344
Antibody Molecules, Genetic Engineering of
avidin to the carboxy-terminus of the heavy
chain of IgG. This approach is similar
to that described in Sect. 4.1 (produc-
tion of tetramers of scFv by fusing it
with streptavidin) based on the fact that
both streptavidin and avidin are tetramers
of four noncovalently linked monomers.
Since each antibody-avidin protein con-
tains two molecules of avidin (one geneti-
cally fused at the carboxy-terminus of each
heavy chain), two independent antibody
fusion proteins bind to each other through
their respective avidins forming a dimeric
structure. One example of this approach is
a recently developed human IgG3-avidin
fusion protein speci±c for the transferrin
receptor (TfR). The anti-TfR IgG3-avidin
wasabletofunctionasauniversalvectorto
deliver different biotinylated compounds
into cancer cells overexpressing the TfR.
Furthermore, it was unexpectedly discov-
ered that anti-TfR IgG3-avidin, but not a
recombinant anti-TfR IgG3 or a nonspe-
ci±c IgG3-avidin, possesses a strong an-
tiproliferative/proapoptotic activity against
hematopoietic malignant cell lines. Stud-
ies con±rmed that anti-TfR IgG3-avidin
exists as a dimer, suggesting that cross-
linking of the surface transferrin receptor
may be responsible for the cytotoxic ac-
tivity. These ±ndings demonstrate that it
is possible to transform an antibody spe-
ci±c for a growth factor receptor that does
not exhibit inhibitory activity into a novel
drug with signi±cant intrinsic cytotoxic ac-
tivity against selected cells by fusing it
with avidin. The antitumor activity may
be enhanced by delivering biotinylated
therapeutics into cancer cells. Further de-
velopment of this technology may lead to
effective therapeutics for
in vivo
eradica-
tion of hematological malignancies and
ex
vivo
purging of cancer cells in autologous
transplantation.
4.4
Antibody Fusion Proteins
Fusion
proteins
with
nonantibody
molecules fused to antibodies can be
produced
using
different
approaches.
Antibody fusion proteins that contain
an intact antigen binding site should
retain
the
ability
to
bind
antigen,
while the attached nonantibody partner
should
be able
to
exert its
function.
Such molecules, which have been called
immunoligands
,c
a
nb
ep
r
o
d
u
c
e
di
n
several different ways (Fig. 8 a–f). When
the
nonantibody
partner
is
fused
to
the
end
of
the
C
H
3d
om
a
i
n(
C
H
3-
ligand) (Fig. 8a), the antibody-combining
speci±city can be used to deliver an
associated biological activity as well as
antibody-related
effector
functions.
An
example is the anti-TfR IgG3-avidin fusion
protein
described
in
Sect. 4.3.
Other
examples are antibodies targeting cancer
cells fused with interleukin-2 and GM-
CSF. The goal of this approach to cancer
therapy is to concentrate the cytokine in
the tumor microenvironment and, by so
doing, enhance the tumoricidal effect of
the antibody and/or the host immune
response against the tumor, while limiting
severe toxic side effects associated with
a high dose of cytokine administration.
Such antibody–cytokine fusion proteins
have shown signi±cant antitumor activity
in
mice
bearing
tumors,
leading
to
clinical trials. Immunoligands with the
nonantibody partner fused immediately
after the hinge (H-ligand) (Fig. 8b) or to the
C
H
1domain(C
H
1-ligand) (Fig. 8c) may be
useful when the antibody-related effector
functions are unnecessary or harmful.
In addition, for many applications such
as tumor targeting, the small size of
the H-ligand and C
H
1-ligand may be an
advantage over the larger C
H
3-ligand.
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