Biomedical Engineering Reference
In-Depth Information
Molecule size has a profound effect on the clearance rate
from the vascular system via the kidneys as demonstrated by
the PEGylation of GH. Another means of increasing the
molecule size of GH is to create a fusion of GH with another
“inert” molecule; the obvious advantage of a fusion mole-
cule is that the increase in size, and hence the reduced
clearance rate, could be achieved without costly processing
of the expressed molecule. Fusion of GH with albumin has
been shown to have delayed clearance [12] as has the
approach discussed in this chapter, the fusion of GH with
its receptor [13,14].
GH has two binding sites for its receptor and acts by
forming a complex with a GHR dimer at the cell surface; this
initiates a conformational change in the complex and trig-
gers intracellular signaling (Figure 15.1A). The extracellular
domain of GHR is proteolytically cleaved and circulates as
GH-binding protein (GHBP). Circulating GH binds to
GHBP as a 1:1 molar complex and this biologically inactive
complex is protected from degradation and has a reduced
rate of clearance [15,16]. A covalently cross-linked complex
of GH-GHR has delayed clearance but is biologically
inactive [17], and an augmentation of GH activity is
observed when separately purified GH and GHBP is admin-
istered at a ratio of 1:1 [18]. Thus, the clearance rate of GH is
reduced due to the circulating reservoir of GH-GHBP
complex, which is in equilibrium with active free GH
[19]. A similar system is utilized in many other hormonal
systems to increase the circulating half-life of the hormones.
This concept of protecting GH from degradation and
clearance by linking it to the extracellular domain of GHR
(GHRec) was the basis of the design of the ligand-receptor
(LR) fusions. Fusion proteins of GH covalently linked end-to-
end with GHRec were designed; based on our observations
that a truncated variant of GHR at the cell surface acts a
dominant negative inhibitor of GHR [20,21] we initially
hypothesized that the LR-fusion would be an antagonist
(Figure 15.1B). Unexpectedly, the GH LR-fusions generated
were potent long-acting agonists (Figure 15.1C).
FIGURE 15.1
(A) The signaling mechanism of GH, a GH
molecule binds to a GHR dimer at the cell membrane (CM) this
causes a conformational change that initiates signaling. (B) A
diagram of how the LR-fusions may act as an antagonist by forming
an inactive complex with a single GHR molecule at the cell surface.
(C) A diagram of how the LR-fusion may act as an agonist by
mimicking the action of GH.
15.2 THE GHLR-FUSIONS
The GH LR-fusions were generated at the genetic level by
cloning the different components of the fusion protein
sequentially into the expression vector. The LR-fusions
were then generated by expression of the protein from these
expression vectors.
Initial testing of the LR-fusions was performed on dif-
ferent combinations of GH, the extracellular domains of
GHR and length of linker between the two constituents of
the fusion protein. The lead LR-fusion chosen for this study
consisted of GH (growth hormone, 1-191) linked to GHRec
(growth hormone extracellular domain, 1-238) by a flexible
(Gly
4
Ser)
4
linker (Figure 15.2). This prototype molecule,
variant 0 (GH-LRv0), consisted of extraneous sequence as
an artifact of the cloning processes required to generate the
LR-fusion gene, for example, restriction sites.
To reduce the immunogenic profile of the LR-fusion, all
nonimportant sequences were removed from the fusion
protein. Extraneous sequence at the N-terminus and the
C-terminus of the gene was removed, to give variant 1
(GH-LRv1). The restriction sites at either end of the linker
region were then removed, to produce variant 2 (GH-LRv2).
The N-terminal region of GHR is disordered in the crystal
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