Biomedical Engineering Reference
In-Depth Information
The alternative protocol that has been developed for the
purification of latent cytokines consists of three chromatog-
raphy steps. Cell supernatants from transfected cells are
dialyzed and applied to a heparin-sepharose column, taking
advantage of the heparin-binding sites in the LAP part of the
molecule (Figure 16.4). The binding of the latent cytokine to
the heparin column is of relatively low affinity and can be
eluted with an increasing salt gradient. The LAP fusion
proteins elute at the beginning of the gradient. This first
affinity chromatography step serves not only to partially
purify the latent cytokine, but also to concentrate the protein
greatly (although further concentration is generally neces-
sary) for application to a gel filtration column.
The gel filtration step is particularly useful as any LAP
protein aggregates are separated from the latent LAP dimers
at this stage. Aggregation is much less problematic as a
result of the Cys
33
mutation (as discussed above). This step
also removes any endogenous LAP that may be present from
the cultured cells. This material can also be purified from the
heparin column, but the difference in size means that it is
effectively removed by the gel filtration column. A final
anion exchange chromatography step is then used to purify
the latent cytokines to homogeneity.
The purified latent cytokines that have been produced
thus far are soluble in aqueous media and are stable when
stored at
There are a whole host of pathological conditions in
which inflammation is a feature and may occur in response
to an exogenous stimulus, for example, in response to an
infectious agent, or as part of an autoimmune response.
Treatment of inflammation with latent cytokines is most
likely to be appropriate in those inflammatory conditions
that are chronic in nature, for example, atherosclerosis, or in
auto-immune diseases that are characterized by chronic
inflammation, such as RA. The central role of cytokines
in regulating inflammatory processes means that these mol-
ecules are of potentially great therapeutic use, notwithstand-
ing the issues surrounding their short half-lives and
pleiotropic effects, as previously discussed.
The onset of inflammation is characterized by the release
of large amounts of proinflammatory cytokines such as
TNF-
a
, IL-6, and IL-1 from leukocytes such as neutrophils
and macrophages that have been recruited to the site of
inflammation. Of course, the roles of cytokines in inflam-
matory processes are not confined to the actions of the
proinflammatory cytokines and it is the cytokines with
anti-inflammatory properties (e.g., IL-4, IL-10, TGF-
b
,
and IFN-
b
) that are the focus of the latent cytokine technol-
ogy for the treatment of inflammatory conditions.
There are already a small number of therapeutic cyto-
kines that have gained approval for clinical use. Examples
include IFN-
b
for the treatment of MS, IFNs for a variety of
conditions including hepatitis B and C, hairy cell leukemia
and AIDS-related Kaposi's sarcoma. Unsurprisingly, given
the potency of these molecules, there are many more cyto-
kine-based therapeutics in development, most of which are
based on the “classic” anti-inflammatory cytokines IL-4,
IL-10, and IFN-
b
. However, the pleiotropic effects and short
half-lives of these cytokines continue to hamper these efforts
in many cases. The development of latent versions of these
anti-inflammatory cytokines could potentially allow the
anti-inflammatory potential of these molecules to be fully
harnessed for the treatment of disease.
80
C for extended periods (
6 months). All of
the experiments using latent cytokines to date have been
conducted in murine models of disease (e.g., the CIA model
of RA, experimental allergic encephalitis (EAE) and the
ApoE knock-out/high-fat diet model of atherosclerosis)
where latent cytokines using either human LAP or mouse
LAP have been well tolerated ([7]; unpublished data).
>
16.5 APPLICATIONS AND POTENTIAL
CLINICAL INDICATIONS
There are many potential clinical applications for latent
cytokines, but the two indications that show particular
promise are inflammatory conditions and cancer. A common
denominator between these two groups of disease is the local
increases in the expression and activity of MMPs at the sites
of disease. As discussed above, this family of proteinases are
essential for normal development, but are also implicated in
a wide variety of pathological processes, where tissue
remodeling takes place, for example, osteoarthritis, auto-
immunity, neuroinflammation, spreading of metastatic can-
cer cells, and atherosclerosis. It is for this reason that the
latent cytokines described here are engineered with a MMP
cleavage site, situated between the LAP and the therapeutic
cytokine. This approach means that these latent molecules
can potentially be used to deliver therapeutic cytokines to
the sites of disease in a range of inflammatory conditions
including cancer.
16.5.1 Multiple Sclerosis
16.5.1.1
As mentioned above, the use of
IFN-
b
as a therapeutic is already in use in the treatment
of MS, a neurodegenerative autoimmune disease. The neu-
rodegeneration in MS is caused by an inflammatory response
(the cause of which has remained elusive) that strips myelin
from neurons in the white matter of the CNS [47]. This
demyelination results in a range of neurological deficits. As
this condition is an inflammatory disease, there is obvious
interest in attempting to treat this disease with anti-inflam-
matory cytokines. Indeed, IFN-
b
is now the treatment of
choice for the relapsing/remitting form of MS and is effec-
tive in
Interferon
b
50% of patients, in whom relapse rates are reduced
by half [48]. Although the mechanism of action of IFN-
b
in
MS has not yet been fully elucidated, there is convincing
Search WWH ::
Custom Search