Biomedical Engineering Reference
In-Depth Information
Despite the impressive preclinical results, in the clinic
systemic administration caused severe side effects. Initially,
TNF alone was used. Since the noncovalent trimer showed
low stability, trimerization through tandem repeats in a
single-chain molecule was chosen. This molecule had
also a better antitumoral activity [65].
First generation TNF fusion proteins received a targeting
domain to specifically address only selected cells exposing a
typical antigen and to increase the local concentration while
keeping the systemic concentration low. A number of clas-
sical TNF fusion proteins containing only a targeting
domain such as a scFv or a peptide are currently in devel-
opment [66].
Consequently, second generation TNF fusion proteins
were designed, which included a targeting domain and a
second domain that could shield TNF. This shielding or
neutralizing domain is equipped with a protease-sensitive
linker that can be cleaved by tumor proteases such as MMP-
2 or uPA, thus releasing the active TNF. As TNF-neutraliz-
ing agent a fragment of the extracellular domain of the TNF
receptor 1 was selected. In order to maintain the natural
trimeric conformation of TNF, the trimerization domain of
tenascin-C (TNC) was included as well. Upon administra-
tion of this complex multidomain protein, elimination of
target antigen negative cells in close proximity could be also
observed which might be beneficial for heterogeneous
tumors. Targeted TNF fusion proteins create a similar
conformation as the original membrane bound TNF, which
is more potent since it not only activates TNF receptor 1 but
also the more important receptor 2 as well. Overall, this
novel class of prodrug fusion proteins contains two levels of
safety, first the targeting and second the target-dependent
activation [67].
On the basis of the experiences with shielded TNF,
molecules were designed that shielded the activity of FasL
while in circulation. Alternative formats were even equipped
with a targeting moiety, in this case a scFv against the tumor
marker fibroblast activation protein (FAP). In order to allow
desired activation of the FasL domain in tumor proximity, the
protective domain was equipped with a protease-sensitive
linker. Experimentally, it was observed that FasL requires a
free C-terminus for optimal activity, and that the shielding
domain consisting of the extracellular part of Fas only inhibits
FasL activity when trimerized. Therefore, the most potent
fusion protein had FasL positioned at the N-terminus, fol-
lowed by the trimerization domain of tenascin-C and the
protease-sensitive linker. Between linker and FasL that
formed the C-terminus, the anti-FAP-scFv was inserted.
On the basis of size exclusion chromatography, this multi-
domain protein forms noncovalent head to tail hexamers. This
engineered protein had a highly specific potency with no
negative systemic effects in a mouse tumor model [71].
Besides antibodies targeting peptides such as the
integrin-binding RGD motif were also used in combination
with FasL. The RGD-FasL fusion protein could suppress
tumor growth of a murine hepatocellular carcinoma more
efficiently than the untargeted FasL [72].
17.5.3 TRAIL Fusion Proteins
The TNF-related apoptosis-inducing ligand (TRAIL) exists
as soluble and membrane bound form similarly as the other
members of the TNF superfamily. TRAIL binds to four
different death receptors (DR4, DR5, DCR1, and DCR2),
but only the interaction with DR4 or 5 triggers apoptosis.
The process follows the aforementioned extrinsic pathway.
Soluble TRAIL was selected as therapeutic molecule owing
to its preference to kill cancer cells but not normal cells, thus
being well tolerated even in systemic administration. Despite
these relatively good prerequisites of TRAIL, efforts were
undertaken to improve specific activity and bioavailability.
ErbB2, which is expressed on a multitude of tumors, was
selected as target for a scFv. In order to simplify manufacture
and to stabilize the required trimer of TRAIL, three TRAIL
molecules were linearly fused together separated only by
short peptide linkers and equipped with one anti-ErbB2-scFv
molecule at the N-terminus. This fusion protein induced
apoptosis in a xenotransplant model more efficiently and
had a longer half-life than the nontargeted TRAIL trimer
lacking the scFv component [73].
Even without the forced trimerization, TRAIL fusion
proteins showed a good dose-dependent activity. One exam-
ple is the addition of an integrin targeting peptide (RGD) to the
amino terminus of TRAIL. This molecule formed monomers,
dimers, and trimers and suppressed tumor growth in mice
bearing COLO-205 tumors more efficiently than untargeted
TRAIL [74].
17.5.2 FasL Fusion Proteins
Another death receptor ligand is CD95L binding to its
receptor, CD95, also called apoptosis stimulating fragment
(Fas). This receptor is the most intensely studied death
receptor. Initially its ligand, FasL, was evaluated and com-
pared with a series of multimerizing domains. It was found
that soluble FasL does not induce cell death. But hexameric
variants of FasL generated either by fusion to the collagen
domain of adiponectin ACRP30 or fusion to an Fc domain
were very cytotoxic and mediated apoptosis [68]. The fusion
protein is also known as Mega-Fas-ligand or APO010 [69].
Of course, targeting FasL with antibody fragments was
also tested. In one example, CD20 binding variable domain
of Rituximab was converted into a scFv and linked to FasL.
This fusion protein had a high cytotoxic activity in a wide
range of malignant B-cell lines. Cell killing by this chimeric
molecule was more efficient than co-treatment with
Rituximab and FasL. Interestingly, this targeted FasL did
not affect normal B-cells or show any systemic toxicity [70].
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