TARGETING INTERFERON-a TO THE LIVER: APOLIPOPROTEIN A-I AS A SCAFFOLD FOR PROTEIN DELIVERY - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 426

clearance of the IFN-a and, therefore, significantly increases
its half-life from about 5 h to almost 90 h, which in turn
allows a reduction in the required frequency of treatments
[15,16]. PEG-IFN-a preparations have replaced the use of
standard IFN-a for the treatment of hepatitis B and C,
because of greater convenience and efficacy. When com-
pared with oral regimens, PEG-IFN-a offers several advan-
tages such as lack of development of drug resistance and
greater rates of HBeAg seroconversion. Unfortunately, the
prolonged peginterferon therapy necessary to control
chronic hepatitis C virus (HCV) or B virus (HBV) infections
was often associated with serious side effects such as
fatigue, fever, and myalgias. These are symptoms of
many acute virus infections, possibly because such effects
are due to the induction of IFNs by the infecting agents.
Usually, these symptoms respond to treatment with non-
steroidal anti-inflammatory agents [17]. In some patients,
treatment with IFNs has also resulted in psychiatric prob-
lems such as depression, anxiety, and excessive irritability,
which may require treatment with psychoactive pharma-
ceuticals. More severe toxicities, such as cytopenias and
autoimmune disorders, also have been reported in patients
treated with IFNs [18].
Another effort to stabilize IFN-a was made by Osborn
et al. [11], when they developed the technology of fusion to
human albumin. This is the most prevalent serum protein in
the circulatory system, with an unusually long half-life
(19 days). They not only fused human albumin to IFN-a
(alb-IFNa), but also to a wide range of proteins to treat human
diseases, such as growth hormone [19], insulin [20], or other
small bioactive peptide hormones, such as the b-natriuretic
peptide [21]. This albumin-IFN-a treatment was developed
by Human Genome Sciences (HGS) and Novartis. On
October 5, 2010 both companies announced that they had
stopped drug development in response to an FDA ruling.
The last strategy of IFN-a stabilization to reach clinical
trial was Locteron 1 . Locteron 1 is a new formulation of
recombinant IFN-a-2b in poly(ether-ester) microspheres
(PolyActive, OctoPlus N.V., Leiden, the Netherlands)
[22]. The recombinant IFN-a-2b component of Locteron 1
(BLX-883) is synthesized in a Lemna aquatic plant expres-
sion system. Phase I and II clinical studies were completed.
The results of these clinical trials indicate that Locteron 1
can be dosed once every 2 weeks. The best advantage of this
new formulation over the pegylated IFN is an improved
tolerability profile with fewer,

virtually all the cells of the organism. IFN-a signaling in
other cell types than hepatocytes may induce unwanted
adverse effects.
The first attempts to target IFN to the liver were per-
formed by Kasama et al. [24]. They produced asialo IFN-b
by treating natural IFN-b with neuraminidase. They dem-
onstrated that the desialylated glycoprotein was recognized
and taken up by the liver via asialoglycoprotein receptor.
However, the half-life in circulation was reduced and the
biological activity compromised. This latter limitation was
solved years later and the benefits of IFN liver targeting were
demonstrated for the first time in a murine model of HBV.
Asialo IFN not only increased the interferon-stimulated
genes (ISGs) expression in the liver but also reduced the
HBV virions in the sera of athymic nude mice transfected
with HBV plasmid [25]. To target the nonglycosylated
IFN-a at the asialoglycoprotein receptor, IFN-a was
linked [26] or complexed [27] in pullulan. Pullulan is a
linear, nonionic polysaccharide with a repeated unit of
maltotriose condensed through a-1,6 linkage. This com-
pound was able to enhance the ISGs expression in the liver
mediated by IFN-a.
Yuan et al. [28] developed a new formulation for IFN-a
that increased the short half-life and solved the lack of liver-
specific affinity. This approach was based on the reversible
lipidation of IFN-a by conjugating palmitoylcysteine to
cysteinyl residues of IFN-a via disulfide bonds. This pal-
mitoylated IFN-a behaved like a controlled release conju-
gation. Upon reduction of the disulfide bonds inside the
body, unmodified IFN-a is slowly released to the circulation,
increasing the half-life. In addition, reversible lipidization
alters tissue distribution by an unknown mechanism. Palmi-
toylated IFN-a accumulates in the liver and exerts an
enhanced activity.
Another strategy to increase the local concentration of
IFN-a in the liver is to synthesize the cytokine in this organ.
The IFN-a gene can be introduced into a gene therapy vector
and this vector will infect the liver and deliver the genetic
material into the hepatocytes. Finally, the heterologous gene
will be transcribed and translated to produce the therapeutic
protein. Proof of the principle of this approach was estab-
lished using a helper-dependent adenovirus vector [29]. This
vector predominantly infects the liver and lacks all viral
coding sequences, minimizing the innate immune response
and allowing long-term expression of the transgene. The
IFN-a gene was under the control of an inducible promoter
regulated by doxycycline. This promoter allowed the authors
to regulate the level of IFN-a produced and to switch the
production of the cytokine on and off.
Unfortunately, experiments performed in the best animal
model of chronic hepatitis infection (the woodchucks
infected with the woodchuck hepatitis virus) revealed sev-
eral drawbacks of the long-term expression of IFN-a in the
liver of infected individuals [30]. In this study, the IFN-a

less severe, and shorter-

lasting flu-like symptoms [23].

29.1.3 IFN- a Liver Targeting

The second major limitation of IFN-a as a drug is the lack of
organ-specific activity. The main indication of IFN-a is the
treatment of viral hepatitis B and C and the target organ is
therefore the liver. However, the IFN receptor is expressed in