Drug Targeting Organ-Specific Strategies

11.9 Recombinant Domains as Building Blocks for Drug Targeting Constructs 303
genetically inserted into a recombinant protein backbone. For some of these peptides, the
surface receptor has not yet been elucidated. Further investigation into the cellular processing
of the receptor and its bound ligand is essential to ensure a rational design for the targeting
constructs that are being developed.
11.9.2 Membrane Translocation Domain
Most of the targeting domains mentioned above are aimed at cell surface molecules for the
obvious reason of accessibility. In many cases these target receptors are able to internalize together
with the bound ligand. However, this process will deliver the construct to the lysosomes,
a compartment in which enzymes and low pH will result in degradation of proteins. In
order to escape this aggressive environment, a membrane translocation domain might be introduced
so that the drug delivery preparation can cross the cell membrane in a receptor-independent
manner.
Membrane translocation domains have been identified in toxins and viruses and derived
from signal sequences of secreted proteins.When derived from a signal sequence the translocation
domain contains hydrophobic sequences [146–148] while the toxin and viral translocation
domains contain mostly basic residues [149,150].
In terms of targeting, membrane translocation domains lack specificity for particular cells
or tissues. Therefore, these domains should be combined with targeting domains such as
those discussed in the previous section. In such a construct, the targeting domain will ensure
a rapid accumulation at the surface of a specific cell type and the translocation domain will
facilitate entry into the cytosol of the target cells.
11.9.3 Assembly Domain
Although feasible and resulting in highly uniform end-products, the construction and synthesis
of a complete drug targeting preparation as one genetic construct has one major disadvantage:
lack of flexibility. If a construct does not show the expected results, the whole
process of designing and production has to be repeated. Therefore, the use of assembly domains
in the individual components of the drug targeting construct can be advantageous. Recently
several studies have reported on the flexibility of such constructs containing an
avidin/streptavidin moiety for non-covalent binding to biotinylated proteins. This approach
was followed for the delivery of biotinylated compounds across the blood–brain barrier using
a genetic fusion protein of avidin and an anti-TfR antibody [151].A similar approach was
used to engineer the RGD cell adhesion sequence into accessible surface regions of streptavidin
without disrupting the biotin binding properties [152].
Even greater flexibility was achieved by genetic fusion of streptavidin with protein A
[153,154]. Protein A specifically binds the Fc domain of IgG immunoglobulins of almost all
mammals without inhibiting the antigen binding activity of the antibody. The
streptavidin–protein A fusion construct was used for the assembly of complexes of biotinylated
β-galactosidase and different monoclonal antibodies specific for tumour cell receptors.
As a result these complexes were efficiently delivered into several cancer cell lines [154].