Biomedical Engineering Reference
In-Depth Information
in patients is varied. For example, MG patients can develop
variable antibody responses to multiple subunits, which can
range from a polyclonal response to a number of subunit
epitopes or a multiple response to a single subunit. Approx-
imately 50% of MG patients develop notable antibody levels
to the a -subunit [12]. Anti- a and anti- b subunit antibodies
are both able to cross-link and downregulate AChR receptors
(antigenic modulation). The anti- a antibodies are approxi-
mately four times more effective than anti- b -subunit anti-
bodies at promoting antigenic modulation [13]. Antibodies
to the a -subunit are able to induce experimental auto-
immune myasthenia gravis (EAMG) in mice, while the
effect of the anti- b antibodies is much weaker [8]. These
data suggest that anti- a -subunit antibodies play a key role in
MG pathogenesis.
Current treatment options for MG patients are invasive
and are not targeted therapies for MG. Thus, novel thera-
peutic approaches, which eliminate the need for surgery and
the complications of reoccurring plasmapheresis are desir-
able. The in vitro studies described in this chapter are a
positive first step in the characterization of one such
approach: specific targeting of a domain specific anti-
AChR antibodies and clearing the antibodies from circula-
tion via a fusion protein. Given the broad range of antibodies
that can cause MG, it is worthwhile to consider a combina-
torial therapeutic strategy. The potential efficacy of a fusion
protein targeted to a domain specific anti-AChR antibodies
suggests the possibility of developing multiple subunit
AChR-transferrin fusion proteins for the treatment of
MG. When used in combination with anti- a -subunit therapy,
this experimental therapeutic approach could address a
range of MG patients.
the surface of almost all cell types and transferrin recycling
mediated via the transferrin receptor occurs very rapidly, on
the order of 5-15 min [15-17]. Serum TF, in the iron bound
form (holotransferrin) binds to its target cell surface receptor
triggering internalization through clathrin-mediated endo-
cytosis into early endosomes. Acidification of the endosome
releases iron from transferrin, and this iron-free form
(known as apotransferrin) in conjunction with transferrin
receptor is returned to the cell surface, via Rab11
recycling
endosomes. Thus, for proof of principle studies to test the
concept of a fusion protein, TF was identified as the fusion
partner to a -subunit AChR to facilitate rapid and specific
receptor-mediated cellular uptake in a wide range of cells.
The targeted mechanism of action (Figure 12.1) is (1)
binding of a -subunit AChR antibodies to a -subunit AChR of
fusion protein, (2) binding of Ab-bound fusion protein to
TFR via TF domain of fusion protein, (3) uptake of fusion
protein and antibody complex into cells (serum clearance),
(4) separation of fusion protein from cargo (anti-AChR
antibody) within the endosome, (5) degradation of cargo
in lysosomes or via the proteasome, and (6) ideally, recy-
cling of fusion protein back to the circulation to bind and
clear a -subunit AChR antibodies once again.
There are several challenges the a -subunit AChR:TF
(SHG2210) fusion protein faces. The fusion protein must
be able to bind to anti- a -subunit antibodies specific to native
antigen; AChR. The antibody-bound form of the fusion
þ
12.2 FUSION PROTEIN SHG2210 DESIGN
Chimeric proteins containing portions of the a -subunit fused
to GST have been shown to effectively bind anti-AChR
antibodies and can protect and treat MG-like disease in both
chronic and acute EAMG models [14]. There are two key
factors that contribute to the design of SHG2210 fusion
protein as a targeted therapy for MG; the target molecule
implicated in disease is known ( a -subunit AChR antibodies)
and TFR-mediated cellular uptake can be exploited as a
means to clear target molecules from circulation.
Although MG patients have an array of antibodies against
various AChR subunits, the majority of patients develop an
autoimmune response to the main immunogenic region
(MIR) region of the a -subunit of AChR. Thus, to test the
concept of a fusion protein, the a -subunit of the AChR
containing the MIR domain was chosen to capture disease
causing anti- a -subunit AChR antibodies.
TFR-mediated cellular uptake is a complex process that is
studied extensively. Transferrin receptors are ubiquitous on
FIGURE 12.1 Schematic representation of proposed mechanism
of action of SHG2210. SHG2210 fusion protein binds to anti-AChR
a -subunit specific autoantibodies in the circulation through inter-
actions with the AChR a -subunit portion of the fusion protein.
Fusion protein:antibody complex is then internalized by cells
expressing the transferrin receptor. Internalized complex may
then be dissociated to its individual components within the endo-
some as it acidifies or remain as an associated complex. Associated
complex or the individual components of the complex may then
follow one of two pathways: default trafficking to the lysosome for
degradation or recycling back to the cell surface via fusion with
Rab11 þ recycling endosomes.
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