Biomedical Engineering Reference
In-Depth Information
wide range o f different cytokines has been fused in a dimer ic
and mono meric form to Fc doma ins [36].
technol ogy is pate nted and calle d NexP fusion (http://
www.alteog en.com /).
6.2.1.3 Tr ansferrin Fus ions Tr ansferrin ( Tf) is under
the 10 most a bundant serum p roteins h av ing the task of
fa cil ita ting i ron tra nspor t fr om intestine and liver into
p r o l i f e r a t i n g c e l l s . I t s s i z e o f 7 9 k D a a l o n e p r even t s f a s t
r e m oval t h r ough kidney filtration. But i n a ddition it is
ca pable of r ec e pt o r- me di at ed re cyc ling as pa rt o f i ts
no r m al f u nc ti on a lit y. T h e o b s erve d half- life of unglycosy-
lated Tf i s 14-17 days and 7-10 days f or the glycosylated
form, respectively. The recycling of Tf through the trans-
ferrin receptor (TfR) is pH independent. The acidic pH in
the e ndosom e dissociates iron form Tf, but not Tf from its
re ce ptor [37].
Tf fusi on prot eins that address a cel l surface receptor will
bind first to the rece ptor of the fusion part ner in the presence
of abundant Tf. Thereaft er, a secon dary binding can occur
between Tf and its rece ptor. A secon dary binding coul d also
happen after endocytosis in the early endoso me. If the fusion
protein is now bound thr ough the Tf part, it retai ns bindin g to
the TfR under acidic conditions and can recycle back to the
cell surface [38] .
Th e Tf-medi ated passage through the endosome can also
be utilize d to activate protein prodrug s by proteol ysis. Th is
was dem onstrated with a proinsulin -Tf fusion that was
processe d to the act ive insu lin-Tf fusion when incu bated
with hepatocytes [39]. Other peptide s to treat diab etes such
as GLP-1 were also fused to Tf, resu lting in a more than
1000-fol d improved half-lif e [40]. However, the GLP-1-Tf
fusion was
6.2.2 Half-Life Extension Through Attachment
to Large Proteins
The superior half-life of albumin has triggered a number of
approaches to exploit this advantage for therapeutic con-
cepts. Many researchers have designed HSA-binding mol-
ecules that can be fused to other proteins.
HSA-binding peptides were identified by phage display
and reached up to 40% of the normal albumin half-life. This
indicates that the noncovalent binding released the peptide
from HSA during the incubation, which is impossible for
covalent binding or fusion. This feature is quite useful for
preparing slow release formulations of small therapeutic
proteins. Since the peptides bound also the albumin from
rodents albeit with a lower affinity, they could be used in
animal studies without substitution [46]. The same peptide
was later used in fusion to human growth hormone (hGH).
Besides the typically prolonged half-life, this study also
revealed, that the peptide at the N-terminus was more active
than at the C-terminus [47]. Other combinations of this
peptide included antibody fragments against different cancer
cell epitopes. The resulting half-life of the molecules was
dependent on the affinity of the peptide to albumin; the
stronger the affinity, the longer the half-life [48]. Using
antibody fragments fused to the HSA-binding peptide, a
much better tumor penetration could be achieved compared
to the full-length antibody. This effect is caused by the
smaller size of the Fab on one hand, but also by the tumor
accumulation of albumin on the other hand [49].
Besides the standard linear peptides also a wide range of
affinity scaffolds has been selected to target albumin. The
obvious first choice was an antibody Fab fragment binding to
albumin. The half-life of a bispecific Fab with albumin
binding capability was increased eightfold compared to
another bispecific Fab without albumin specificity [50].
Another variation of this theme is a HSA-targeting domain
antibody (AlbudAb). In the first example, fusing the inter-
leukin-1 receptor antagonist (IL-1Ra) to AlbudAb, the half-
life was extended 100-fold. This was lower than what could
be achieved with the AlbudAb alone, and results probably
from losses during recycling through the endosome [51].
Another example connecting the same AlbudAb with inter-
feron
a
2b (IFN-
a
2b) prolonged the circulation time almost
20-fold [52].
Of course, other novel scaffolds such as Affibodies
1
have
been screened for albumin binding abilities as well. The
selected molecule consisting of 46 amino acids only, had a
subnanomolar affinity to albumin and the complex between
the Affibody
1
and albumin had a half-life of several days
[53]. The same molecule could be further engineered at the
side that does not bind to albumin to obtain specificity for
recently withd rawn
from clini cal
stud ies,
because it increas ed puls e and blood pressure [41] .
Be sides the positive effects on half-life of fusion prot eins,
Tf has anot her important feature. Iron is essential for
neuronal functi on, but has to be kept at a specific equilibriu m
to avoid dangerous side effects. The home ostasis is regulated
through iron transporte rs such as the TfR at the blood brain
barrier (BBB) between blood and cerebros pinal. There fore,
Tf can be used as vehicle to transfer cargo acro ss the BBB.
Early examples compri sed the fusion of nerve growth factor
(NGF) to Tf [42]. Anot her appro ach focused on the treat-
ment of brain tumors with Tf toxin fusion protei ns [43]. The
unique featur es of Tf have also been utilize d in gene therapy.
A genetic construc t coding for a Tf
a
-
L
-iduronidas e (ID)
fusion to treat Hurler syndr ome was injected into mice. The
Tf-ID protein could be detected in the brain of the trans-
fected animals and reduced cerebella glycosaminoglycans
storage in MPS I mice [44]. The specific case of Tf fused to
the acetylcholine receptor to treat myasthenia gravis is
presented in Chapter 12.
The success with abundant serum proteins brought
a
1
antitrypsin (
a
1AT) into the focus as fusion partner to
increase half-life. A Fab fragment targeting CD28 fused
to
a
1AT extended blood circulation twofold [45]. This
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