346
Antibody Molecules, Genetic Engineering of
molecule can be fused to an scFv molecule
as described in Sect. 4.1. Moreover, two
antibody Fc fragments can be genetically
fused resulting in a molecule with novel
biological properties. An example of this
approach is the protein GE2, which is the
product of the fusion between the Fc frag-
ments of human IgG and human IgE
(Fc
γ
–Fc
ε
fusion protein). GE2 does not
have an Fab and, as a consequence, is un-
able to target an antigen. However, GE2
was able to form complexes with both
Fc
γ
RII and Fc
ε
RI, resulting in an inhi-
bition of mast cell and basophil activation
that results in the blocking of the anaphy-
laxis in transgenic mice expressing human
Fc
ε
RI
α
. This approach has therapeutic
potential in IgE- and Fc
ε
RI-mediated dis-
eases such as allergic asthma, allergic
rhinitis, chronic urticaria, angioedema,
and anaphylaxis.
5
Expression Systems
A large variety of expression systems have
been used for the production of geneti-
cally engineered antibodies and antibody
fragments. These expression systems in-
clude bacteria, yeast, plants, baculovirus,
and mammalian cells.
Antibody fragments are commonly ex-
pressed in bacteria and yeast. The bac-
terium
E. coli
is a frequently used expres-
sion system owing to its rapid growth and
easy genetic manipulation. However, pro-
teins expressed in
E. coli
are frequently
insoluble and/or inactive, and refolding
may be required to obtain functional frag-
ments. Secretion of fragments into the
bacterial periplasm or culture supernatant
provides an alternative means to obtain the
desired functional fragments without the
need for refolding. Although the results
are highly antibody-dependent, there are
many examples in which the bacterial ex-
pression system is successful.
Complete functional antibodies have
been most successfully expressed in mam-
malian cells, as these cells possess the
mechanisms
required
for
correct
im-
munoglobulin assembly, posttranslational
modi±cation (glycosylation), and secre-
tion. Posttranslational modi±cations can
influence the biologic properties and ef-
fector functions, important considerations
especially when the antibody is to be
used for therapy. Examples of mammalian
cells that have been successfully used
to express properly assembled and gly-
cosylated antibodies and antibody fusion
proteins are the mouse myeloma cell lines
P3X63Ag8.653, Sp2/0-Ag14, and NS0/1.
These three myeloma cell lines have lost
the ability to produce endogenous H and
L chains and are derived from the parent
myeloma. Antibodies produced in nonlym-
phoid cell lines such as Chinese hamster
ovary (CHO), HeLa, C6, and PC12 are also
properly assembled and glycosylated. Ow-
ing to the slower growth of mammalian
cells, mammalian expression requires a
longer time frame and higher costs than
bacterial or yeast expression, but it is pre-
ferred when complete functional antibod-
ies with proper glycosylation and disul±de
bonds are required. Other expression sys-
tems that have been extensively used to
produce complete functional antibodies in-
clude insect cells and plants. There is no
‘‘universal’’ expression system – each sys-
tem has its advantages and disadvantages.
6
Conclusion
Rapid
progress
has
been
made
in
producing
genetically
engineered
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