Drug Targeting Organ-Specific Strategies

264 10 Phage Display Technology for Target Discovery in Drug Delivery Research
When the target molecules are not easily purified, or are cell surface antigens that require
the presence of a lipid bilayer for maintaining their native form, selection on complex antigen
mixtures is necessary. To avoid selection of non-relevant phage and to select antigen or,
in the case of cell selections, to obtain cell type specific phage ligands, depletion and/or
subtraction methods may be employed. Depletion is achieved by the incubation of the phage
library with non-target cell population(s), which do not display the target antigen, previous
to the incubation with the target cell population. Subtraction, on the other hand, is performed
when the phage library is incubated with both target and non-target cell populations
simultaneously, and the target cell population with the bound phage is subsequently isolated.
Competitive elution with an antibody or the antigen itself [67] can be used to elute only those
antibodies homing to the desired antigen. Selection with alternating different sources of
antigen to select only those ligands that bind to antigen(s) which are common to all sources
[68], may also be employed.Alternatively, a method named ‘pathfinder’ has been devised for
guided selection on complex antigen sources, using ligands directed towards the target
molecule. These ligands, conjugated to horseradish peroxidase (HRP) can be used as
pathfinders during the panning procedure. In the presence of biotin tyramine these molecules
catalyse biotinylation of phage binding in close proximity to the target antigen, allowing
specific recovery of ‘tagged’ phage from the total population using streptavidin. In this
way, phage binding to the target itself, or in its immediate proximity, are selectively recovered
[69]. This technique has been applied for the selection of phage antibodies against antigens
including carcinoembryonic antigen (CEA), E- and P-selectins, and for the selection of novel
antibodies which recognize immobilized purified antigen [70].This technique could also be
applied in the future for the selection of ligands to novel targets such as unknown receptors
of known ligands or any molecules involved in other protein–protein interactions (Figure
10.4).
10.3.2 Phage Display for Target Identification
10.3.2.1 In Vitro Selections on Complex Antigens
We have seen the versatility of phage display technology for the selection of ligands directed
to known target molecules. However, one of the major advantages of this technology is based
on its applicability for the selection of ligands directed to novel targets present on certain tissues,
cell types, or cells in a specific stage of differentiation or in disease-induced states. The
selection methods developed for defined targets may now be used to detect, by virtue of the
phage, novel ligands or epitopes in the antigenic mixture. In addition, specific protocols have
been developed to direct the selection process towards the isolation of tissue- or cell typespecific
ligands (Figure 10.2).
In principle, whole live cells or crude preparations derived from cells or tissues can be used
as an antigen source to identify novel targets. For example, measles virus-infected cell lysates
have been used to select antibodies which reacted specifically with measles virus-infected
cells [71]. Protein mixtures from cells or tissues may first be separated to direct the selection
to specific cell compartments or antigens. By SDS-PAGE, the antigen(s) of interest can be
identified and isolated, e.g. antigens detected in 2-D gels of cell lysates of target cells but not