Biomedical Engineering Reference
In-Depth Information
construct-contained SAK at the N-terminus linked by a
factor Xa (FXa) sensitive spacer to HIR. This fusion protein
combines three features: presence of active FXa in proximity
to a thrombus, attenuation of HIR activity by an N-terminal
extension, and therefore low systemic anticoagulant activity
[46]. A variant of this molecule was equipped with a
thrombin-sensitive spacer replacing the FXa recognition
domain. This fusion protein benefits from the high concen-
tration of thrombin at blood clots and the ability of free HIR
to inhibit thrombin. So, the release of HIR is efficiently
regulated by the amount of thrombin present. This fusion
protein demonstrated an improved thrombolytic effect and a
lower bleeding risk in animal studies [47].
complicated process was established to artificially modify
the glycosylation of glucocerebrosidase (GCD), the enzyme
lacking in Gaucher's disease, to obtain terminal mannose
residues to facilitate enzyme internalization. Alternatively,
highmannose glycosylation could be achieved by changing to
a plant system as expression host and forcing the trafficking of
the recombinant enzyme to the storage vacuole where the
mannosylation is generated. This requires the fusion of GCD
with a C-terminal targeting and retention signal encoding the
sequence DLLVDTM [49]. It could be demonstrated that
macrophages frompatients with Gaucher's disease efficiently
internalized this GCD fusion protein and maintained its
functionality in the cells. Currently, this protein has completed
Phase III trials and awaits marketing approval [50].
An alternative entry point for enzyme replacement ther-
apy (ERT) is the low-density lipoprotein receptor (LDLR)
family. Bound ligands of these receptors are delivered
directly to the lysosome. During the expression, LDLRs
bind the receptor-associated protein (RAP) to prevent any
association with their normal ligands during their passage
through the secretory pathway toward the cell surface. The
high affinity of RAP and the absence of RAP in the plasma
can be exploited as a tool to mediate specific protein uptake
via the LDLR to the lysosome. For this purpose, RAP was
fused to the N-terminus of
a
-
L
-iduronidase (IDU) and acid
a
-glucosidase (GAA), the enzymes missing in Hurler/
Scheie syndrome and in Pompe syndrome, respectively.
For the production of these constructs, mutant Chinese
hamster ovary (CHO) cell lines had to be used to prevent
association of RAP to endogenous LDLR already during the
expression. Both RAP-GAA and IDU fusions were 70-fold
and 43-fold better internalized by primary fibroblasts from
Pompe and Hurler disease patients than the enzymes without
RAP fusion. Both fusion constructs had a high stability in
serum, but were processed in the lysosome, which resulted
in cleavage from the RAP domain. The much higher endo-
cytosis capacity of the LDLR system is certainly an advan-
tage compared to mannose receptors. Owing to the trans-
epithelial trafficking ability of some LDLR, the RAP fusion
system could even enable ERT over the BBB [51].
Despite the success with mannose 6-phosphate (Man6-P)
glycosylated enzymes, this approach suffers sometimes
from manufacturing issues or rapid clearance from the
bloodstream by cross-reactive receptors. Therefore, another
approach for ERT was established, the glycosylation-inde-
pendent lysosomal targeting (GILT). It is based on the ability
of insulin-like growth factor 2 (IGF-2) to bind to the
bifunctional, IGF-2 cation-independent Man6-P receptor.
This is particularly useful if cells must be targeted that
lack the mannose receptor. Further advantages are that IGF-2
binds to a different site on the receptor than Man6-P and that
the entry point to the endocytic pathway for lysosomal
targeting is identical to Man6-P glycosylated proteins.
IGF-2 had to be truncated to the amino acids 8-67 to avoid
25.3
INTRACELLULAR DELIVERY
The vast majority of currently approved and marketed anti-
bodies is directed against extracellular targets. This follows
the natural task of humoral response to neutralize infectious
agents or foreign objects such as bacteria and viruses by
capturing them. Binding of antibodies to surface antigens
labels the cells for later destruction by the immune system.
Owing to their large size of
150 kDa antibodies would
usually not be able to move across the cell membrane.
However, it would be attractive to utilize the high specificity
of antibodies also to address intracellular targets. In many
cases, they are internalized through receptor-mediated endo-
cytosis. It is not always obvious which type of receptor
supports internalization; therefore, a functional screen was
established to identify both targets and antibodies that allow
intracellular delivery. For this purpose, a Fab library was
created that links the antibody fragment to the intracellularly
active Pseudomonas exotoxin A. Only Fab fragments bind-
ing to internalizing epitopes were toxic to the cells. This
technique was called fusogenic and served to find new
epitope-antibody pairs that might be useful to treat cancer
and allow the characterization of cellular entry points [48].
25.3.1 Enzyme Replacement Therapy
A special case for targeted therapy is enzyme replacement. In
a number of rare diseases, a single enzyme is dysfunctional
and immediate cure can only be achieved by supplying cells
with the required enzyme from external sources. However to
accomplish that, the respective enzyme has to be transferred
into the cell to be localized in organelles such as the lysosome.
This type of enzymatic diseases is therefore termed lysosomal
storage disease (LSD). The uptake of the enzymes is
often mediated through the mannose/N-acetylglucosamine
(Man/GlcNAc) receptor. Unfortunately, heterologous protein
expression in mammalian systems does not lead to that
glycosylation of the enzyme, which is required to be
efficiently taken up by the defective cells. Therefore, a
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