Drug Targeting Organ-Specific Strategies

266 10 Phage Display Technology for Target Discovery in Drug Delivery Research
10.3.2.2 In Vivo Selections and Selections for Functional Activity
In an original approach named ‘in vivo selection’ [19], phage capable of selective homing to
different tissues, such as the vasculature of lung, skin, and pancreas [78], were recovered from
a phage display peptide library following intravenous administration of the library to a living
mouse. In vivo selections have also been carried out in animals with a human tumour
xenograft. Phage specifically bound to the murine tumour vasculature could then be recovered
from the tumour tissue and amplified to yield tumour-specific endothelial cell binding
peptides [79]. So far in vivo selections strategies have been limited to the selection of peptides
directed to murine endothelial cell markers. This technique is producing ligands that
may have extensive application in vasculature targeting. This was demonstrated with in vivo
selected anti-integrin peptides coupled to the anti-cancer drug doxorubicin. The targeted
drug–peptide complex enhanced the efficacy of the drug against human breast cancer
xenografts in nude mice with reduced toxicity [79].
All the selection methods described are applicable for the generation of ligands with biological
activity. Often ligands selected by conventional techniques can be screened for a fortuitous
biological function: immunoneutralizing antibodies, receptor agonists or antagonists
can be identified from a pool of selected ligands when screened for biological function. Alternatively,
selections may be targeted towards biologically active sites of the antigens.
Some selection methods have been specifically designed for the selection of ligands with a
particular biological effector function. Such ‘selection for function’ has been used for the retrieval
of catalytic antibodies [80] and cell-internalizing phage antibodies [81], the latter being
highly useful molecules for the delivery of drugs, toxins, or DNA into the cytoplasm of
mammalian cells. Selection of antibody or peptide ligands for their function may, in the future,
be directed towards cell survival or killing upon ligand binding, cell transfection, inhibition
of cell surface molecules such as drug transport molecules and inhibition of viral entry
and receptor cross linking or triggering [7].
10.4 Engineering and Optimization for Targeting
Once the specific ligand has been selected, large arrays of possibilities are available in order
to reshape the ligand to obtain the best targeting results. In drug targeting applications, pharmacokinetics,
biodistribution, penetration, and bioactivity are strictly governed by the characteristics
of the ligand.
Certain therapeutic applications require high affinity ligands. Ligands such as peptides
with intrinsic low affinity for target antigens can be affinity matured. Secondary libraries of
the selected peptide can be created by selectively incorporating mutations, and variants with
higher affinities can be selected. Often the display format will change from multi- to monovalent
display to aid a genuine affinity selection [82].
Affinity maturation of antibodies has been achieved by the introduction of diversity into
the V-genes, which then creates diversity within the antigen binding sites. This secondary library
is then subjected to a selection that will enrich high affinity variants. More or less random
diversity may be introduced by altering variable domain pairings in a process called