CELL-SPECIFIC TARGETING OF FUSION PROTEINS THROUGH HEPARIN BINDING - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 407

FIGURE 27.4 NRG1's heparin-binding domain targets HBD-S-H4 to cell surfaces through
heparan sulfate interactions. (A) CHO-wt and CHO-pgsd677 (mutant) cells, with deficient heparan
sulfate synthesis, were incubated with biotinylated HBD-S-H4 or H4 (50 nM) with or without
heparinase treatment. Compared to the other conditions, HBD-S-H4 treated CHO-wt cells had the
highest green fluorescence intensity that appeared to concentrate in ECM between cells. Scale bar is
50 m m. (B) A binding assay of protein constructs to CHO cells shows the enhanced binding ability
of HBD-S-H4 to wild-type CHO cells (CHO-wt), but not to mutant cells. The H4 construct did
not bind to these cells. and indicate significant differences at P

< 0.001 and < 0.005, respectively.
(C) Increasing concentrations of biotin-conjugated HBD-S-H4, H4, or IgG were added to heparin
coated 96-well plates to produce a saturation-binding curve. The amount of HBD-S-H4 bound to
heparin increased until saturation of binding sites. A calculated dissociation constant (Kd) is 14.7 nM.
Source: This research was originally published in J. Biol. Chem. Reference 79.

specificity that leads to NRG1 deposition on specific tissues
during chick embryonic development is preserved with the
HBD-S-H4 antagonist. Within the developing spinal cord,
NRG1 is highly expressed in and released by motor and
sensory neurons where upon it adheres to specific tissues and
cell types [60,67,80,81]. The tissue distribution of exoge-
nously added HBD-S-H4 was compared to NRG1 tissue
distribution as well as the H4 construct in developing
embryos. Protein distribution of HBD-S-H4 was determined
using a specific antibody that only recognizes the human
HBD domain of NRG1 and does not cross-react with
endogenous chicken NRG1 [82] and compared to a chicken
NRG1 specific antibody staining (Figure 27.5). HBD-S-H4
became concentrated in the same regions of the spinal cord
and peripheral nerve as endogenous NRG1. In addition, the

fusion protein also accumulated outside the nervous system
in regions that do not normally express NRG1, most likely
because of high concentrations of HSPGs in these regions as
well. To demonstrate that HBD-S-H4 is bound through ionic
interactions to HSPGs, we showed that we could remove it
from histological sections by high salt treatment [67,79]. In
fact, a double-labeled region of the ventral nerve root shows
that the same regions of peripheral nerve where chicken
NRG1 is concentrated is also enriched with high levels of
HBD-S-H4 (Figure 27.5b).
Tissue-specific targeting of HBD-S-H4 to the exact same
regions where the NRG1 agonist is targeted in HS-rich cell
surfaces thus places the NRG1 antagonist fusion protein on
an even footing with the natural agonist. This novel, entirely
“humanized” fusion protein could therefore be a powerful