Biomedical Engineering Reference
In-Depth Information
100
100
Remaining G-S-S-T
Released G-CSF
G-C-T
10
10
1
0.1
1
0
50
100
150
200
0
50
100
150
200
Time (min)
Time (min)
FIGURE 4.2
In vivo cleavage of the disulfide linker. (a) In vivo release of free G-CSF from G-CSF-
S-S-Tf (G-S-S-T) with a cleavable disulfide linker. (b) In vivo elimination of G-CSF-cyclo-Tf
(G-C-T) with a stable dithiocyclopeptide linker. G-S-S-T (thrombin-treated G-C-T) or G-C-T was
administered intravenously to CF1 mice via the tail vein at a dose of 4mg/kg. After administration,
the collected blood was analyzed by nonreducing SDS-PAGE followed by anti-G-CSF Western blot
analysis to measure the intact fusion protein and free released G-CSF. The y-axis represents the
concentration of each protein in mouse plasma, quantified using Quantity One software (Bio-Rad
Laboratories, Hercules, CA, USA). No free released G-CSF was detected following administration of
G-C-T. Source: Adapted from Reference [45].
Figure 4.2, free G-CSF could be detected in the blood as
early as 5min after injection of G-S-S-T, with a peak at
is widely used to prolong half-life of protein drugs and to
improve their efficacy. In the first attempt, a fusion protein of
rFIX and albumin with a flexible GS linker exhibited a poor
FIX activity. The bulkiness of albumin was thought to be
affecting the interaction of FIX with other coagulation
factors (e.g., factor VIII and factor FX) and thereby limited
its potency. To overcome this limitation, a proteolytically
cleavable sequence (VSQTSKLTRAETVFPDV) derived
from the N-terminal activation region of FIX was tested
as a linker to connect FIX with albumin. The idea was to take
advantage of the activation of FIX by either tissue fac-
tor/factor VIIa or factor XIa. Both activation sites on FIX
and the linker would be simultaneously cleaved. This cleav-
able linker significantly improved the clotting activity of the
cleavable fusion protein by 10- to 30-fold compared to that
of the fusion protein with noncleavable linkers. At the
meantime, since this linker was only cleavable during clot-
ting, the fusion protein exhibited prolonged half-life com-
pared to rFIX.
In a similar study, Vessillier et al. fused IFN-
b
with the
latency-associated peptide (LAP) of transforming growth
factor (TGF)
b
to create a latent cytokine that was covered
by the shell structure provided by the LAP [47,48]. Between
the two protein moieties, a linker containing two flexible
sequences (GGGGS) flanking a matrix metalloproteinase
(MMP) cleavage site was designed. The release of IFN-
b
would occur only through the cleavage by MMPs that were
overexpressed at the disease site during a variety of patho-
logical conditions such as arthritic diseases [49], cancer
[50], and inflammation [51,52]. Since the cleavage of the
linker was very limited, IFN-
b
could be masked by LAP and
15 min postinjection. A rapid release of G-CSF from the
fusion protein and a quick elimination of free G-CSF due to
its short in vivo half-life were observed. In contrast, no
detectable amount of free G-CSF was present in the blood of
CF1 mice treated with G-C-T (Figure 4.2). These results
demonstrated that the disulfide linkage created by the
dithiocyclopeptide linker could indeed be cleaved in vivo.
This study demonstrates that the reversible nature of the
disulfide bond can be utilized to design an in vivo cleavable
linker in a recombinant fusion protein to ensure the
release of free functional domain into the blood circula-
tion. This in vivo cleavable linker can be applied to a wide
variety of fusion proteins when the in vivo separation of
the domains is required for achieving optimal activity,
desirable biological functions, or independent actions/
metabolism of individual domains.
4.3.3.2
In Vivo
Cleavable Protease-Sensitive Linkers
Another type of in vivo cleavable linkers takes advantage
of proteases that are expressed in vivo under specific
pathological conditions, in specific types of cells or tis-
sues, or are constrained in certain cellular compartments.
Linkers in recombinant fusion proteins can be designed to
be in vivo cleavable specifically where the proteases are
present by incorporating a protease sensitive sequence into
the linker.
Schulte reported the construction of a recombinant coag-
ulation factor IX (rFIX) and albumin fusion protein for the
treatment of hemophilia B [46]. Albumin-fusion technology
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