemented by a more rapid antibody generation cycle, this will broadly change the manner in which antibodies will be made and used for research in the near future.

Figure 2

In vitro selected antibodies can recognize minute differences in small molecules. A) Antibodies against 6-monoacetylmorphine, the major heroin metabolite, do not recognize the closely related morphine. B) Many different antibodies have been selected and subsequently had both affinity and specificity matured to recognize each of the represented steroids (for references see Table 1). C) Antibodies against tyrosine sulfated modified proteins do not recognize proteins containing either tyrosine or tyrosine phosphate,.

Figure 3

Mechanisms for blocking or activating receptor signaling using antibodies. The EGF receptor is used to exemplify mechanisms by which antibodies can block signaling in different classes of receptor. The EGF receptor is a single trans-membrane domain with multiple extra-cellular domains (represented as different colored ovals) having different functional domains. In this example, binding of ligand (green circle) occurs at domain 3, receptor dimerization occurs through domain 2 and interactions between domains 2 and 4 stabilize the “closed” conformation of the receptor. Antibodies can block signaling by a. binding to the ligand and preventing interaction with receptor, b. binding the ligand binding site of the receptor and preventing interaction with ligand, c. preventing dimerization by binding the dimerization domain or sterically blocking the interaction d. stabilising the closed conformation of the receptor. e. Activation can occur by binding the ligand-binding site typically with bivalent antibodies.

Figure 4

An engineered dual specificity synthetic Fab. The bH1 Fab binds to both Her2 (orange, PDB entry 3BDY) and VEGF (red, PDB entry 3BE1). The heavy and light chains of the Fab are colored cyan/grey or blue/black respectively, with the different colors derived from structures of bH1 binding to either Her2 or BEGF.