Biomedical Engineering Reference
In-Depth Information
microenvironment. Hence, they tend to work in pharmaco-
logical doses, and once secreted by the producing cell, have
very short half-lives, often in the order of a few minutes. The
pleiotropic nature of these molecules means that they are
exceptionally potent; it is therefore necessary for their
secretion to be carefully regulated to avoid cytokine-medi-
ated tissue damage. One of the best-characterized examples
of deregulation of cytokines leading to tissue destruction is
in rheumatoid arthritis (RA), where up-regulation of tumor
necrosis factor-alpha (TNF-
a
), and the interleukins IL-1 [2],
IL-6 [3], and IL-15 [4] have been demonstrated.
Despite the challenges that cytokine biology presents as
discussed above, insights into the biological functions of indi-
vidual cytokines have emerged through molecular, bio-
chemical, and immunological analyses so that sufficient
information pertaining to the structure and function of these
molecules has enabled the development of a number of thera-
peutic cytokines and cytokine antagonists. One of the best-
known and most successful groups of therapeutic cytokines to
date is the interferons (IFNs). The IFNs are a groupof naturally
occurring cytokines that are induced in response to viral infec-
tions. IFN-
b
was one of the first cytokines to be purified and
producedinrecombinant formforclinicalapplication[5]andis
now used in recombinant form to treat a number of conditions
including relapsing forms of multiple sclerosis (MS), chronic
hepatitis C (Hep C), and some forms of cancer [6].
However, research continues to focus on ways to increase
the serum stability and half-life of recombinant cytokines as
the short serum half-life makes frequent administration
necessary and increases the possibility of side-effects,
thus limiting their clinical uses. Strategies such as modifi-
cation with polyethylene glycols (PEG) or by fusing the
cytokine to an antibody Fc fragment to improve the phar-
macokinetics have been in use for some time.
known as the latency-associated peptide (LAP). There are a
number of isoforms of TGF-
b
, but all are produced as large
precursor proteins that are 390-412 amino acids in size. This
precursor protein is then processed intracellularly prior to
secretion from the producing cell. A critical processing step
is the proteolytic digestion of the precursor protein by the
endopeptidase furin. This proteolysis cleaves the TGF-
b
precursor protein to yield two products that then form a
complex. The N-terminal portion is the LAP, while the
C-terminal 25 kDa dimer constitutes the mature TGF-
b
.
Crucially, after cleavage of the precursor protein, the mature
TGF-
b
remains noncovalently attached to the LAP and it is
this association that renders the secreted TGF-
b
latent, due
to the fact that mature TGF-
b
cannot interact with its cellular
receptors while associated with LAP in this manner.
It is this LAP portion of TGF-
b
that we harnessed to
make latent cytokines. Therapeutic cytokines are engineered
with the LAP at the N-terminus (the same arrangement as
that found in native TGF-
b
) so that the expressed protein
consists of the recombinant therapeutic cytokine, sur-
rounded by the LAP (Figure 16.1), which shields the cyto-
kine from interacting with its cellular receptors, thus
rendering it biologically inactive. The presence of the
LAP also increases the molecular weight of the cytokine
and improves serum stability and half-life. A further aim of
the design and engineering of these latent cytokines was to
address the side-effects that are all too common features of
treatment with these pleiotropic molecules. Hence, the latent
cytokines are designed to be released from their protective
LAP shell only at the sites of disease. This was achieved by
cloning in a specific cleavage site for matrix metalloprotei-
nase (MMP) between the LAP and the active cytokine.
Therefore, at sites where the latent cytokine is exposed to
MMP activity, the LAP is cleaved from the cytokine, thereby
allowing the therapeutic cytokine to interact with its cellular
receptors and exert its biological effects (Figure 16.2).
MMPs are a family of zinc-dependent endopeptidases
that are responsible for the timely degradation of the extrac-
ellular matrix (ECM) [9,10,11]. This degradation is essential
for tissue repair and remodeling, morphogenesis, and wound
healing. Indeed, the levels of MMPs in normal steady-state
tissues are extremely low [12], with expression under tran-
scriptional control by pro-inflammatory cytokines, cell-
matrix interactions, and growth factors. The activity of
MMPs is also subject to further regulation via the activation
of precursor zymogens and by the presence of endogenous
inhibitors, known as tissue inhibitors of MMP (TIMPs).
There are 23MMPs in humans, most of which can be
grouped according to substrate specificity [13] (Table 16.1).
MMPs are extracellular proteins, although at least three
(MMP-1, MMP-2, and MMP-11; [14]) have been shown
to be present intracellularly and may act on intracellular
proteins. However, these enzymes are also implicated in a
number of pathological processes, including the destruction
of arthritic joints, atherosclerosis, spread of metastatic
16.2 DESCRIPTION OF CONCEPT
The recent development of “latent” or “shelled” cytokines
[7,8] aims to rectify not only the short serum half-life of
recombinant cytokines, but also dramatically reduce the risk
of undesirable side-effects. The idea underpinning this
technology is to produce cytokines that are biologically
inactive (i.e., latent) until they arrive at the site of disease,
at which point they are released so that they can exert their
biological effects. These latent cytokines are engineered by
using a naturally occurring “shell” structure from the cyto-
kine transforming growth factor-
b
(TGF-
b
). The release of
cytokines in vivo is usually tightly regulated at the transcrip-
tion and translation stages to prevent cytokine-related tissue
damage. However, in the case of TGF-
b
, a different and
unique strategy is employed. TGF-
b
is secreted from cells in
a latent form; this is achieved by “packaging” of the active
cytokine inside a disulfide-linked shell-like structure formed
by dimerization of its long precursor amino terminal end,
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