SIGNAL CONVERTER PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 443

“loop-back” mechanism of action is indeed operative [90].
The latter included the demonstration, through both soft agar
clone formation and cell dilution analysis that CD40

some kind of coordinate signaling might be capacitating the
Fas receptor. The molecular underpinnings for this capaci-
tation turned out to be the ability of CTLA-4

FasL
inhibition does not require intercellular contact. Moreover,
the notion that cis loop-back signaling can be artificially
elicited using fusion proteins derives support from another
line of investigation. Our group has demonstrated that when
costimulators are painted on to the surfaces of T cells, in the
form of CoS

FasL, but not

CTLA-4 Ig, agonistic anti-Fas antibody, or the two in com-

bination, to abrogate the usual increase in expression of the
anti-apoptotic protein, c-FLIP. This framed a model for
CTLA-4

FasL action wherein there is coordinate triggering
of a death receptor (Fas, CD95) and suppression of activa-
tion-driven induction of a pivotal anti-apoptotic protein
(c-FLIP) that otherwise dampens the activity of that death
receptor [93]. This coupled effect offers an explanation not
just for CTLA-4

Fc g 1 :palmitated protein A conjugates, they can
trigger their cognate costimulator receptors on the same
cells, thereby yielding auto-activating T cells [92]. Paren-
thetically, this finding also establishes that it is not just
autoinhibition that can be artificially elicited with fusion
proteins, but also auto-costimulation.
The feasibility of using fusion proteins to autotrigger
receptors at cell surfaces raises an intriguing cis/trans
paradox. Given that ligands and their cognate receptors
are often naturally co-expressed on the same cell surfaces
(LFA-3 and CD2 on T cells, as but one of many examples),
why is there no constitutive receptor triggering, or alterna-
tively, competitive blockade of incoming trans signals? And
on the flip side, why do painted ligands, whether proteins
paints or CLBP, elicit autotriggering so readily, while the
natural ligand:receptor pairs seem to ignore each other?
While autocrine signaling by soluble secreted ligands, such
as growth factors, has been dealt with extensively in
the literature, it is curious that this cis/trans paradox for
membrane-associated ligands has been essentially ignored.
As the catalog of CLBP grows, new insights into the
determinants of, and constraints upon, autotriggering will
likely be forthcoming.

FasL's high efficacy, but also for its
distinctive capacity to promote early T-cell apoptosis.
This CTLA-4 FasL example captures a recurring theme

that has emerged from SCP studies to date. Chimerization
provides functional dividends that cannot be recapitulated
by simply delivering the fused components as a combination
of separate molecular entities. New mechanisms and func-
tions are emergent within fusion proteins. The growing list
of chimerization dividends for SCP includes abilities to

elicit new, sometimes unexpected, functionalities (as
exemplified by CTLA-4

FasL's effect on c-FLIP)
co-localize targeted molecules on cell membranes,
sometimes generating macromolecular assemblies
associated with enhanced function
deliver ligands to preselected cell types and organ sites
in vivo, thereby concentrating therapeutic agent at the
right location (as exemplified by Fn14 TRAIL deliver-

ing TRAIL to inflamed TWEAK þ endothelium)
tether ligands to membranes, thereby eliciting higher
order ligand function (as exemplified by the various
FasL-containing SCP that yield membrane-anchored
FasL)
enhance functional avidity by enforcing multimeriza-
tion (discussed later in this chapter).

30.6 MECHANISTIC DIVIDENDS
OF CHIMERIZATION

While signal conversion is the initial design focus for each
SCP, greater functional dimensionality is often revealed.
Intriguingly, an SCP can function in ways that its component
parts cannot replicate, alone or even in combination. The
emergence of such chimerization-dependent properties has
pointed to an important fusion protein principle—an SCP
can be more than the sum of its parts.
CTLA-4

FasL:c-FLIP connection represents a situa-
tion in which a unique SCP mechanism of action could not
have been readily predicted based upon known functions of
its components. In other instances, however, SCP mecha-
nistic insights flow in a more straightforward manner from
new information that becomes available on protein func-
tional repertoires. CTLA-4

The CTLA-4

FasL is a case in point. As set forth earlier,

FasL is illustrative here again, in
light of new insights into the CTLA-4 component's func-
tional repertoire. For example, though largely studied as an
inhibitory receptor on effector T cells, CTLA-4 has subse-
quently been linked to regulatory T cells. In particular,
soluble CTLA-4 (in the form of CTLA-4

CTLA-4 FasL, our paradigmatic TSCP, exhibited exception-

ally high efficacy, when compared to its soluble CTLA-4 or
FasL components, alone or in combination. This observation
prompted deeper investigation into mechanisms of action
unique to chimeric CTLA-4 FasL [52]. The absence of a

Ig) can indirectly
drive the generation of CD4 þ CD25 hi regulatory T cells, by
reverse signaling through B7-2 on dendritic cells and
thereby eliciting from them the potential to induce regula-
tory T cells [94]. This new functional dimension for one

CTLA-4 FasL effect on cell cycle entry and progression

pointed away from the kind of classical anergy associated
with CTLA-4

Ig. However, an intriguing mechanistic clue
came from the further observation that CTLA-4 FasL elicits

apoptosis as early as 24 h after T-cell activation, hinting that