Drug Targeting Organ-Specific Strategies

10.4 Engineering and Optimization for Targeting 267
chain shuffling [38], by error prone PCR [62], by using bacterial mutating strains [83], or by
DNA shuffling [84].Alternatively, the antigen binding region may be mutated using oligonucleotide-directed
mutagenesis or by the introduction, with limited frequency, of random mutations
using PCR. The highest affinity increases in antibodies have been achieved when directing
the mutations to the complementary determining region 3 (CDR3), yielding in some
cases picomolar affinity antibodies [85,86]. Similar methodologies can be used with different
ligands. In addition, it is not only library-derived ligands which can be affinity matured. Hybridoma-derived
monoclonal antibodies, as well as other proteins or protein fragments may
be affinity matured by some of the methods mentioned above.Affinity maturation of the ligand
can also result in high affinity molecules with improved in vivo biodistribution. This was
demonstrated by the anti-human CEA specific scFv, isolated and affinity matured by phage
display technology. Both the original and affinity-matured antibody showed targeting to tumour
xenografts. However, although no difference was detected in tumour uptake, the affinity-matured
antibody with improved off-rates, was retained in the tumour for a longer period.
[87]. Occasionally, maximizing the affinity in vitro may result in modification of the antibody
specificity which could complicate the use of the resulting ligands for therapeutic applications
[88].
The possibility of specifically tailoring antigen-binding properties can also be directed
towards the engineering of avidity and valency. Antibody fragments are known to have
more rapid tissue penetration than full antibody molecules, as demonstrated in tumour targeting
[89]. Phage display has made easier the use of recombinant DNA technology for the
development of multi-specific and multivalent molecules, previously generated using
non-recombinant methods. Dimeric antibody fragments, or ‘diabodies,’ can be designed
for bivalent or bispecific interactions [90]. Phage libraries displaying bivalent bispecific antibody
fragments have also been constructed. Diabody libraries enable the selection of the
most appropriate bispecific molecules, with the highest affinity for binding, epitope recognition
and stability [91]. Multi-specific ligands can be used to direct targeted drugs to one or
more cellular antigens which may be present in either a particular cell type or diverse cell
types, or to stromal or secreted proteins found in the target tissue. Multivalent ligands have
been exploited mainly for immunotherapy, in the stimulation of cytotoxic pathways in vivo
to treat cancer [92]. The use of phage display in immunotherapy has been recently reviewed
[93].
Ligands can be tailored to improve their in vivo stability. To improve the in vivo thermal
stability of the tumour targeting monoclonal anti-epithelial glycoprotein-2 (EGP-2) antibody,
the antigen binding residues were grafted onto the framework of a highly stable scFv
resulting in increased serum stability at 37°C [94].The stability of peptides for in vivo use has
been approached in a different manner, using selection of peptide libraries on the D-form of
the antigen [95]. This technique named ‘mirror image phage display’ has recently been employed
in the generation of D-peptide inhibitors of HIV-1. Peptides directed against the mirror
image (chemically synthesized with D-amino acids) of a pocket of a viral protein involved
in viral entry, were first selected. The D-peptide mirror images of the selected consensus sequences,
binding to the natural ‘L’ form of the target, were then chemically synthesized. Because
these D-peptides are not subject to degradation by naturally occurring enzymes, they
can be used as the starting point for the development of new drugs or as effective orally administered
pharmaceuticals [96].