Drug Targeting Organ-Specific Strategies

262 10 Phage Display Technology for Target Discovery in Drug Delivery Research
latter being important in angiogenesis (see Chapters 9 and 7), with possible application as human
therapeutics [46,46a].
10.2.2.5 Engineering Proteins with Phage Libraries
Phage display technology offers a valuable tool for the in vitro evolution of molecules. By targeted
or random mutagenesis, libraries containing variants of a protein can be constructed,
and selection of these libraries under controlled conditions can result in the generation of
variants with improved characteristics.A large variety of proteins have been successfully displayed
on phage with the purpose of selecting desired variants. Hormone variants with higher
affinity for their receptors [47], with similar biopotency and reduced size [48], or with improved
specificity for one of its receptors [49] have been selected from phage libraries. Proteins
with higher affinity or specificity for their receptor [50] or enzymes with higher or new
catalytic activities [51,52] have also been generated.
Minimizing proteins into significantly smaller polypeptides has also been achieved via
both rational design processes and selection from vast combinatorial phage display libraries.
Such ‘mini-proteins’ represent a potential intermediate step toward the development of
drugs targeted to protein–protein interfaces [53]. Finally, binding ligands with desired function
and specificity can also be generated using a combination of phage display technology
and semi-rational protein design. In this way peptides that bind to the extracellular domain
of the fibroblast growth factor (FGF) receptor, were selected from a peptide library. Guided
by the knowledge that agonist activity of FGF is conferred by the ability to cause receptor
dimerization, the selected peptide was expressed as a fused protein coupled to domains that
undergo spontaneous dimer formation, enhancing binding affinity to the receptor.
Furthermore, one of these fusion proteins also possessed agonist activity in vitro. This has
generated a small protein with no homology to FGF which binds to the FGF receptor reproducing
the biological activity of FGF [54].
10.2.2.6 cDNA Expression Libraries
The display of cDNA libraries was first achieved on pIII by an indirect display method, based
on the interaction between the Jun and Fos leucine zippers [55,56]. This approach was successfully
applied for the selection of allergenic proteins from Aspergillus fumigatus using
serum from affected patients [57].An alternative approach was later described where cDNA
fusion occurred at the C-teminal domain of the minor coat protein pVI [58]. cDNA libraries
are being used for the screening and selective isolation of genes by specific gene-product/ligand
interaction. For the identification of disease-related targets a cDNA fragment display library
can be selected against homogeneous ligands [59], such as natural ligands or ligands selected
from antibody or peptide libraries against novel targets, or heterogeneous ligands such
as patient sera or polyclonal antibodies. The latter application has been validated by our
group for the selection of products from a colorectal carcinoma cDNA library using both homogeneous
and heterogeneous ligands [10]. Display of cDNA or cDNA fragment libraries
on lambda phage is complementary to the filamentous phage display libraries. With M13