FUSION PROTEINS WITH TOXIC ACTIVITY - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 255

Apoptosis induction through TRAIL can even be accom-
panied by a secondary inhibition of proliferation if the
targeting molecule contains this functionality. This was
demonstrated with an epidermal growth factor receptor
(EGFR) blocking scFv fused to TRAIL. The synergistic
effect of downregulating EGFR signaling in combination
with apoptosis induction eliminated EGFR positive tumor
cells very efficiently. The binding of the fusion protein to the
EGFR on cell surfaces converted TRAIL from the soluble
into a membrane bound form that cross-links agonistic
TRAIL receptors to create an even stronger apoptosis effect.
This works also through a bystander effect on EGFR
negative cells [75].

Mice obtaining the DNA by intramuscular injection sup-
pressed HER2 positive tumor growth and showed a pro-
longed survival [78]. Replacing the translocation domain by
a short furin-sensitive sequence of diphtheria toxin reduced
potential immunogenicity and maintained full activity of the
fusion protein [79].
A key molecule upstream in the apoptosis pathway is the
tumor suppressor p53. Many tumors lack functional p53, so
the introduction of active p53 could restore apoptosis.
Combination of full-length p53 with a nucleus targeting
scFv fragment reached in the majority of tested cancer cells
a killing efficiency of 90% [80]. The same fusion protein
significantly reduced liver metastases in a colon cancer
model in mice in vivo [81].

17.5.4 Intrinsic Apoptosis Inducing Fusion Proteins

Besides the extrinsic pathway, apoptosis can also be medi-
ated through the intrinsic pathway that is primarily stimu-
lated by DNA damage caused by irradiation or cell
permeable drugs. A key step of intrinsic apoptosis is the
release of cytochrome C from the mitochondria. The two
molecules Bak (Bcl-2 homologous antagonist/killer) and
Bax (Bcl-2-associated X protein) act on mitochondrial
permeability by forming pores after oligomerization.
Therefore, attempts were made to introduce these proteins
into malignant cells to trigger apoptosis. In any case,
proteins that initiate intrinsic cell death must be internalized
to execute their function.
In a conceptual study, IL-2 receptor positive cells were
targeted by a chimeric protein containing IL-2 and Bax.
After internalization, the IL-2-Bax molecule is found first in
the nucleus. Then it stimulates the expression of endogenous
Bax while suppressing apoptosis inhibitors such as Bcl-2.
Human T-cell lymphoma (HUT-102) cells incubated with
IL-2-Bax were killed in a time-dependent manner [76].
Other proapoptotic members of the Bcl2 family such as
Bik (Bcl-2-interacting killer) or Bak were combined to
gonadotropin releasing hormone (GnRH) since its respective
receptor is expressed in many tumors of hormone-dependent
tissue such as the prostate. Exposing GnRH receptor positive
adenocarcinoma cells with either GnRH-Bik, -Bak, or -Bax
fusion protein killed the cells in a time- and dose-dependent
manner through intrinsic apoptosis [77].
Both extrinsic and intrinsic apoptosis pathways are con-
nected and the protein BH3-interacting domain death ago-
nist (BID) is positioned at the interface between them.
Therefore, a novel BID containing molecule was designed.
It included an antihuman epidermal growth factor receptor-2
(HER2) scFv connected via a Pseudomonas exotoxin A
derived translocation domain. Since BID is cleaved by
caspase 8 to obtain the active truncated BID, the construct
was designed to contain only the shortened carboxyterminal
domain. Cells transfected with this DNA construct secreted
the fusion protein and selectively killed HER2 positive cells.

17.6 Fc-BASED TOXICITY

To some respect, antibodies already fulfill the promise of a
“magic bullet” by being able to selectively address a mole-
cule through the variable domain and induce ADCCor
complement-dependent cytotoxcitiy (CDC) [82]. Dissecting
the antibody into these two parts, molecular targeting/
binding and Fc receptor mediated effector functions, a
new recombination with other molecules becomes possible.
On one hand, the binding arms in form of a Fab fragment
could be connected to a powerful toxin as described in the
many examples earlier. On the other hand, the ADCC/CDC
conferring Fc domain can be connected to a nonantibody
derived binding moiety. However, it has to be taken into
account that IgG1 and IgG3 immunoglobulins are the most
potent mediators of ADCC or CDC, whereas IgG4 cannot
stimulate CDC at all. This lack of cytotoxic activity in IgG4
variants could be demonstrated with OX40-Fc fusion
proteins and swapping IgG1 against IgG4 regions [83].
The importance of the Fc domain triggered approaches to
optimize effector functions by amino acid sequence adapta-
tion or specific glycosylation [84]. In Figure 17.4, all different
Fc mediated cell killing mechanisms are summarized.

17.6.1 Antibody-Derived Cellular Cytotoxicity
(ADCC)

Antibody-derived cellular cytotoxicity is particularly useful
to deplete cells with certain surface antigens. This mecha-
nism acts in three steps. First, the antibody binds to the
respective antigen on the target cell. Then NK cells with
their CD16 Fc receptor (Fc g RIIIa) bind to the CH3 domain
of antibodies on the cell surface. In the last step, the cross-
linking of the CD16 receptors leads to degranulation at a
so-called lytic synapse. The release of GrB and perforin
ultimately causes apoptosis of target cells [85].
Recently, a Fc domain double mutant S239D/I332E
was designed, which had a 40-fold higher affinity to