Biomedical Engineering Reference
In-Depth Information
TABLE 4.5 Comparison of IC 50 Values from Competitive Receptor Binding Assays and Plasma-Half Lives of
GH-Tf and G-CSF-Tf Fusion Proteins
Protein
IC 50 , GHR (nM)
IC 50 , G-CSFR (nM)
IC 50 , TfR (nM)
t 1/2 (h)
GH-LE-Tf
17.7
/
21.2
4.97 0.34
GH-cyclo-Tf
8.2
/
4.2
1.76 0.27
GH-(H4) 2 -Tf
7.0
/
8.7
1.87 0.44
G-CSF-LE-Tf
/
38.0
7.5
4.15 0.75
G-CSF-cyclo-Tf
/
39.5
0.9
5.69 0.46
G-CSF-(H4) 2 -Tf
/
31.2
4.5
4.84 1.18
IC 50 values were determined via competitive receptor binding assay on receptor-bearing cells. Unlabeled fusion proteins were used to
compete with radiolabeled free GH, G-CSF, or Tf for the respective receptor binding. Smaller IC 50 value represents higher receptor
binding affinity. Half-life values represent mean SD from three to four mice.
The impact of linker insertion on receptor binding affinities
resulted in dramatic differences in the PK profiles of three
hGH-Tf fusion proteins. hGH-LE-Tf exhibited an almost
threefold longer plasma half-life compared to the others,
despite the three fusion proteins having very similar size
and sequence (Table 4.5). A strong correlation between
hGHR binding affinity (but not TfR binding affinity) and
plasma half-lifewas identified for the hGH-Tf fusion proteins.
hGH-LE-Tf, which had the weakest binding for hGHR,
exhibited the longest plasma half-life. hGH-cyclo-Tf and
hGH-(H4) 2 -Tf, which exhibited comparable hGHR binding
affinities, had similar plasma half-lives.
These results suggested that binding of hGH-Tf fusion
proteins to hGHR likely led to endocytosis and lysosomal
degradation of the fusion proteins as reported for free hGH [70].
Like many other hormones and cytokines, hGH and hGHRwill
be endocytosed into endosome following receptor binding,
where hGHR is ubiquitinated [71]. The hGH-hGHR complex is
then further sorted to lysosome and gets degraded. The receptor
binding of hGH domain in hGH-Tf probably leads to similar
intracellular processing as free hGH. As a result, hGH-LE-Tf
exhibited longer half-life because of its lower binding affinity to
hGHR and consequently less degradation via receptor-medi-
ated endocytosis. This result indicated that linker insertion
could greatly affect the receptor binding and subsequent
intracellular processing, and ultimately alter the plasma half-
lives of bifunctional fusion proteins. It also suggested that
hGHR bindingwas the primary binding site for determining the
plasma half-lives of the hGH-Tf fusion proteins.
To further test whether linker insertion could affect PK
profiles of other bifunctional fusion proteins, the same
three linkers were adopted to construct G-CSF-Tf fusion
proteins. The impact of linker insertion on receptor binding
affinities and PK of fusion proteins was observed again in
G-CSF-Tf as in hGH-Tf (Table 4.5). Interestingly, different
from hGH-Tf, the G-CSF receptor (G-CSFR) binding
affinities of three G-CSF-Tf fusion proteins were quite
similar, whereas their Tf receptor (TfR) binding
affinities were statistically different. The plasma half-lives
of G-CSF-Tf fusion proteins followed the ranking of
G-CSF-cyclo-Tf (5.69 h)
G-
CSF-LE-Tf (4.15 h). Stronger TfR receptor binding affinity
of G-CSF-Tf fusion protein was correlated with longer
plasma half-life.
These results suggested that TfR binding of G-CSF-Tf
fusion proteins might follow the classic Tf-TfR recycling
pathway [60,71]. For endogenous Tf, its receptor binding to
TfR at cell surface usually leads to the endocytosis of the Tf-
TfR complex. With the acidification of endosome, Tf releases
Fe 3 รพ and becomes Apo-Tf, which retains high binding affinity
to TfR at acidic pH within endosome, until the complex is
recycled back to the cell surface. The neutral extracellular pH
promotes the release of Apo-Tf from the TfR. Taking advan-
tage of this property; Tf can be recycled and reused by the cells
for intracellular iron delivery. These results suggested that TfR
binding of G-CSF-Tf may lead to the recycling of the fusion
protein through the Tf-TfR recycling pathway and prolongs
the plasma half-life of the fusion proteins. As a result, the
stronger TfR binding affinity, the longer plasma half-life was
exhibited by the G-CSF-Tf fusion proteins (Table 4.5).
A mechanistic PK model was proposed for hGH/G-CSF-
Tf fusion proteins (Figure 4.4). Owing to the competition
from high level of endogenous Tf, the fusion proteins likely
bind first to the protein drug receptor (e.g., hGHR/G-CSFR)
on the plasma membrane of target cells. This binding is
considered the primary binding, which enriches the fusion
proteins onto the target cells for the drug action, and may
also increase the accessibility of the Tf domain to TfR. TfR
binding, which likely occurs after hGHR/G-CSFR binding
either on the plasma membrane or inside the endosomes, is
then referred to as secondary binding. Following the
hGHR/G-CSFR binding, signal transduction is initiated,
and the fusion proteins are endocytosed into the early
endosome, where TfR is also present [72]. Within the acidic
endosome, the fusion proteins may dissociate from
hGHR/G-CSFR, and bind tightly to TfR because of the
high binding affinity of Tf to TfR at acidic pH [60]. The
receptor binding inside the endosomes dictates different
fates for the fusion proteins: The hGHR/G-CSFR binding
leads to the lysosomal degradation while the TfR binding
G-CSF-(H4) 2 -Tf (4.84 h)
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