Combinatorial Phage Antibody Libraries
77
antibodies using hybridoma technology
and Epstein–Barr virus transformation of
human B-cells has met with only limited
success, particularly when one considers
the size of the available antibody reper-
toire in man. Consequently, much effort
has been directed toward ‘‘humanizing’’
existing mouse monoclonal antibodies
through grafting murine antigen-binding
sequences (complementarity-determining
regions) into a human antibody frame-
work. This methodology is useful insofar
as the immunogenicity of the Fnal con-
struct is considerably lower than that of the
original murine molecule. SigniFcantly
though, extra manipulation of framework
residues is often required to attain the
afFnity of the original mouse antibody,
and the process is labor-intensive and
time consuming.
Nevertheless, humanized monoclonal
antibodies are being increasingly used
in the clinic including in the treatment
of breast cancer, lymphomas, Crohn’s
disease, rheumatoid arthritis, and graft re-
jection and in the prevention of respiratory
syncytial virus (RSV) infection in prema-
ture infants. Combinatorial antibody li-
braries provide a rich source of diverse and
fully human antibodies without the need
for manipulation and so are expected to be
used increasingly for generating antibod-
ies for the clinic. Indeed, the Frst human
antibody developed from phage libraries
has recently been approved for human use.
2
Combinatorial Antibody Libraries
2.1
The Early Libraries: The Lambda Phage
System
In the search for an alternative approach
for generating human monoclonal anti-
bodies, two developments were critical.
The Frst was the demonstration that the
antibody antigen-binding fragments ±ab
and ±v could be cloned and expressed in
bacteria. To ensure correct folding and
assembly of the antibody fragments (essen-
tial for retention of the binding characteris-
tics of the parent molecule), the translated
proteins were shepherded to the bacterial
periplasm under the guidance of appro-
priate bacterial leader sequences. Second,
and equally important to the emergence of
combinatorial antibody libraries, was the
use of the polymerase chain reaction (PCR)
and a family of oligonucleotide primers to
amplify antibody genes from a mixed pop-
ulation of antibody-producing cells. Taken
together, these techniques meant that one
could, by using an appropriate vector,
express in bacteria the antigen-binding do-
mains of an array of antibodies of any class
and from any animal for which one had
the antibody sequence for the construc-
tion of appropriate PCR primers: in effect,
antibody repertoire cloning.
The
Frst
combinatorial
antibody
li-
braries were prepared from hyperimmu-
nized mice and were constructed in the
lytic bacteriophage lambda. Total RNA
taken from mouse spleen was reverse-
transcribed, and antibody heavy chains
(±d of IgG1, V
H
and C
H1
domains) and
light chains (V
L
and C
L
domains) were
PCR-ampliFed and independently cloned
into modiFed lambda phage vectors. These
heavy and light chain libraries were com-
bined, following restriction digestion and
religation, to create a single vector capa-
ble of expressing ±ab fragments derived
from the randomly scrambled antibody
light and heavy chain genes. To identify
±ab fragments speciFc for the immuniz-
ing antigen, the combinatorial library was
screened using a Flter lift plaque assay.
Here, soluble ±abs, released during plaque
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