Combinatorial Phage Antibody Libraries
81
Drawing from the studies of antibody ex-
pression in bacteria described earlier, it
seemed reasonable that an antibody fused
to the N-terminus of pIII should be able
to fold correctly in the environment of the
periplasm. The tethered antibody would
then be assimilated into the extruding
virion and displayed on the surface.
In the earliest phage display experi-
ments, peptides and proteins were fused to
pIII by cloning directly the corresponding
genes into the phage genome. Conse-
quently, every copy of pIII on the phage
surface carried the fusion. In the context
of antibody display, the result is multiva-
lent binding and a signiFcant avidity effect,
which dramatically hampers the efFciency
of selection between antibody clones of
different afFnities. Thus, monovalent dis-
play is preferable for selecting clones with
the highest afFnity. However, multivalent
d
isp
layo
fan
t
ibodyhasp
roveduse
fu
lin
selecting antibodies against a number of
targets including those on the cell surface
wherein the phage becomes internalized
much more efFciently.
To
regulate
the
number
of
fusion
molecules displayed per phage particle,
phagemid systems have been developed.
Phagemids are simply plasmids contain-
ing Flamentous phage regulatory elements
(typically f1 is used). This sequence con-
tains a Flamentous phage DNA origin of
replication and a DNA packaging signal.
Upon coinfection of a cell with an M13
helper phage, the regulatory element of
the phagemid sequesters the replicative
machinery provided by the helper phage to
package single-stranded phagemid DNA
into an infectious phage particle. By in-
troducing into the phagemid a segment
of DNA encoding the pIII fusion, sub-
sequent superinfection with the helper
phage results in the production of na-
tive pIII encoded by the helper phage and
the pIII fusion encoded by the phagemid.
Tagging a bacterial leader sequence to
the front of the pIII fusion serves to di-
rect the translated protein to the bacterial
inner membrane. A competition is there-
fore established between these two forms
of the coat protein for incorporation into
newly formed phage particles. The resul-
tant phages generally carry one copy of
the fusion.
Phagemid vectors have also been con-
structed to generate antibody fusions with
pVIII. This coat protein is present in
greater abundance than pIII and covers the
entire length of the phage particle; display
of up to 24 copies of ±ab fused to pVIII has
been described. However, there are signif-
icant limitations in the size of the protein
that can be displayed by pVIII, which are
presumably imposed by steric constraints
during the phage assembly process. Thus,
certain antibodies may not get displayed
by pVIII, or the number of copies of an-
tibody per phage may vary considerably
leading to poor discrimination of antibod-
ies of high and low afFnity. The limits of
pVIII display and the associated lack of
control over antibody valence during selec-
tion have made pVIII display a less favored
choice for antibody library construction.
The Frst vector system developed to
display ±ab fragments on the surface of
Flamentous phages via pIII fusion was
pComb3. Similar but improved versions
of this vector, pComb3H and pComb3X,
have since been described, and their use
in constructing combinatorial antibody li-
braries will be examined here by way of
example. During the construction of an
antibody library, the light and heavy (±d)
chains are cloned sequentially into the vec-
tor, effectively randomizing the light and
±d pairings of the recombinant ±abs. In
pComb3H, both antibody chains are un-
der the polycistronic control of a single
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