Biomedical Engineering Reference
In-Depth Information
insect Triatoma infestans, the main Chagas' disease vector
in Brazil [83]. One inhibitory domain of infestin, infestin-4,
has been cloned, expressed and purified, showing remark-
able inhibitory activity toward the activated human coagu-
lation factor XII [84]. However, infestin-4 has a very short
half-life, reducing its efficacy and bioavailability. As an
approach to prolong its half-life, infestin-4 has recently been
fused to rHA and analyzed in vitro [42,85]. The resulting
protein, rHA-infestin-4, was created by inserting a flexible
(GGS)
3
linker between rHA and infestin-4. The fusion
protein was expressed in HEK-293 cells. rHA-infestin-4
demonstrated a prolonged half-life compared with that of
(His)
6
-tagged infestin-4 in rats (4.6 h vs. 0.3 h). In addition,
the fusion protein inhibited FXIIa and prolonged the acti-
vated partial thromboplastin time in human, mouse, and rat
plasma. In mouse and rat models of pathological arterial
thrombosis, rHA-infestin-4 prevented the formation of arte-
rial thrombosis while leaving hemostasis (tail-bleeding
times) fully intact. In addition, rHA-infestin-4 moderately
inhibited fibrinolysis, and was highly protective in a mouse
model of ischemic stroke [42].
to that of T
a
1, rHA-T
a
1, and rHA-L-T
a
1 demonstrated
longer half-lives of 32-36 h following subcutaneous or
intravenous administration.
10.3.2.14 Antibody Fragments
An increasing number of
small recombinant antibodies and their fragments are being
developed and investigated for clinical applications, such as
for the treatment of cancer or inflammatory diseases [4].
However, as with many proteins with low molecular weight,
the half-life of antibody fragments is very short, hampering
their therapeutic efficacy. The use of recombinant albumin
fusion technology is a strategy, that is proving to be suc-
cessful in prolonging the half-life of several antibody frag-
ments [4,19,32]. For example, fusion of albumin to the
single-chain variable fragment (scFv), tandem scFv mole-
cules (taFv), and single-chain diabodies (scDb) prolonged
the half-lives of the fragments by 2-10-fold compared to the
unfused antibody fragments [19,32].
10.4 SUCCESSES AND FAILURES IN
PRECLINICAL AND CLINICAL RESEARCH
10.3.2.12 Thioredoxin
Thioredoxin (Trx) is a small
redox-active protein with anti-inflammatory effects [45].
Several reports have indicated protective effects of this
protein against various pathological conditions. However,
its short half-life of around 1 h necessitates multiple admin-
istrations or a constant infusion rate to maintain the required
dose. In an attempt to extend its half-life, Ikuta et al. [45]
created a thioredoxin recombinant human albumin fusion
protein (Trx-rHA) using the P. pastoris expression system
and examined its characteristics. The half-life of Trx-rHA
was prolonged to 10.3 h, a 10-fold increase over the unfused
thioredoxin. However, in an insulin-reducing assay, the
activity of the fusion protein was only 60% of that of
thioredoxin, despite a 10 amino acid linker. Trx-rHA was
also highly protective in a mouse model of endotoxic shock;
survival rate was twice as high with Trx-rHA compared with
thioredoxin.
The development of recombinant albumin fusion proteins
has produced successes as well as failures that have con-
tributed to our understanding of fusion proteins in general.
The recombinant albumin fusion proteins currently in devel-
opment were often selected from multiple variants because
they had the most favorable pharmacokinetics and biological
activity. Other areas of potential improvement, such as
immunogenicity and stability, have led to further modifica-
tions as the optimization of fusion protein technology
remains an ongoing process.
10.4.1 Interferon-a
Alb-IFN is in late-stage development for the treatment of
CHC, but it tends to dimerize and form covalent aggrega-
tions in aqueous solution [86]. The observed heterogeneity
and instability is thought to be caused by incomplete
disulfide bridges between Cys1 and Cys98 of the IFN
moiety. These characteristics have led to a low recovery
rate (10%) and necessitated lyophilization. To restore
disulfide pairing and improve the stability of alb-IFN,
researchers created a unique protein by switching the orien-
tation of the two proteins, placing IFN-
a
-2b at the
N-terminus [86]. Compared to alb-IFN, the new protein
(IFN-
a
-2b-rHA) was relatively more homogenous (i.e., it
migrated as a single band in SDS-PAGE) and was easier to
purify and more stable (i.e., recovery was increased 2.5-
fold). The antiviral activity of IFN-
a
-2b-rHA in vitro was
increased compared to IFN-
a
-2b and alb-IFN.
Unfortunately, the new fusion protein was also suscepti-
ble to forming disulfide-linked aggregates in response to
10.3.2.13 Thymosin-a1
Thymosin-
a
1(T
a
1) is a natu-
rally occurring thymic peptide with immunomodulatory
functions [18,46]. It is indicated for the treatment of certain
viral infections, immunodeficiencies, and malignancies.
Unfortunately, the very short half-life of T
a
1of
2 h means
that frequent injections are necessary to maintain the thera-
peutic effect. Recombinant albumin fusion technology has
recently been employed to prolong the half-life and efficacy
of the peptide [18]. Two fusion proteins, expressed in P.
pastoris, were created (rHA-T
a
1 and rHA-L-T
a
1): both
were composed of rHA fused at its C-terminus to the N-
terminus of recombinant T
a
1, but one included a linker
peptide (GGGGS) joining rHA and T
a
1 (rHA-L-T
a
1).
While the bioactivity of the two fusion proteins was similar
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