338
Antibody Molecules, Genetic Engineering of
high afFnity for human antigens and
suitable for repeated administration to hu-
mans. To date, these engineered mice can
produce only some of the human isotypes,
but further genetic modiFcation of these
mice promises to expand their potential.
3
In Vitro
Antibody Production by Phage
Libraries
The production of monoclonal antibodies
frequently requires several injections of
antigen, which can take weeks to months.
Hybridoma construction and screening
require additional time. In addition, the
immune response is often biased toward
certain ‘‘immunodominant’’ epitopes of
the antigen, making it difFcult or impossi-
ble to produce monoclonal antibodies with
the speciFcity desired for a particular use.
Moreover, the production of antibodies
against antigens conserved among species
may be difFcult or impossible. Phage
display, a technology that allows the ex-
pression of immunoglobulin genes in bac-
teriophages (viruses that infect bacteria)
without the need for developing hybrido-
mas, provides an alternative approach that
overcomes many of these problems.
In phage display, the heavy and light
V-gene obtained from the spleen of an
immunized mouse or from the peripheral
blood of a naive or immunized donor
are expressed as ±ab or single-chain ±v
(sc±v) on the surface of Flamentous phages
(f1, M13, and fd). Phage libraries can be
generated using variable antibody gene
repertoires from any species including
humans, or even synthetic sequences.
±igure 5 shows the development of sc±v
libraries using a human donor.
Both sc±v and ±ab fragments can be
expressed on the surface of Flamentous
bacteriophages (f1, M13, and fd) as either
single or multiple copies of the anti-
body of interest depending on the phage
protein used for fusion. Expression of mul-
tiple copies facilitates the identiFcation
and isolation of low-afFnity antibodies.
When functional antibody V domains are
displayed on the surface of Flamentous
phages, the resulting phages bind specif-
ically to antigen, and rare phages can be
isolated on the basis of their ability to
bind antigen. Multiple rounds of enrich-
ment consisting of binding to immobilized
antigen, expanding the bound phage, and
further enriching by again binding to
immobilized antigen can yield speciFc
phages even if the desired speciFcity was
present on less than 1 in 10
6
of the original
phages in the library. The selected variable
regions will generally have afFnities sim-
ilar to monoclonal antibodies and can be
expressed as antibody fragments in the
bacterium
Escherichia coli
or they can be
used to produce complete antibodies and
can be expressed in mammalian hosts. In
addition, speciFc variable regions can be
mutagenized and phages that express vari-
able regions with increased afFnity can
be selected. The bacteriophage expression
systems are designed to allow the genes
encoding for heavy and light chains to
undergo random combinations, which are
tested for their ability to bind the desired
antigen.
The decision of whether to produce
sc±v or ±ab libraries depends partly on
the intended use. The single gene for-
mat of the sc±v is an advantage for the
construction of fusion proteins such as
‘‘immunotoxins’’ (antibodies or antibody
fragments fused to a toxin) or for targeted
gene therapy approaches where the
scFv
gene is fused to a viral envelope protein
gene. The use of sc±v also appears to
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