Biomedical Engineering Reference
In-Depth Information
single-dom ain DARPi ns, tumor accum ulation and tumor-to-
blood ratios were all the better, the highe r the affini ty was
[30]. This was les s p ronounced for larger construc ts. These
results showed that in cont rast to antibody fragme nts, the
DARPins can efficient ly be used for tumor targeting, show-
ing enhanc ed tumor penetra tion and allowing for very high
tumor loading. These are ideal prer equisites to generating
powerful-targeted drugs .
Anot her application, which is strai ghtforwar d for DAR-
Pins, is the generat ion of anta gonist drug candidat es. Indeed,
with a powerful best-in-c lass candidate VEGF -antagon istic
DARPin for the treatme nt of ophthalmi c neovascular ization
diseases, Molecular Partners is curr ently conduc ting clini cal
trials in different indicati ons in o phthalmol ogy.
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Overall, DARPi ns are versatile therape utic biol ogics
candidates with clearly favorable molecu lar prope rties, hav-
ing a wea lth of therapeu tic opportuni ties to venture [38,39 ].
be equippin g a h ormone with a bisp ecific targeting doma in
and with a moiety for half-life extension, or combining several
receptor antagonists to a multispecific superantagonist. These
are only two simple possibilities of many more possibilities.
To exploit these possibilities, a versatile, robust, and easy-to-
handle fold is ideal. DARPins fully fit these requirements and
are thus perfectly suited for this task [38,39].
34.3.2 Fusion Prote ins with Nonant ibody Binding
Proteins
The rese arch field of alter natives to antibodi es is compara bly
new. It is thus clear that only a limit ed amount of data has
been publishe d especia lly for fusion protei ns. The following
provides a brief overview on the pres ent status of publi shed
examples on fusion prot eins with alter natives to antibodi es.
The field is current ly very active, and as the technol ogies
around alter natives to antibodies mature, more and more
applicati ons are expected to be publishe d in the near fut ure.
A strengt h of DARPins and othe r scaffolds is the fact that
they can easily be fuse d head-to- tail to form bidomain or
multidom ain proteins, without com promis ing their bio-
physical or expression prope rties to a grea t extent. Several
such examples have been publishe d and are explained in the
following (Figure 34.2).
34.3 NEW THE RAPEUTI C CONCEP TS WIT H
NONANT IBODY BINDING PRO TEINS
34.3.1 Natural Fusion Prote in Exampl es
The immu noglo bulin fold is often found naturally in com-
bination with other domains or effector molecu les. Anti-
bodies are just one o f several examples of immu noglo bulin
fold fusi on protei ns. Th ey are natu rally bivale nt binding
molecules combine d with an effector funct ion. Th e varia-
tions in domain numbers and domain com position s of
human antibodi es and shar k or llam a antibodies indicate
that the fold is rather flexible. This is further underli ned by
the diversity of protei ns that cont ain varyin g numb ers of
immunogl obulin domains .
A similar flexibility is observed for repeat prot eins.
Ankyri n repeat proteins, for example, are found naturally
to exist in multivalent and multispe cific formats in a large
number of genes [29]. Typic ally, ankyrin repeat proteins are
also found as natural fusion proteins in combina tion with
various effector doma ins. Given this flexibility, they can
fulfill the diverse set of functi ons that have been reported in
the lite rature. Similar ly, leucine-r ich repeat proteins also
occur as fusion protei ns to effector moiet ies. Since protei n A
and fibrone ctin doma ins are also found in natural fusion
proteins, nature appea rs to give us a hint that many natural
folds are very flexible regardi ng fusion protei ns.
Lo oking at the natu rally occur ring com binations of bind-
ing doma ins with effector doma ins reveals the flexibility of
nature and indi cates basical ly any fusion protein to be
feasible. Th e ticket to novel therape utics, however, rather
should be sought in looking at wha t natu re has no t com-
bined. Such non explored combina tions could, for example,
34.3.2.1 Bivalent and Bispecific Molecul es If two
identical binding proteins are com bined in one binding
moiety, the resulting binder will exhibit a highe r appar ent
affinity to the target, as rebindi ng or parallel binding of two
targets may occur. In case of a homo -dimeri c target, b iva-
lency will lead to double bindi ng and thus highe r affinity. On
the othe r hand, there are vari ous reas ons to com bine two
differ ent binding doma ins in one binding moiet y. For exam-
ple, two different targets coul d be bound at the same time
using a classical bispecifi c molecu le. Taking a biparatopi c-
binding molecu le, one target could be bound at two different
epitope s, and thus highe r affinity coul d b e achieved or two
different activities of a molecule could be blocked simulta-
neously, which could be interesting for, for example, mol-
ecules that interact with receptors via distinct interfaces.
Both bispecific and bivalent DARPins made of single-
domain DARPins targeting different epitopes on the
a
chain
of the high-affinity receptor for human immunoglobulin E
(Fc
RI
a
) were generated [40]. Importantly, the multidomain
DARPins did not exhibit altered biophysical behavior com-
pared to the single-domain DARPins. By screening the
individual combinations of DARPins favorable combina-
tions could be identified, leading to inhibitors which blocked
IgE-mediated signaling (measured as inhibition of mediator
release) to completion, showing equivalent results as oma-
lizumab [40].
With single-domain DARPins, we can achieve affinities
in the low picomolar range without the need for affinity
e
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