FUSION PROTEINS WITH A TARGETING FUNCTION - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 379

TABLE 25.1 Endosomal Escape Mechanisms

Protein Mechanism Sequence Length [aa]

Hemagglutinin (HA2) Fusion GLFGAIAGFIEGGWTGMIDG 20

Diphtheria toxin Fusion INLDWDVIRD-----QVVHNSYNRP 177

Pseudomonas exotoxin A Pore GGSLAALTAH-----TGNDEAGAAS 112

Melittin Pore GIGAVLKVLTTGLPALISWIKRKRQQ 26

GALA peptide Fusion WEAALAEALAEALAEHLAEALAEALEALAA 30

The opposite is true for large, protein-based therapeutics that
reach their targets by invasive procedures such as sub-
cutaneous or intravenous injections. A major limitation is
the sensitivity of proteins to proteolytic degradation during
the passage through the digestive tract. Besides proteolysis,
low absorption and bioavailability are other major obstacles
that cause dosing problems [75]. Encapsulation would rep-
resent an obvious solution. Alternative strategies focus on
transferrin as carrier molecule relying on receptor-mediated
transcytosis because TfRs are highly expressed in the gas-
trointestinal tract, and Tf is resistant to trypsin and chymo-
trypsin. The first payload was insulin, conjugated to Tf. Rats
receiving this molecule orally displayed a slow but pro-
longed hypoglycaemic effect [76]. Later, G-CSF was fused
to the amino terminus of Tf. Replacing chemical conjuga-
tion by genetic fusion generated homogeneous proteins that
are more suitable for therapy. Initially, a very short dipeptide
linker consisting of leucine and glutamate was used. The
bioactivity of the G-CSF-Tf fusion protein was 90% less
than for G-CSF alone, whereas the half-life was much
improved. Interestingly, only the fusion protein caused a
measurable effect in mice when administered orally. This
effect lasted twice as long as subcutaneous injection [77].
The efficacy of this G-CSF-Tf fusion protein could be
further increased by optimizing the length and sequence
of the spacer connecting both molecules. The best variant
with a 10-fold higher activity contained a long helical linker
of 50 AA [78].
The same helical linker was used to generate an orally
administrable human growth hormone (hGH) transferrin
fusion protein. As expected, hGH-Tf showed a weight
gain effect in rats, but the effect was lower for the oral
administration than for the subcutaneous injection [79].
The intestine epithelia also contain the FcRn that remains
present in adult humans. The FcRn fulfills its primary role in
the uptake of maternal immunoglobulin present in milk. As
already described earlier, binding of the CH2 and CH3
domains of the Fc part mediates transcytosis of the globulin
from the intestine to the bloodstream. Initially, polarized
T84 cells were used as model system to verify FcRn-
mediated transcytosis of hGH-Fc fusions. A molecule con-
taining a hinge region between hGH and Fc was better
uptaken than a similar variant with only a short flexible
linker. It could also be demonstrated that a dimeric structure

of the Fc is required for optimal binding to the FcRn [80].
Previously, the ability of FcRn-induced intestinal transcy-
tosis of large, orally administered molecules was proven
with follicle stimulating hormone (FSH) fused to Fc. New-
born rats absorbed FSH-Fc after oral dosing. Two types of
molecules were tested: one connected the b - and a -domain
of FSH via a linker and another the hinge region with the
heavy IgG1 chain in a single polypeptide, ultimately form-
ing a homodimer with two FSH units. The other variant
contained either the a -or b -domain of FSH fused to a heavy
chain, generating a heterodimer with only one FSHmolecule
per fusion protein. At an age of 10 days, rats received daily a
single oral dose of the different fusion proteins for 14 days.
Both molecules achieved a half-life of more than 60 h, but
the heterodimer was significantly more potent [81].

25.5 CONCLUSIONS AND FUTURE
PERSPECTIVES

Much progress in the field of drug targeting has been made in
the last decades. Owing to the advancement of antibodies,
now a broad selection of binding entities is available to
address extracellular targets. The reduction of antibody
specificity to small binding fragments is now an essential
part of many fusion protein constructs. In addition, the
understanding of disease-related changes of surface mole-
cules and organ-specific receptors has contributed a lot to the
refinement of targeting techniques. Certainly, the field of
targeted therapy that addresses cancer is the most advanced
but probably also the most dispersed. Cancer seems to be the
primary testing ground for fusion proteins because it com-
bines the need for targeting with aim of toxicity, two
activities that are nicely covered by bifunctional molecules.
But it is still very difficult to transfer large protein
molecules into cells or across membranes such as the
BBB. Some of the entry points are well understood now
and will be exploited extensively in the future. However,
more work is needed to establish alternatives and to identify
more tissue- and organ-specific surface markers. Oral deliv-
ery that would allow a more patient friendly administration
of therapeutic protein is still in its infancy. But maybe other
concepts such as encapsulation instead of fusion protein
approaches can cover that gap.