Biomedical Engineering Reference
In-Depth Information
the virus to cells expressing the target molecule. This
concept is even more interesting, as the ErbB2-targetting
DARPin could in principle be exchanged by any other
targeting DARPin, thus generating targeted adenovirus at
will. Importantly, the constructs exhibited no substantial
decrease in expression properties and also the general
biophysical behavior was comparable to the one of a sin-
gle-domain DARPin [44]. A projecting calculation in that
report estimated the amount of a 1-L shake-flask culture to
be sufficient to fully supply a midscale Phase I gene therapy
trial with this system.
Multispecific binding proteins are certainly very promis-
ing beyond bispecific or individual binding proteins. Similar
to this, a multitude of functionalities can be combined.
Half-life engineering domains can be combined with two
or three targeting domains and one or several disease path-
ways can be tackled simultaneously.
could be expressed in soluble form in the cytoplasm of
E. coli allowing for simple production and purification,
enabling fast evaluation of this novel combination.
Similarly, we have generated cytokine and interleukin
fusions to DARPins. The favorable DARPin properties
enabled high-level expression (Binz H.K., unpublished
results). For example, TNF-
a
-fusions could be expressed
in soluble form at levels of more than 200-mg/mL shake-
flask culture with an ordinary E. coli XL1-blue strain in
LB/Glucose medium. Purification was thus straightforward.
In case of interleukin-2 (IL-2), the expression level was even
higher and the protein was expressed quantitatively in
inclusion bodies, which enabled to reach high purity of
fully active protein in a single refolding step. The fusion
proteins are typically fully active independent of the pres-
ence of the DARPin. Thus again, the platform of a nonanti-
body binding protein is offering a rapid way to assess the
therapeutic possibilities of effector fusion proteins.
34.3.2.3 Critical Advantage of Multispecific Nonanti-
body Binding Proteins Bispecific and multispecific
approaches are especially interesting as they may open
new therapeutic modalities. This has very well been recog-
nized by antibody companies and strong efforts are put in
this area of research (see also other chapters in this topic).
The critical advantage of nonantibody binding molecules
over antibodies is their flexibility in genetic engineering,
ease in production, and high protein stability, making
research fast-forward. Such approaches are more difficult
to tackle with antibody platforms, as they might require
elaborate and extremely streamlined cloning and production
processes to work efficiently. Furthermore, protein propert-
ies of the final products will be very different, and especially
protein stability might hamper the use of the resulting
products in the case of antibody constructs. It is thus to
be expected that multispecific nonantibody binding protein
do have a critical advantage and will allow to rapidly assess
the possibilities in multitargeting, both regarding efficacy
and toxicology.
34.4 SCAFFOLD-FUSION PROTEINS BEYOND
ANTIBODY POSSIBILITIES
With the limited background of antibody research, we
are just about to explore the vast possibilities of fusion
proteins to scaffolds. Currently, the scaffold-fusion protein
approaches are very close to the antibody approaches. It is
just a question of time until novel approaches will appear.
One important advantage of scaffolds such as the DARPins
is their flexibility regarding multispecificity and formatting,
allowing basically generating any type of fusion protein with
comparably little effort. Similar to this, novel combinations
of several antagonists or targeting molecules with several
agonists might become feasible, which were either not
feasible with immunoglobulin domains, or which were
only achievable with major investments of both time and
money in production optimization. Here, an important step
will be to not only look at cancer treatment but also apply the
multifunctionality to other disease types.
Another important advantage of scaffolds such as
DARPins is their robustness and chemical flexibility. This
should facilitate chemical modification in a directed and
much more controlled way than was possible for immuno-
globulins. Depending on the fusion protein, one could thus
add chemical modifications on top to create multifunctional
molecules. On the other hand, stable scaffold-fusion proteins
could be tested for novel routes of administration or pene-
tration of, for example, the blood-brain barrier. The flexi-
bility of the scaffolds should allow testing more approaches
than could be tested with antibodies, increasing the chances
for success dramatically.
Alternatives to antibodies as such will undoubtedly lead
to a number of innovative therapeutics, enabling improved
treatments of diseases or tackling unmet clinical needs.
34.3.2.4 Combining Scaffolds with Other Effector
Proteins Instead of combining two or more targeting
moieties, the combination of a targeting moiety with inno-
vative effector moieties is alluring. For antibodies, many
different possibilities of fusion proteins have been explored
to generate novel therapeutics approaches. Fusions to inter-
leukins, interferons, cytokines, Pseudomonas exotoxin A
(ETA) [45], RNase, and other have been reported [46]. Often
these combinations were used for tumor therapy and good
preclinical results have been achieved. Importantly, several
of these constructs are indeed in clinical development.
In case of DARPins, ETA-fusions have been generated,
which could efficiently be used for the treatment of cancer in
tumor xenograft models in mouse, similarly to what has been
shown with antibodies [45,47]. Importantly, the proteins
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