Biomedical Engineering Reference
In-Depth Information
to improve physical, chemical, and pharmacological prop-
erties will be the key to its success in future development,
manufacturing, and clinical use.
better binding affinity to the receptor than the free peptide
and was 100-fold more active than EMP1 in an in vitro UT7
cell proliferation assay (Figure 8.2). By further carefully
engineering a linker region between the peptide and the Fc,
we were able to improve the activity of CNTO 528 an
additional twofold. The new molecule, CNTO 530, demon-
strated significantly more activity in vivo as measured by its
ability to increase hemoglobin levels in normal rats relative
to its parent protein [95].
8.6.1 Improving Activity
In most cases, peptides will lose potency upon fusion to
either the N- or C-terminus of an Fc domain. This could be
due to any one or a combination of the following factors: the
loss of translational/rotational entropy associated with
fusion to a much larger partner, steric hindrance, conforma-
tional changes of the peptide, loss of interactions with the
N- or C-terminus which may be involved in binding, or
differences in posttranslational modification (e.g., amidation
of the carboxy terminus). In many cases, protein-engineer-
ing efforts to restore some of the activity or to minimize the
activity losses are necessary. For example, direct fusion of
DPP-IV-protected GLP-1 analog (V8-GLP-1) to the Fc-
hinge region dramatically reduced in vitro activity (by
8.6.2 Improving Stability
Many unconstrained peptides are flexible enough to be
easily accessed and digested by tissue or serum proteases
in vivo. Improving their in vivo stability and making them
more resistant to oxidative stress, changes of temperature,
pH, and solution condition are crucial for their use as
therapeutic drugs. We routinely evaluate peptide stability
by incubating peptides or peptide-Fc-fusion proteins in
plasma, tissue homogenates, or cell lysates at 37 C for a
period of time and then test their biological activity and
molecular integrity. In many cases, we have observed that
peptides were significantly better protected from protease
cleavage when they are fused to an Fc domain. However, for
those more metabolically labile fusions, additional engineer-
ing efforts are needed to stabilize the peptide region. In this
scenario, several approaches should be considered, includ-
ing constraining the peptides, modifying the labile sites, or
introducing carbohydrate moieties nearby to shield the labile
sites [87,93]. For example, one of our MIMETIBODY
constructs contained a protease-sensitive peptide. Multiple
cleavage products were observed after this protein was
purified from tissue culture supernatants. We performed a
disulfide scan through the entire peptide region and
95%) compared to that of free V8-GLP-1.
However, by insertion of a linker with optimal length
and sequence between the C-terminus of the peptide and the
N-terminus of the hinge, the newly engineered peptide-Fc-
fusion protein demonstrated approximately fourfold greater
in vitro potency over that of free peptide [87].
Careful engineering of a peptide-Fc fusion, in some
cases, can make the fusion protein significantly more potent
than the free peptide as illustrated in the example of EPO
MIMETIBODY, CNTO 528. As discussed in Section
8.5.2.1, CNTO 528 contains an EPO mimetic peptide,
EMP1, which binds to EPO receptor and exerts effects
similar to the native ligand, EPO. However, EMP1 binds
to EPO receptor with more than 1000-fold weaker affinity
than the native ligand [94]. CNTO 528 showed significantly
FIGURE 8.2 Supernatants obtainedby transient transfection forCNTO528 (EC 50 ¼ 2.28 10 10 M)
or CNTO530 (EC 50 ¼ 5.00 10 11 M) were tested for proliferative activity in UT-7 cells. Viable
cells monitored by MTS absorbance at 490 nm are plotted as a function EPO receptor agonist
concentration in log scale. Human recombinant EPO (EC 50 ¼ 2.14 10 11 M) was used as positive
control and cell supernatant fromamock transfectionwas used as negative control. Data are presented as
the mean SD (n ¼ 2).
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