Biomedical Engineering Reference
In-Depth Information
specificities can be generated. The outcome of this process
and the analysis of the resulting binding molecules can help
to refine a library design, and thus improve the process of
generating binding molecules. Generating binding biologics
from scratch using computer methods is currently not pos-
sible, as these methods are limited by the high degree of
variability of biologics. Certain scaffolds, such as ankyrin
repeat proteins, show binding sites on very rigid secondary
structures. This should reduce the number of variables
dramatically and actually might be the most attractive target
for computer-designed binding proteins [25].
The following paragraph describes the process of tailor-
ing nonantibody binding molecules using DARPins as a
successful example to illustrate how to combine database
knowledge, structural knowledge, published know-how,
experimental tests, protein engineering, and library methods
to yield one of the best platform technologies for the
generation of binding molecules.
of highly stable molecules [18]. With increasing repeat
numbers, the molecules get more and more stable, and
very often, a four-repeat protein has a midpoint of thermal
denaturation close to 100
C, and the analysis of larger
constructs requires the addition of chemical denaturants
to determine a midpoint of thermal unfolding. This high
stability could be explained in part by a highly regular repeat
fold, where the repeats stack perfectly to form an elongated
repeat domain with a continuous and compact hydrophobic
core in combination with a regular pattern of H-bonds [18].
A further important feature of DARPins is the absence
cysteines in the fold, enabling specific introduction of
cysteines at designated positions at will, allowing for easy
chemical modification of the protein, including coupling of
toxins or PEG-like molecules [30]. Also, DARPins are not
glycosylated, which clearly facilitates production.
The analysis of the target interactions of natural ankyrin
repeat proteins permitted the generation of libraries of
DARPins [4]. We generated libraries of four or five repeats,
with theoretical diversities of 5.2
10
23
,
respectively. Using ribosome display or phage display,
sampling between 10
10
and 10
12
variants, we could select
single digit nanomolar to midpicomolar affinity binders to
various targets just by doing regular selection rounds [11].
When increasing the selection pressure by, for example, off-
rate selection, we could, still without any affinity maturation
or additional randomization, select binders in the low pico-
molar range. Importantly, the selected DARPins exhibited
high thermal stabilities and showed favorable expression
properties equivalent to the nonselected DARPins, indicat-
ing that the DARPin scaffold is able to sustain a large
number of modifications [11,31]. The regular ankyrin repeat
fold is maintained in all selected DARPins whose structure
has been determined so far [32].
The versatility of the DARPin platform has been proven
at various instances. DARPins have been used as co-
crystallizing agents [32], where their properties of high
expression yield, ease of purification and rigid structure
come into play very favorably. As diagnostic tools, the
high specificity and affinity of DARPins is ideal, allowing
for very sensitive assays [33]. DARPins have also been
selected to act as intracellular kinase inhibitors [34], drug
transporter (membrane protein) inhibitors [35], or enzyme
inhibitors [36], whereby it became obvious that DARPins
can be transformed to various types of inhibitors depending
on the selection pressure, including competitive or allosteric
inhibitors. Importantly, DARPins have all the favorable
properties you would expect for a therapeutic biologic agent.
Like for antibodies, DARPins can also be used to target
tumor antigens. For example, high-affinity DARPins have
been generated against the tumor targets EGFR [37] and
ErbB2 [31]. By mutating a picomolar-affinity ErbB2-bind-
ing DARPins to variants of varying affinity and of varying
size, we could show that for very small molecules such as
10
15
and 3.8
34.2.4 DARPins—Designed Ankyrin Repeat Proteins
For various reasons, repeat proteins are attractive scaffolds
for the generation of alternatives to antibodies [20]. They are
among the most frequent natural protein-protein interaction
mediators across all phyla and are found in human at high
frequency. Nature uses them in a similar fashion as anti-
bodies, applying them in innate and adaptive immune
systems, and involving them to inhibiting other proteins.
Ankyrin repeat proteins are one prominent type of repeat
proteins. They are involved in various cell-regulation mech-
anisms such as transcription factor inhibitors or cell signal-
ing pathways [26] and are also important structural elements
for cell integrity [27]. Interestingly, they also act as key
molecules for information management, for example, in our
hearing system [28]. An important feature of ankyrin repeat
proteins is their occurrence as natural multivalent or multi-
specific proteins and very often, ankyrin repeat proteins are
combined with other effector molecules combining different
functionalities to a diverse set of possibilities [29]. In that
respect, ankyrin repeat proteins are an expanded variant of
antibodies. Importantly, they interact with their targets with
high affinities and specificities, just as antibodies.
Using a consensus-design approach, we have generated
designed ankyrin repeat proteins (DARPins) of varying
repeat numbers (Figure 34.1) [4]. The design process
involved sequence database analyses, structural analyses,
computational design, as well as the use of experimental
data [4]. Important features of the DARPins are the capping
repeats and the consensus-designed repeat module. The
design process had an important impact on the properties
of DARPins. They can be expressed in soluble form in the
cytoplasm of Escherichia coli at high levels, allowing for
straightforward purification. The ankyrin repeat fold in
combination with consensus design permitted the generation
Search WWH ::
Custom Search