Biomedical Engineering Reference
In-Depth Information
hydrodynamic coinjection of a plasmid encoding LacZ
together with pIFN or pIA or pApo in wild-type mice
and in SR-BI
þ
/
or SR-BI
/
animals. In agreement with
previous data, pIA showed an eightfold increase in the
specific lysis compared to that induced by pIFN in wild-
type mice. However, this difference was reduced to twofold
in SR-BI
þ
/
animals and the cytotoxicity values were
similar for IA and IFN-a in SR-BI null mice. Thus, IA
requires interaction with SR-BI to display maximum adju-
vant activity.
[84], hamster ovary cells [85,86], and more recently
plant-based expression systems [87,88]. Regarding IFN-a,
the recombinant protein produced in Escherichia coli was
approved for clinical use since 1986. Most of the human
IFN-a species are devoid of carbohydrates and therefore,
bacteria are still the most widely used production system
although many other systems have been tested [89-91]. The
production of the recombinant fusion protein has been
successfully performed both in E. coli and in yeast using
standard technology.
29.2 TECHNOLOGICAL ASPECTS
29.3 TYPICAL APPLICATIONS AND
INDICATIONS
When the fusion protein is produced directly in the liver by
gene therapy vectors, most of the protein is able to complex
in HDLs. This high incorporation is probably because the
protein is produced inside the transduced hepatocytes, fol-
lowing the natural apo A-I pathways, and therefore only
2-8% of apo A-I is found lipid-free in plasma, as it occurs
physiologically. When administered intravenously,
IFN-a was first discovered for its antiviral activity [1] but its
promising antitumor activity was soon appreciated [92]. The
differential effects of IA versus IFN-a make this novel
molecule a candidate, not only for the conventional IFN-
a-based therapies, but also for new applications.
The major indication for using a liver-targeted IFN-a
formulation such as IA is viral hepatitis. Nowadays, the
treatment of HCV patients is based on combination of PEG-
IFN and ribavirin plus/minus direct-acting antivirals [93-
95]. The reduced toxicity of the new fusion protein could
increase the response rate to these combinations and could
perhaps rescue those patients, who failed to respond to
conventional therapy. On the other hand, IA might be
particularly useful in chronic HBV infection. In this infec-
tion, the viral genome remains in hepatocytes in the form of
covalently closed circular (ccc) DNA maintaining transcrip-
tional activity despite inhibition of replication with nucle-
oside/nucleotides analogs. Only an effective immune
response generated against hepatocytes containing ccc
HBV-DNAwill be able to clear the infection. In this regard,
the enhanced immunostimulatory activity of the new fusion
protein can be crucial.
IFN-a also displays antitumor activity through its ability
to halt cell proliferation and to induce apoptosis of tumor
cells [96,97]. In addition, IFN-a may also block tumor
progression by acting on stromal cells where it has been
shown to inhibit angiogenesis by suppressing the prolifera-
tion and migration of endothelial cells [98,99]. Most impor-
tantly, IFN-a plays a central role in the stimulation of
antitumor immune responses, being able to enhance the
trafficking, expansion, and lytic activity of NK cells and
T lymphocytes [100,101]. Although these properties make
IFN-a very attractive for cancer therapy, clinical use of this
cytokine is limited by its toxicity. An additional limitation is
due to the fact that IFN-a plays a dual role in the biology of
the T lymphocytes. On the one hand, it stimulates clonal
expansion but, on the other hand, it sensitizes T cells to the
antigen-induced cell death normally occurring at the end of
an immune response [68].
10% of
the recombinant protein can also be detected in the HDL
enriched fraction, and this percentage of incorporation is
also observed when IA is administered by intratumoral
injection of a viral vector expressing IA, and therefore
delivered to plasma after being produced by tumor cells.
An important question is, therefore, whether HDL incorpo-
ration of the fusion protein IA could be playing an essential
role. All the important functions of IA (long half-life in
circulation, liver targeting, reduced hematological toxicity,
and enhanced immune stimulation) have been demonstrated
both with the recombinant protein and with HDL-containing
IA. In addition, to avoid the need for IA complexing to
HDLs to be immunologically functional, an in vivo killing
assay was performed in Apo A-I knockout mice (that lack
HDLs) and in wild-type mice receiving rIA and ovalbumin
protein as antigen. In both cases, rIA was able to act as an
effective adjuvant increasing the cytotoxic T-cell-mediated
specific lysis.
Therefore, the recombinant fusion protein can be used
directly and the in vitro reconstitution of synthetic HDLs is
not required. However, there is a lot of experience concern-
ing the feasibility and security of this approach due to the
clinical trials that have used reconstituted HDLs for the
treatment of cardiovascular diseases [71-80]. Incubation of
the recombinant fusion protein with phospholipids may lead
to the formation of nanoparticles that resemble natural
HDLs and which can be infused to patients.
Both components of the new fusion molecule have been
produced and purified previously using different technolo-
gies. In spite of the amphiphilic character, autoaggregation,
and degradation of apo A-I, a wide variety of heterologous
protein production systems have been used successfully.
They include bacteria [81,82], yeast
[83],
insect cells
In addition,
IFN-a causes
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