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S. R. Schmidt
factors or the ribosome. Ribonucleic acid (RNA) is the key component for the translation process involved either as messenger RNA (mRNA) or as amino acid carrying transfer RNA (tRNA). This highly sensitive molecule can be easily damaged by
RNA degrading enzymes, the RNases. So in principle targeted RNases, the socalled ImmunoRNases can act just like toxins [166]. However, a limiting factor to
the efcacy of RNAses is the presence of RNase inhibitor (RI) in the cytosol that
protects RNA from enzymatic destruction. Therefore, besides human pancreatic
RNase, angiogenin (ANG) or amphibian RNases, as, for instance, Onconase®
(ONC), that is insensitive to RI, have been utilized. Alternatively, mutations can be
introduced that abolish the inhibition by RI or multimerization of RNases can prevent RI blockade simply by steric hindrance. A further hurdle to overcome is the
difculty of design as RNases either need a free N- or C-terminus for activity. ONC
must be preferably fused to its C-terminus, while ANG can be connected to its
N-terminus as the opposite end contains the active center. Efcacy can be further
improved by designing a fusion protein based on a diabody that could bear two
ANG moieties [167]. This type of construct can help to counteract the depletion of
the target antigen on the cell surface. It could be demonstrated that a traditional
(G4S)3 linker combining an anti-CD22 scFv with ANG increased the activity, as it
probably improved folding when expressed and secreted from mammalian cells
[168]. In general, it seems that the folding apparatus of mammalian cells can better
cope with the complexity of RNase fusion proteins as microbial expression systems.
But nevertheless, production cost and production scale still represent a limitation to
the clinical application of ImmunoRNases. Additionally, as RNA is an intracellular
target, internalizing surface markers must be selected for the targeting fusion partner to be efciently translocated across the cell membrane. Interestingly ONC does
not require a targeting moiety, as it binds to tumor cells through a still unidentied
receptor which is the starting point in the routing of ONC to the cytosol via the
Golgi apparatus. Therefore, ONC can also be used as fusion partner to afnity binders against noninternalizing to increase the local concentration of ONC on cancer
cells. The proper pathway into the cytosol is another important prerequisite for the
cytotoxic activity as the default transfer to endosome results in much lower activity.
To avoid potential immunogenicity of amphibian RNases, human RNases are being
used more frequently nowadays.
Optimizing cell killing potency through increasing the catalytic activity or resisting inhibition while reducing collateral toxicity are the key parameters for therapeutic success. Although ImmunoRNases are a research topic for more than 20years,
none of the proposed molecules has reached clinical trials yet [169].
Cellular proliferation is regulated through signal transduction cascades relying
on phosphorylation. Aberrant phosphorylation often causes uncontrolled cell expansion; therefore, an obvious countermeasure to trigger self-destruction or to halt proliferation would be to introduce kinases. Ideally a kinase is chosen that can trigger
death signals, as, for instance, death-associated protein kinases (DAPK). Within
cancer cells DAPK is often silenced; therefore, the restoration of its activity could
initiate cell death. Pairing a constitutively highly active DAPK mutant at its
C-terminus with a scFv targeting CD22 a so-called immunokinase could be created.
11 Fusion Proteins: Current Status andFuture Perspectives
331
When internalized in malignant B-cells, this fusion enzyme successfully caused
apoptosis and autophagy. To avoid any negative effect on the HEK293T cells that
were used for the production, a strong secretion signal was used that immediately
pushed the fusion protein to the supernatant of the cell culture. The immunokinase
showed efcacy on malignant B-cells isolated from CLL patients [170].
Another natural factor that is used by immune cells to specically destroy cancer
cells is Granzyme B (GrB) which is released from cytoplasmic granules of natural
killer (NK) cells and cytotoxic T lymphocytes (CTLs). It enters cells with the help
of co-secreted pore forming proteins, the so-called perforins. The dependency on
perforin to reach the cytosol is probably the major hurdle to overcome, as new ways
to facilitate endosomal escape must be established. This could be achieved by incorporating the translocation domain of bacterial toxins. Granzyme B is a protease that
activates multiple caspase family members by proteolytic cleavage, thus inducing
apoptosis. Unfortunately, GrB contains several positively charged amino acids that
could lead to unspecic accumulation on any negatively charged cell surface.
Furthermore, GrB activity could be inhibited by a protease inhibitor (PI-9) that can
be produced by cancer cells to evade destruction. Therefore, mutant variants of GrB
resistant to PI-9 inhibition and without the basic amino acids were developed and
fused to anticancer scFv molecules, creating immunoproteases or cytolytic fusion
proteins (CFP). A further modication that allows cancer cell specic activation and
thus reducing unspecic side effects is the replacement of the N-terminal Gly-Glu
dipeptide by a sequence recognized by proteases highly expressed in cancer
cells [171].
Alternatively, to using GrB as activator of caspases, these proteases themselves
can be used directly to initiate apoptosis. Several attempts have been made with
Caspase 3 or 6, and the most advanced molecule is a fusion of an anti-HER2 scFv
connected to a constitutively active caspase 3 via the translocation domain of a bacterial toxin [172].
11.5.3.5 Apoptosis Induction
Instead of utilizing surface markers to identify cancer cells and then translocate
apoptosis inducing agents like proteases or RNases, the natural function of death
receptors (DR) that belong to the tumor necrosis factor (TNF) receptor superfamily
can be utilized. The DR transduce an extracellular signal triggered by binding a corresponding ligand through their cytoplasmic death domain (DD) into the cytosol.
Suitable ligands are TNF, apoptosis stimulating fragment ligand (FasL), and the
tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) which all induce
receptor oligomerization. Initially these ligands were utilized in their natural conformation, but due to short half-life and high unspecic activity most clinical programs were abandoned. In the meantime, fusion proteins that, on the one hand,
target the ligands to cancer cells while enabling multimerization of receptors have
become the molecules of choice. The rst progress was to design trimeric single
chain ligands that are connected through short linkers in a linear fashion which also
332
S. R. Schmidt
simplied their manufacturing. Then either a scFv or full IgG was fused to enable a
targeting function. In the case of TNF, it was fused to an anti-L19 antibody and
tested in clinical trials in melanoma [173]. Molecules harboring FasL showed promising results in preclinical studies when fused to scFv and targeting CD40, CD20,
CD7, or TAG72, but did not yet reach clinical studies. FasL fusion proteins require
the ligand positioned at the N-terminus. Most data exist for TRAIL fusion proteins
that exert their activity by binding to DR4 and DR5. Again the typical fusion partners are scFvs, addressing, for instance, EGFR, ErbB2, CD40, CD27, or other
cancer- specic antigens. A hexameric single chain TRAIL variant (ABBV-621)
resulting from a fusion with the Fc domain of IgG1 with improved clustering of
TRAIL receptors is currently in clinical phase 1 trials against solid tumors and
hematologic malignancies [174].
As in all cases the apoptotic activity is induced through receptor clustering, the
crosslinking can be further enhanced by using full antibodies instead of scFv. This
can either happen through binding on the same cell (autocrine activation) or through
neighboring cells (paracrine activation) Sometimes even a bystander effect could be
observed, killing cells that are not presenting the corresponding antigen. This observation could on the one hand be benecial for heterogeneous tumors, but might be
a safety concern on the other hand. A further safety control can be establishing by
so-called prodrugs that shield the active ligand through receptor fragments included
in the molecule but being able to be released through the cleavage by tumor associated protease. Additional modications can be introduced through modifying afnity and selectivity to the respective receptors through mutations in the ligands [175].
As alternative to the extrinsic pathway also an intrinsic pathway can be utilized
to trigger apoptosis. The two molecules Bak (Bcl-2 homologous antagonist/killer)
and Bax (Bcl-2–associated X protein) destroy mitochondria by forming pores, thus
destroying cells. Therefore, it was attempted to introduce Bak or Bax into malignant
cells. One example is the fusion of Bax to endostatin (ES) that targets endothelia
cells and supports translocation into the cytoplasm. The combination of ES and Bax
is more powerful in destructing cancer cells invivo and invitro compared to ES
alone [176]. Both extrinsic and intrinsic apoptosis pathways are interlinked, and the
protein BH3-interacting domain death agonist (BID) is positioned at the interface
between them. A molecule consisting of an anti-HER2 scFv connected by a minimal endosomal translocation sequence derived from Pseudomonas containing a
furin cleavage site and truncated BID resulted in efcient cell killing of HER2 positive cells [177]. Figure11.12 shows how the different pathways can interact to
induce apoptosis.
11.6 Summary
Currently there are22 original fusion proteins approved and additionally numerous
biosimilar variants have reached the market. Most frequently, the Fc-domain of IgG
has been utilized as the fusion partner of choice. Nevertheless, there are in principle
11 Fusion Proteins: Current Status andFuture Perspectives
Fig. 11.12 Apoptosis induction as utilized by some fusion proteins. Binding of death receptor
(DR) ligands such as tumor necrosis factor (TNF), TRAIL, or FasL, trigger the generation of a
death-inducing signaling complex (DISC) at their death domains (DD) that interact with FASassociated death-domain (FADD) and activate caspase-8. This protease cleaves BID to form tBID
which connects the extrinsic with the intrinsic pathway. tBID or the p53-induced oligomerization
of Bax and Bak causes cytochrome C (CytC) release which triggers ultimately the activation of
caspase-9. This protease links the intrinsic with the extrinsic pathway by activating caspase-3 that
degrades ICAD to release CAD which in turn causes cell death by DNA fragmentation. Several
important molecules such as BID, caspase-3, and ICAD can also be activated by granzyme B
(GrB) that can enter the cell during inammation through perforin pores
333
endless choices to design fusion proteins from natural building blocks. Some potential limitations of a widespread use are the still not completely understood mechanisms of aggregation and the risk of immunogenicity due to unnatural combinations
or novel epitopes. Manufacturing of fusion proteins is benetting from the huge
body of knowledge generated from the production of antibodies but still has its
unique challenges such as low expression levels. Fusion proteins are often designed
to solve some shortcomings of their unfused building blocks. For instance, many
fusion proteins address the issue of short half-life or low bioavailability due to lack
of targeting. Particularly in the context of oncology there is a wide range of therapeutic concepts trying to eliminate malignant cells by different means. Despite the
various approaches, currently immunotoxins, representing the “magic bullet” idea,
are the only examples that have reached approval.
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11.7 Future Perspectives
Fusion proteins represent a highly successful but heterogeneous class of recombinant therapeutics. A lot of progress has been achieved in the last twenty years. Some
modules like Fc or HSA are nowadays well established and are simple tools to
generate biobetters. But every year a vast range of new variants is created. In parallel to the positive development, other modalities have been catching up. Particularly
interesting is the boom of bi- or multispecic antibodies and antibody drug conjugates that partly resemble or even rene the concept of fusion proteins. They share
the complexity of manufacturing and require much attendance during development
but give new and better treatment options. In parallel the emerging approaches in
cell therapy and gene therapy must be considered. These drugs promise complete
healing. Currently not all effects are fully understood and the manufacturing process is quite costly, but it will be an exciting experience to watch the race of multiple
molecule classes side by side to offer new treatment concepts for a wide range of
patients.
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