Drug Targeting Organ-Specific Strategies

256 10 Phage Display Technology for Target Discovery in Drug Delivery Research
PlacZ
ColE1
ori
L L
r M13 intergenic region
H
H1
κ H1 H6 MYC A
+ IPTG
induction
+ Helper
Phage
Soluble Fab
Phage displayed
Fab
Figure 10.1. Display of a Fab fragment on filamentous phage. Fab fragments can be displayed on phage
using phagemid vector pCES1 which expresses the heavy chain fragment containing the variable domain
and the first constant domain fused to the coat protein gene III, in combination with separate expression
of the partner (light) chain. Bacteria harbouring this vector are infected with helper phage to drive the
production of phage particles carrying the Fab fragment as a fusion product with the phage coat protein
pIII on the surface, while the immunoglobulin encoding genes reside within the phage genome.
Alternatively IPTG induction drives the generation of the soluble Fab fragments on the bacterial
periplasm. AMPr, ampicillin resistance; H6, histidine tag for purification purposes; MYC, myc tag for
detection purposes; A, amber stop codon (TAG) which allows expression of the soluble antibody
fragment in non-suppressor strains; gIII, phage gene III; rbs, ribosomal binding site; S, signal sequence
directing the expressed protein to the bacterial periplasm; ColE1 ori, E. coli origin of replication; PlacZ,
LacZ promoter.
material resides within the phage genome. When the cloned DNA encodes variants of a certain
ligand, a phage display library is created. Phage display was first achieved in 1985 by the
expression of a peptide on the surface of bacteriophage M13 [1]. Five years later the first random
peptide libraries [2–4], and antibody fragment libraries [5] were constructed. Today a
large number of moieties have been successfully displayed on the surface of filamentous
phages. These include peptides (reviewed by Cesarini et al.) [6], antibody fragments (reviewed
by Hoogenboom) [7], enzymes (reviewed by Soumillon et al.) [8], protein scaffolds
(reviewed by Nygren and Uhlen) [9], cDNA libraries, (reviewed by Hufton et al.) [10], protease
inhibitors [11], transcription factors [12], cytokines [13], and extracellular domains of
receptors [14].
In order to retrieve ligands with the desired specificity, phage display libraries are enriched
for target binding clones by subjecting the phage libraries to repetitive rounds of selection.
This includes incubation with antigen or ‘biopanning’, washing of non-bound phage, elution
and re-infection of selected phages into bacteria (Figure 10.2).Antigen binding phage is generally
eluted by low or high pH treatment, which drives the dissociation of the ligand from its
target without substantially altering the infectivity of the phage for bacteria. A selected filamentous
phage is propagated in bacteria, which secrete multiple copies of the phage displaying
a particular insert. Selection is repeated until a population of binding clones is enriched
gIII
V H
C H1
V L
C L