Biomedical Engineering Reference
In-Depth Information
antibodies [15]. One particularly interesting approach is the
removal of A
b
amyloid fibrils through a specific single-
chain antibody fragment (scFv) connected to the IR anti-
body. This fusion protein reaches the brain through IR-
mediated import. In the brain, the scFv binds to the amyloid
plaque, disintegrating the fibrils. A
b
molecules captured by
the antibody are then exported via a neonatal Fc receptor
(FcRn) mechanism. This asymmetric reverse transcytosis of
antibody molecules reduces the A
b
concentration in the
brain significantly [16].
Although antibodies against the TfR have proven to be
less efficient for RMT, a new approach was launched quite
recently. Here the affinity of the antibody against the TfR
was modulated, and it was demonstrated that lower affinity
anti-TfR antibody variants reach higher brain concentra-
tions. The underlying mechanism is that high affinity anti-
bodies remain bound to the TfR at the BBB, whereas low
affinity ones are released and can penetrate the brain. The
selected antibody was configured as a bispecifc molecule
whose other arm binds and inhibits
b
-secretase (BACE1).
This enzyme digests the amyloid protein into the A
b
peptides causing Alzheimer's disease (AD). The new mole-
cule could be a novel therapeutic for AD [17].
Another mechanism for BBB transport involves the low-
density lipoprotein receptor-related protein (LRP). In a
transcytosis process, LRP shuttles other proteins containing
a Kunitz protease inhibitor (KPI) domain across the BBB.
Initially, this concept was validated by demonstrating the
transfer of aprotinin, which contains a KPI, to the brain.
The transport was 10 times more efficient than transferrin
uptake into the brain. By a bioinformatic approach, further
natural peptide sequences were identified sharing homology
to the C-terminal region of aprotinin. Some were further
optimized by site-directed mutagenesis and had up to
sevenfold better efficacy in transcytosis than the original
aprotinin [18]. The involvement of LRP suggests a receptor-
mediated transport mechanism through the low-density
lipoprotein (LDL) receptor. So far, no fusion proteins using
these peptide sequences have been reported, but this seems
to be a logical next step.
Recently, an Apolipoprotein A1 and interferon
a
(ApoA1-IFN
a
) fusion protein was described that is capable
to cross the BBB in a saturable manner involving a receptor
[19]. Therefore, ApoA1 might prove to be a valuable addi-
tion to the arsenal of BBB crossing protein motifs.
Besides the RMT over the BBB, other strategies to target
the brain rely on viral or bacterial proteins that are involved
in infections of the central nervous system. For example, the
neurotropic virus coat glycoprotein of rabies virus can be
split into peptides. One such 43 amino acid containing
peptide (RDP) is sufficient for brain targeting. The mecha-
nism utilizes retrograde axonal transport via the peripheral
nerves into the brain. To demonstrate the ability of brain
targeting, a fusion protein comprising
b
-galactosidase
(
b
-Gal) and the RDP peptide was generated. After 1 h after
i.v. injection, enzymatic activity could be detected in the
hypocampus of mice [20]. A shorter peptide from rabies
virus coat glycoprotein with only 39 residues was tested with
b
-Gal, luciferase, and brain-derived neurotrophic factor
(BDNF) in a similar setting. The uptake into neuronal cells
was very fast and the BDNF fusion protein protected nerves
in a stroke animal model [21].
Another class of neuronal targeting proteins can be
derived from tetanus toxin C (TTC). The full length Tetanus
neurotoxin has a high specificity for the synapses of motor
neurons. Similarly, to RDP, the Tetanus toxin is carried to the
central nervous system by retrograde axonal transport. TTC
represents the carboxy-terminal domain of the post-transla-
tionally processed heavy chain. TTC is nontoxic and the key
domain required for internalization of the toxin. A wide
range of large molecules has been fused to TTC and injected
into animals [22]. Several studies were undertaken with a
glial-derived neurotrophic factor (GDNF)-TTC fusion.
GDNF-TTC was transported fast to the central nervous
system of mice after intramuscular injection [23]. A similar
GDNF-containing construct increased survival of mice
being symptomatic for amyotrophic lateral sclerosis
(ALS) by 9 days when administered intramuscularly [24].
For the sake of completion, it should also be mentioned
that cell-penetrating peptides such as TAT are also able to
cross BBB. This was demonstrated with a number of fusion
partners, the earliest being
b
-Gal [25]. A more recent
example is GDNF that was used to protect mice from
ischemic stroke [26]. Further details on CPP can be seen
in Section 25.3.2 and Chapter 26.
25.2.2 Liver
The liver as the major organ for metabolism is involved in a
number of diseases and is the target for a wide range of
drugs. However, so far, only limited success could be
reported for targeted delivery of large biological therapeu-
tics to the liver. Frequently, specific receptors of glycopro-
teins such as the asialoglycoprotein receptor (ASGP-R) are
used as a means to address this organ [27]. Other approaches
utilize cyclic peptides containing an Arg-Gly-Asp (RGD)
motif that binds to the collagen type VI receptor on hepato-
cytes [28]. The highly upregulated platelet-derived growth
factor
b
receptor (PDGF
b
R) in cystic fibrosis represents
another molecule for targeted intervention. Cyclic peptides
with a high affinity to PDGF
b
R were coupled to albumin
and interferon-
g
. These conjugates were specifically taken
up by hepatic stellate cells (HSC) in an acute liver injury
mouse model and improved vital functions in liver [29].
Despite the increasing understanding of binding motifs,
none of these strategies has led to the design of liver-specific
fusion proteins yet.
Interferon-
a
(IFN-
a
) that is applied to treat chronic viral
hepatitis is one of the therapeutic proteins that would benefit
from liver targeting. Recently, a newmolecule was described
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