Biomedical Engineering Reference
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protein must be able to engage the transferrin receptor, and
the fusion protein:antibody complex must be efficiently
internalized by cells. The fusion protein:antibody complex
must either dissociate within the cell, or the entire complex
must be degraded within the cell. This latter scenario, in
which the fusion protein does not release its cargo and
recycle, would impact dosing but, may still be a feasible
therapeutic strategy. Further, if the antibody or fusion
protein:antibody complex is recycled back into circulation,
the fusion protein will lack efficacy or may exacerbate
disease. Finally, as MG is caused by antibodies to other
AChR domains, SHG2210 would not be able to treat all
cases of MG.
Specific uptake of SHG2210
in HeLa cells
40
SHG2210
SHG2210 + HTF14
30
20
10
0
0
200
SHG2210 added (nM)
400
600
FIGURE 12.2
Uptake of SHG2210 in the presence or absence of
antitransferrin neutralizing antibody HTF14 HeLa cells were incu-
bated with increasing doses of SHG2210 or SHG2210
þ
fivefold
molar excess of anti-TF neutralizing antibody HTF14 for 2 h at
37
C. Following trypsin treatment to remove nonincorporated
protein, cell lysates were made and the amount of SHG2210 taken
into cells quantified by human transferrin ELISA. Values are the
mean of triplicate wells extrapolated from a standard curve gener-
ated with recombinant SHG2210.
12.3 CHARACTERIZATION OF SHG2210
There are two critical elements to the fusion protein strategy;
SHG2210 must bind to anti-
a
-subunit antibodies within the
circulation and SHG2210 must bind transferrin receptor,
which facilitates cellular internalization. A corollary to these
two prerequisites is that the antibody bound form of
SHG2210 must also bind TFR and be internalized by cells.
In vitro studies were performed to assess the efficacy of
SHG2210. Key assays and technology platforms include
cell-based binding and uptake assays, molecular imaging
and confocal microscopy studies, and antigenic modulation
assays. In vitro studies show (1) SHG2210 binds to anti-
AChR antibodies, (2) SHG2210:antibody complexes are
specifically targeted to the transferrin receptor on cells,
(3) fusion protein:antibody complex is taken into cells
and traffics to the lysosome, and (4) SHG2210 has a
protective effect on the antigenic modulation of the
AChR induced by serum from select MG patients. These
findings suggest that a fusion protein approach may be an
effective therapeutic for treating MG.
total cellular uptake occurring through nonspecific (i.e.,
nontransferrin receptor mediated) mechanisms. ELISA
results were further verified with molecular imaging data,
which demonstrated that fusion protein is specifically inter-
nalized in HeLa cells through transferrin receptor and that
fusion protein and transferrin receptor colocalize in the cell
cytoplasm [19]. Importantly, neither free fusion protein nor
fusion protein:antibody complex were appreciably internal-
ized through AChR receptor itself. Although
a
-
a
-subunit
interactions are known to occur, SHG2210 is not internal-
ized via AChR. Cross-linking and internalization of native
receptor could potentially lead to its trafficking to the
lysosome for degradation, which would exacerbate disease
rather than have a therapeutic effect.
12.3.1 SHG2210 Binding and Internalization
Studies in Cells
12.3.2 SHG2210 Anti-AchR Antibody Binding Studies
To characterize transferrin receptor specific binding and
uptake, SHG2210 uptake assays were performed in HeLa
cells, which are known to express high levels of transferrin
receptor and have been shown to internalize and recycle
transferrin [15,16]. Cells were treated with increasing doses
of SHG2210 for 2 h at 37
C in the presence or absence of
monoclonal antibody HTF14, which specifically binds to
human transferrin at an epitope required for recognition by
transferrin receptor [18]. Cells were washed to remove
surface bound but noninternalized protein, lysed in standard
cell lysis buffer, and assayed for fusion protein uptake using
a human transferrin enzyme-linked immunosorbent assay
(ELISA). As depicted in Figure 12.2, fusion protein uptake
occurs primarily through transferrin receptor, with
Binding to
a
-subunit specific anti-AchR antibodies was first
established using the well-characterized anti-AchR mono-
clonal antibodies mAb198 [20] and mAb 35 [21]. A com-
petition ELISA using immobilized torpedo fish AChR as
antigen was used to measure SHG2210 binding to anti-
AChR Ab (mAb 198). SHG2210, but not human transferrin,
inhibited binding of mAb 198 to AChR, with an IC
50
¼
0.37
0.15
m
M [19]. mAb 35 is highly confirmation specific
[21] and coimmunoprecipitation studies were used to verify
binding of mAb35 by SHG2210. Antibody binding was also
verified using serum from patients undergoing treatment for
MG disease. SHG2210 bound to anti-AChR antibodies in
the sera of patients known to have high anti-
a
-subunit titers,
while antibody binding was weak or absent in patients with
20% of
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