340
Antibody Molecules, Genetic Engineering of
be a better option for ‘‘intracellular im-
munization,’’ a procedure in which the
gene encoding the antibody binding site
is delivered intracellularly to achieve phe-
notypic knockout. However, the use of
Fab fragments offers the advantage that
heavy- and light-chain libraries can be
produced independently and reassorted.
The use of two vectors also facilitates
the construction of hierarchical libraries
in which a ±xed heavy or light chain is
paired with a library of partners. In the-
ory, Fab libraries may be preferred where
the±na
lp
rodu
c
tw
i
l
lbeacomp
le
tean
-
tibody, since, in some instances, removal
of the scFv linker might alter the antigen-
binding properties. However, scFvs have
been successfully converted into complete
antibodies.
Phage display has proved to be very
effective in generating a variety of anti-
bodies that are dif±cult to obtain using
the hybridoma technology. For preexist-
ing mouse monoclonal antibodies, phage
display can be used to obtain antibodies
that are entirely human in sequence, but
which bind to the same part of the anti-
gen (epitope) as the mouse monoclonal
antibodies. Antibodies to targets previ-
ously inaccessible using immunization
approaches (for example, self-antigens,
ubiquitous
compounds, or
toxic com-
pounds) have been isolated by selection
of phage on antigen
in vitro
.Bu
i
ld
ingon
the concept of phage display, alternative
display strategies such as ribosome dis-
play and cell-surface display have been
recently developed.
4
Further Genetic Modifcations o± Antibodies
4.1
Engineering Antibody Fragments:
Monovalent, Bivalent, and Multivalent
scFvs
As explained in Sect. 3, scFv fragments
consist of the variable domains of heavy
and light chains (V
L
and V
H
) genetically
fused by a flexile linker peptide. scFvs
are much smaller than intact antibodies
(Fig. 6) (25–27 kDa vs around 150 kDa for
intact IgG), yet can retain the speci±city
and the af±nity of the parental molecule.
The small size of scFvs is a tremendous
C
H
1
C
H
2
C
L
C
H
3
Intact antibody
scFv
scFv dimer
diabody
scFv-C
H
3
minibody
V
H
V
H
V
L
V
L
V
H
V
L
C
H
3
V
L
V
L
V
H
V
H
V
L
V
H
Fig. 6
Schematic representation showing the relative size and domain
relationships between intact IgG (around 150 kDa) and engineered
single-chain Fv (scFv, 25–27 kDa), scFv dimer or diabody (55 kDa), and
minibody (scFv-C
H
3, 80 kDa) fragments.
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