Drug Targeting Organ-Specific Strategies

10.3 Generation of Ligands Amenable for Targeting 265
in lysates of non-relevant cells. The target antigen(s) can then be blotted onto a membrane,
or eluted from the gel for later use as an antigen source for selection. This principle has been
proven by the selection of antibodies recognizing the native form of the ED-B domain of fibronectin,
a marker of angiogenesis [72].
Selection techniques are often adapted to conditions where antigenic expression is as close
as possible to the in vivo situation.Antigen expression on cells is highly dependent on the cellular
environment and on cell–cell and cell–matrix interactions. In order to maintain some
cell–cell and partial cell–matrix interactions, cell panning can be carried out on in vitro cultured
cell monolayers, where culture conditions can also be further modified in order to mimic
the antigenic expression in vivo. Cell panning can also be carried out with cells in solution.
Unbound phage can be removed after the cells and bound phage are separated from the supernatant
by centrifugation or by magnetic retrieval with magnetically labelled beads which
bind to the cells (reviewed by Mutuberria et al.) [73].
A highly pure cell population, with its specific bound phages, can be isolated from complex
cell mixtures by the use of magnetic activated cell sorting (MACS) [74] or by fluorescent activated
cell sorting (FACS) [75,76]. Cells bearing the antigens of interest are magnetically or
fluorescently labelled via cell-specific antibodies. Following incubation of the cell mixture
with the phage library, the antigen-positive cell population and bound phage is retrieved
from the antigen-negative cells using MACS or FACS. When the phage library is incubated
with the cell mixture prior to target cell isolation (subtraction), selection of target cell-specific
antibodies may be favoured, since negative non-target cells will compete for antibodies
that bind to common antigens. MACS selections on a model system has shown clear advantages
over other cell selection methods such as selections on monolayers [73].This may be attributed
to the improved interaction between cells and phage, the efficient elimination of
non-relevant phage by washing the cells immobilized on a magnetic column, and the high cell
viability and integrity during this mild selection procedure. We have applied this selection
method using a large naive antibody library on mildly fixed endothelial cells immobilized on
a magnetic separation column.Angiogenic factors and tumour-conditioned media were used
to induce tumour angiogenesis-associated antigen expression on cultured human umbilical
vein endothelial cells. A large number of endothelial cell binders have been selected by this
approach, most of which bind preferentially to angiogenic vasculature, and therefore tumour
vasculature. A further advantage of the MACS selection approach is that it is based on the
isolation of positive target cells on magnetic columns, from which irrelevant phage can be simultaneously
washed off, using simple, efficient and gentle methods.
As an alternative to selections on cells, other antigen sources, which better maintain the in
vivo antigen expression profile and that allow appropriate in vitro selection procedures, are
available. Selection on tissue cryosections may result in antibodies directed to intra- and extracellular
antigens on any cell type present in the tissue section, as well as antibodies binding
to matrix components [77].
Model selections, in which phage antibodies with defined specificity are mixed with nonspecific
phage and the enrichment and yield for a selection procedure is measured, provides
a rapid experimental approach for studying such complex selection procedures. In order to
compare different selection approaches on complex preparations, and to determine the best
selection parameters for each approach, an extensive study of various in vitro and in vivo
model selections was recently carried out by our group [73].