Biomedical Engineering Reference
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FIGURE 9.2
Rilonacept as an example of a heteromeric ligand trap engineered to mimic the high-
affinity binding observed with the natural receptor complex. (A) Interleukin 1 (IL-1) has two receptor
binding sites (light and dark gray). The IL-1 receptor type I (IL-1R1, also referred to as IL-1-RI) binds
to one site and IL-1 receptor accessory protein (IL-1RAP, also referred to as IL-1-RAcP) to the other,
and binding of IL-1 initiates signaling. (B) The IL-1 Trap (rilonacept) is constructed as an “in-line”
fusion of the extracellular domains of IL-1RAP and IL-1R1, followed by the Fc domain of human
IgG1. The two Fc domains drive dimerization of each trap monomer, and the resulting homodimer is
further stabilized and held together via two disulfide bonds. IL-1 binds to rilonacept with high
affinity, similar to the binding seen with the receptor complex on the cell surface. (C) The naturally
occurring IL-1 receptor antagonist (IL-1RN) binds to only one site and as a result prevents complex
formation and receptor activation. In contrast to the rilonacept
IL-1 interaction, which displays a
low K
d
in the picomolar range, the binding of IL-1RN to IL-1R1 forms a low-affinity complex.
Source: Reprinted by permission from Macmillan Publishers Ltd Nature Medicine (Dinarello Nat.
Med. 2003 Jan, 9(1) 20-22), copyright (2003).
These two groupings are artificial, yet they are useful in
illuminating the challenges that have to be overcome when
attempting to use receptor ectodomains to design specific
and high-affinity blockers. For the second type of receptor
system, it is usually not possible to utilize the traditional
receptor-Fc methodology to create high-affinity blockers
for their cognate ligands because simple dimerization of
one receptor subunit will either result in a “promiscuous”
blocker with relatively low affinity (see, e.g., gp130-Fc,
discussed below), or worse yet generate a protein that will
act as a carrier for and potentiator of cytokine activity. The
latter property is best reflected in the ciliary neurotrophic
factor (CNTF) and IL-6 receptor systems, where it has
been elegantly demonstrated that the soluble or shed forms
of IL-6R [126] and CNTFR [123] enable signaling in cells
that do not express them, but which express the shared
receptor subunits (e.g., gp130 for IL-6/IL-6R; and
gp130 plus LIFR
b
for CNTF/CNTFR). This process has
been termed “trans-signaling” [23,96] (Figure 9.3). In fact,
we have used this property to design cell type-specific
potentiators of cytokine activity and achieve exactly the
opposite of a blocker, that is, to selectively convert IL-6- or
CNTF-unresponsive cells to responsive ones by addressing
IL-6R-Fc and CNTFR-Fc to the surface of IL-6R- and
CNTFR-negative cells [127].
9.3.1 Proof-of-Concept for High-Affinity Traps for
Ligands Utilizing Multicomponent Receptor Systems
As the earlier discussion indicates, the mechanism of
receptor assembly for factors and cytokines utilizing multi-
component receptor systems imposed unprecedented limi-
tations on the design of receptor ectodomain-based blockers,
and therefore required the development of a new kind of
technology. A general solution to the problem of generating
receptor-based blockers for these types of ligands was
provided by the invention of Trap technology [53]. Traps
are fusion proteins that are composed of more than one
receptor ectodomain (or part thereof) fused to the constant
region of immunoglobulins. The first sets of Traps to be
developed were for IL-6 and CNTF, but their successful
engineering was rapidly followed by development of Traps
for multiple other cytokines and growth factors.
The methodology for engineering Traps for ligands uti-
lizing multicomponent receptor systems reflects the mecha-
nism by which certain ligand
receptor signaling complexes
form (Figures 9.1b and 9.3). Two key observations informed
the design:
1. Signaling receptor complexes form in a sequential,
ordered fashion where the ligand binds to a first
receptor subunit, usually the specificity-determining
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