Biomedical Engineering Reference
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FIGURE 16.3
The LAP technology cannot be used to make latent molecules that bind soluble
ligands. (A) LAP-dTNFR detected on Western blot after transient expression from 293 T cells. Lanes
1 and 2 are media from nontransfected cells; 3 and 4 are from cells expressing LAP-dTNFR. Media in
2 and 4 was treated with MMP-1 for 18 h. After digestion with MMP-1 the individual components
LAP and dTNFR are clearly released. (B) Nonlatent activity of LAP-dTNFR. 57A HeLa cells
respond to hTNF-
a
stimulation with luciferase expression from an NF-
k
B responsive promoter.
Supernatants from 293 T cells containing LAP-MMP-TNFR2 were either treated with MMP-1 for
18 h or kept untreated prior to addition to 57A HeLa cells along with TNF-
a
(10 ng/mL). Both MMP
treated and untreated LAP-dTNFR equally inhibit the activity of TNF-
a
. (C) Latency of LAP-IFN-b.
Measurement of IFN-b-mediated inhibition of the cytopathic effect of EMC virus on L929 cells
before and after treatment with MMP.
from an NF-
kb
-responsive promoter. Supernatants from 293T
cells containing LAP-MMP-dTNFR, with or in the absence of
treatment with MMP-1, were added to HeLa cells along with
TNF (10 ng/mL). The results showed that both MMP-treated
and untreated LAP-dTNFR equally inhibited the activity of
TNF (Figure 16.3b), therefore indicating that, contrary to
expectations, LAP-dTNFR was not in fact latent. We believe
that the failure of the LAP to provide latency in this instance is
because the ligand for the molecule inside the LAP is soluble.
LAP confers latency to cytokines by blocking access to
cellular receptors, rather than completely enclosing the cyto-
kine within the LAP shell. Hence, in the case of LAP-dTNFR,
TNF-
a
in solution is able to access the dTNFR inside the LAP,
nullifying the latent properties of the presence of LAP. We
believe that these observations indicate a limitation of the
latent cytokine technology, in that it is not suitable for
molecules with soluble receptors or ligands.
Hence, we were puzzled by the report of a “latent” human
soluble TNF receptor 1 (hsTNFR1) engineered by fusing LAP
to the hsTNFR1 via an MMP site to treat endometriosis [24].
The initial phase of endometriosis, involving the ectopic
implantation of endometrial tissue, is an invasive event, requir-
ing breakdown of the ECM [25]. In a similar way to cancers,
MMP activity is increased in endometriosis, allowing endo-
metriotic cells to invade the ECM. These increases in MMP
activity are stimulated by proinflammatory cytokines such as
TNF-
a
and IL-1 [26], so blocking the biological effects of
TNF-
a
could be an effective therapy for this condition.
Cleavage of the LAP-MMP-hsTNFR1 by peritoneal fluid
from endometriosis patients is reported [24], indicating that
there is sufficient MMP activity to release the hsTNFR1
from the LAP, and is similar to our results with biological
fluids from a number of inflammatory conditions [7]. How-
ever, it is also claimed that the LAP-MMP-hsTNFR1 is fully
latent on the basis of in vitro MTT assays with L929 cells.
On the basis of our experience of LAP-MMP-dTNFR (and
the data shown in Figure 16.3), it is not possible that LAP-
MMP-hsTNFR1 is latent. The conclusions drawn by Xiong
et al. [24] are based on data from cytotoxicity protection of
L929 cells treated with TNF-
a
. However, the differences
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