Drug Targeting Organ-Specific Strategies

ies may be generated from such non-immune ‘naïve’ libraries built with the V-genes of B cells
from human donors [35], or from synthetic libraries built with in vitro rearranged variable
germline genes [36].Alternatively, for some applications, antigen-biased antibody repertoires
can be created using V-genes from immune sources (reviewed by Hoogenboom et al.) [7].
Today several large single pot libraries with over 10 10 independent clones have been generated.
From these libraries high affinity antibodies against virtually any antigen can be isolated
[7]. The size of the library significantly improves the likelihood of identifying antibody
fragments with high specificity and high affinity due to the sampling of a larger diversity.
Phage displayed antibody libraries have been successfully used to isolate antibodies
against a variety of antigens such as self-antigens [37], haptens [38], carbohydrates [39], DNA
[40] and RNA [41]. Selected antibodies may also have remarkable specificity for the antigen,
for example, antibodies which are able to distinguish between two variant forms of a native
antigen which differ in a single amino acid, have been selected [42].
The selected antibody fragments have been used in many applications, varying from antibody-based
biochemical assays to in vivo imaging of tumours. The relative ease with which
they form multimers and fusion proteins, and the possibility of high levels of expression of
these molecules in a variety of different hosts, makes them ideal protein-based diagnostic and
possibly future therapeutic reagents (reviewed by Hudson and Kortt) [43] (Figure 10.4).
10.2.2.4 Protein Scaffolds
10.2 Phage Display Technology 261
Until the advent of the phage display era, antibodies were traditionally the sole source of
antigen binding molecules. By variation of surface residues of a protein, it is in principle possible
to build a library of proteins each with a different topological surface. From these libraries,
variants with a particular binding specificity can be selected using phage display. Indeed,
protein display libraries based on proteins with very different folding patterns and thus
varying structural frameworks, have been constructed. Ideally protein scaffolds to be used in
drug targeting should: (1) be small single domains, (2) be of human origin, (3) have predictable
pharmacokinetics (for human therapeutics), (4) have available sites or surfaces for
the introduction or transfer of functional sites, and, (5) have a sufficiently large antigen-binding
surface for affinity or specificity maturation. They should also be (6) suitable for engineering
into multivalent, multi-specific molecules, or molecules with effector functions, and
(7) yield high level expression in multiple hosts with the capacity to fold in vitro and in vivo.
Although antibodies fulfil most of these requirements, there are applications where other
proteins may be more suitable.
Scaffold libraries have been created for several applications in affinity chromatography, or
for the generation of ligands to disease-related targets. The most developed scaffold is a two
alpha helix-containing variant of protein A from Staphylococcus, protein Z. so called ‘Affibodies’
[44], showing selective binding to respiratory syncytial virus (RSV) G protein, have
been selected [45]. Some libraries have been used for the transfer of bioactive peptides or
functional residues from one protein to a new protein scaffold. In our laboratory, human cytotoxic
T lymphocyte associated protein-4 (CTLA-4) has been used as a protein scaffold to
display the 14mer somatostatin hormone, or RGD sequence-containing peptides. This has
produced a series of ligands to the somatostatin receptor and αvβ3 integrin respectively, the