Biomedical Engineering Reference
In-Depth Information
TABLE 16.2 Latent Cytokines Engineered to Date
mutagenesis (see [94]). This could make the latent cytokines
produced even more specific to a particular disease or
disease stage, given that the overexpression of certain
MMPs is disease-specific. Examples include the overexpres-
sion of MMP-1 and -3 in RA, MMP-1, -8, and -13 in
atherosclerosis and MMP-2, -9, and -14 in tumors.
There is also the possibility of exchanging the MMP
cleavage site for a site that is cleaved by other enzymes that
are overexpressed at the site of disease. Work is in progress
on an alternative latent cytokine that is produced with an
aggrecanase cleavage site, rather than an MMP cleavage
site. Aggrecanases are a family of proteinases that degrade a
large proteoglycan called aggrecan [95,96]. Aggrecan is the
most abundant proteoglycan in the soft tissues of the joint,
where it is found interspersed within the collagen network of
the cartilage. The function of aggrecan is to endow the
cartilage with the ability to resist compression on load-
bearing. One of the main manifestations of arthritis is the
loss of type II collagen and aggrecan, causing the cartilage to
become thin and weak [97]. It is now well-established that
degradation of aggrecan in articular cartilage is due to the
action of aggrecanases [98]. Cleavage sites for aggrecanases
were identified via phage display by Hills et al. [99] and it is
one of these sequences that has been inserted into a latent
IFN-
b
molecule in between the LAP and the cytokine.
Although the efficacy of LAP-MMP-IFN-
b
administered
to mice with CIA has been demonstrated [7], it is hypothe-
sized that use of the aggrecanase cleavage site will prove just
as effective in the treatment of disease, but will also make
this therapy even more “arthritis specific”. Preliminary
experiments have shown that these new latent proteins are
efficiently cleaved by synovial fluid from arthritic joints
(unpublished data).
Therapeutic
Application
Molecule
Reference
LAP-MMP-IFN-
b
RA
[7]
LAP-agg-IFN-
b
RA
Unpublished
LAP-MMP-VIP
RA
[82]
LAP-MMP-
a
MSH
RA
[82]
LAP-MMP-
g
MSH
RA
[82]
LAP-MMP-IL-4
Atherosclerosis
Unpublished
LAP-MMP-IL-10
Atherosclerosis
Unpublished
LAP-MMP-TGF-
b
MS
Unpublished
LAP-MMP-EPO
MS
Unpublished
LAP-BMP-7
OA
Unpublished
LAP-IGF-1
MS
Unpublished
native TGF-
b
, as shown by the production of latent IFN-
b
[7], which is a significantly larger molecule. A number of
LAP fusion proteins have now been produced and are listed
in Table 16.2, along with the potential applications of these
molecules. It is of note that this technology works just as
well for the production of much smaller molecules than
native TGF-
b
, such as vasoactive intestinal peptide (VIP),
a
-melanocortin (
a
-MSH), and
g
-melanocortin (
g
-MSH),
and that the LAP technology has been extended to molecules
outside of the classical cytokines to include other anti-
inflammatory agents such as those aforementioned.
VIP is a neuropeptide/hormone that was first identified as
a vasodilator and hypotensive peptide [86,87], but over the
last decade has been shown to have potent anti-inflammatory
effects on both innate and adaptive immune responses [88].
A potential problem with the use of VIP as a therapeutic
agent is that its neuromodulatory functions are widespread,
including heart, lung, thyroid gland, urinary tract, genitals,
kidney, and immune system [89] and so increases the risk of
causing unwanted side-effects. Both
a
- and
g
-MSH are parts
of the proopiomelanocortin (POMC) system, which consists
of the POMC preprohormone and its numerous products.
These peptides have anti-inflammatory effects [90] as well
as effects on cardiovascular function such as decreasing
blood pressure that may be useful in treating cardiovascular
disease. The production of these much smaller signaling
molecules (VIP has a molecular mass of about 3.6 kDa,
while the MSH peptides are
16.7 CHALLENGES (PRODUCTION
AND DEVELOPMENT)
It is clear that the latent cytokine technology provides a
novel and efficient method for targeting therapeutic cyto-
kines to the sites of disease (see Reference [7]). The next
step in the development of this technology is to be able to
treat disease by administering purified latent cytokines,
rather than having to rely on a gene therapy approach.
The production of sufficient quantities of recombinant latent
cytokines is likely to present the greatest challenge to the use
of this technology in a therapeutic setting.
The LAP fusion proteins are not easy to purify using
conventional protein purification techniques [7] and there
appear to be two reasons for this. The first is that LAP
demonstrates a propensity to aggregate in standard cell
culture conditions and, although this tendency can be abro-
gated to a large extent by the mutation of Cys
33
to Ser, some
slight aggregation does still occur. The second is the
1 kDa) as LAP fusion proteins
is instructive in demonstrating that full latency, and subse-
quent activation following cleavage of the LAP can be
achieved with a wide range of peptides and proteins, regard-
less of size.
A further variation to the basic latent cytokine technology
is to change the MMP cleavage site so that the resultant
latent molecule is more sensitive to particular MMPs. The
original MMP cleavage site used was derived from a colla-
gen substrate of MMPs [91-93], but this cleavage site could
be made more specific for individual MMPs by site-specific
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