Drug Targeting Organ-Specific Strategies

phage there will be a bias to display naturally secreted cDNAs or extracellular domains (intracellularly
expressed cDNAs can also be expressed on phage, e.g. zinc finger proteins).
Phage lambda display has the advantage of cytoplasmic expression of the cDNAs and higher
display levels [60]. Selection of cDNA or cDNA fragments using cells or tissues, and the
rapid cloning of cDNAs expressing proteins specific for these ligands should facilitate the
search for novel targets and biologically important antigens (Figure 10.4).
10.3 Generation of Ligands Amenable for Targeting
In the following sections, procedures and approaches for retrieving ligands specific for welldefined
targets will be discussed, followed by discussions relating to the application of refined
selection methods for the identification of novel targets.
10.3.1 Selection of Ligands to Defined Targets
10.3 Generation of Ligands Amenable for Targing 263
Most phage selection strategies rely on the availability of purified or recombinant antigen.
This allows common selection strategies such as biopanning on immobilized antigen coated
onto solid supports [35], or on columns [38]. However, coating/immobilization of the antigen
may alter the conformational integrity of the antigen and as a consequence, antibodies that
do not recognize the native form may be selected. To circumvent this problem, indirect coating
methods can be applied. Antibodies may be used to capture the antigen, and non-specific
Ig of the same species and isotype may be added during the selection procedure to avoid
selection of antibodies to the capturing agent [61]. However, the most frequently used
method today, is based on selection with labelled soluble antigen, such as biotinylated antigen,
followed by capture on a biotin-binding surface [62]. After incubation of phage with the
biotinylated antigen, unbound phage can be removed and the antigen-bound phage can be
retrieved with streptavidin-coated paramagnetic beads and a magnet. This selection method
allows for the accurate control of selection parameters such as antigen concentration, which
may result in selection according to the affinity for the antigen, e.g. decreasing the antigen
concentration to favour the selection of high affinity antibodies [63]. Phage libraries can also
be incubated with biotinylated antigen and diluted into excess unlabelled antigen for variable
times prior to capture on streptavidin-coated magnetic beads. This allows for selection
on the basis of the kinetics of dissociation (off-rate) from the antigen; e.g. longer incubation
times with unlabelled antigen will favour selection of slow off-rate antibodies [62].
Phage display allows further variations to favour the selection of ligands directed towards
specific epitopes present in the target antigen. Similar to the presence of immunodominant
epitopes in vivo, selection-dominant epitopes exist in vitro [64]. Ligands directed to immunodominant
epitopes can be included during the panning procedure. By such ‘epitope blocking
panning’, a broader range of specific antibodies from combination libraries can be
rescued. This approach has been used to isolate neutralizing human antibodies to weakly
immunogenic epitopes of human immunodeficiency virus 1 (HIV-1) gp120 [65] and RSV
[66].