Biomedical Engineering Reference
In-Depth Information
of 28 amino acids and the O-linked oligosaccharides. These
data may indicate that the O-linked glycosylation
recognition site of the C-terminal region is preserved
even though the sequence is fused to different proteins.
13.3.3.2 Thyrotropin (TSH). Thyrotropin is a member of
the glycoprotein hormone family, which includes lutropin
(LH), follitropin (FSH), and chorionic gonadotropin (hCG).
These hormones are heterodimeric proteins that share a
common a -subunit but differ in their hormone- b specific
subunit, which is unique and confers biological specificity.
These hormones activate the target cells via G-protein coupled
receptors that, in the case of TSH, lead to increased production
of the thyroid hormones, triiodothyronine (T 3 ), and thyroxine
(T 4 ). Thyroid hormones act on organs throughout the body to
regulate growth and various metabolic processes.
Recombinant TSH is important in clinical studies of
thyroid cancer. Because of its therapeutic potential, a longer
acting analog of TSH was constructed by fusing the
carboxy-terminal extension peptide (CTP) of hCG b onto
the coding sequence of TSH b -subunit. When co-expressed
either with a -subunit complementary DNA or alpha mini-
gene in African green monkey (COS-7) or in human embry-
onic kidney (HEK293) cells, the chimera was fully bioactive
in vitro and exhibited enhanced in vivo potency associated
with a prolonged plasma half-life. Ligation of the CTP to the
C-terminal of TSH b -subunit did not affect the assembly and
secretion of chimeric TSH. Wild-type and chimeric TSH
secreted by COS-7 and 293 cells displayed wide differences
in their plasma half-lives, presumably due to the presence of
terminal sialic acid and SO 4 on their oligosaccharide chains,
respectively. Chimeric and WT-TSH secreted by both cell
lines demonstrated similar bioactivity in cAMP production.
Chimeric TSH appears to be more effective in COS-7 cells
than in 293 cells, as judged by growth assay. COS-7-
produced chimeric TSH showed the maximum increase in
half-life, indicating the importance of sialic acid in prolong-
ing half-life and in vivo potency. Sulfation of both subunits,
predominantly beta and to a lesser extent alpha, appears to
be responsible at least in part for the increased metabolic
clearance of WT and chimeric TSH secreted by HEK293
cells. Apart from its therapeutic potential, chimeric TSH
produced in various cell lines can be used as a tool to
delineate the roles of sulfate and sialic acid in the in vivo
clearance and, thereby, the in vivo bioactivity [21].
Assembly of the glycoprotein hormone subunits is the
rate-limiting step in the production of functional hetero-
dimers. To bypass the problem of dimerization of deglyco-
sylated subunits, hTSH b - and a -subunits were genetically
fused in a single chain hormone with or without the CTP as a
linker between the subunits. Single chains of hTSH retained
a biologically active conformation similar to that of the wild-
type heterodimer [22,23].
13.3.3 Bioactivity of Designed Long-Acting
Glycoproteins
13.3.3.1 Follicle-Stimulating Hormone (follitropin,
FSH) is a pituitary glycoprotein hormone essential for main-
tenance of ovarian follicle and testicular tubule development.
Because FSH specifically induces aromatase enzyme in
granulosa cells, in vitro signal transduction of the modified
FSH dimers was assessed in the granulosa cell aromatase
bioassay by measuring hormone-stimulated estrogen produc-
tion. The detected steroidogenic activity in vitro of FSH-CTP
chimeras was comparable to that of wild-type FSH. Thus,
ligation of CTP to the coding sequence of FSHsubunit, did not
affect receptor binding affinity or in vitro bioactivity. The in
vivo biopotencies of wild-type FSH and the chimeras were
examined by determining ovarian weight augmentation and
granulosa cell aromatase induction. It was found that ovarian
weight increased significantly between animals treated with
wild-type FSH and the FSH chimeras. In addition, estrogen
production by granulosa cells from chimera-treated rats
increased threefold to fivefold over that seen in rats treated
with wild-type FSH.
Because the increased biopotency of the chimeras may
reflect a change in their in vivo longevity, the circulatory
half-life of the hormones was detected. The clearance
patterns of the wild-type and chimera reveal multiple phases.
It seems that the clearance of the chimera is much slower
than that of wild-type FSH, presumably. RIA determinations
show that a high level of the chimera is still detectable in
serum after 24 h and yet injected wild-type hFSH reaches
basal level between 8 and 24 h [16,17].
The safety, pharmacokinetics, and pharmacodynamics of
FSH-CTP were studied in hypogonadotrophic hypogonadal
male subjects as a Phase I multicenter study. The results
indicated that FSH-CTP use is safe and does not lead to
detectable formation of antibodies. Furthermore, pharmaco-
kinetic and dynamic profile of FSH-CTP seemed to
be promising. Compared with recombinant FSH-WT
(Puregon 1 ), the half-life of FSH-CTP was increased two
to three times [18].
Further studies in in vitro fertilization (IVF) patients,
indicated that a single dose of FSH-CTP is able to induce
multifollicular growth compared to daily injection of
FSH-WT for 7 days [19,20]. According to the promising
results described earlier in clinical trials, on January 28,
2010theEuropeanCommission(EC)gaveMerck&Co.
marketing approval with unified labeling valid in all
European Union Member States for FSH-CTP, now
branded as Elonva 1 .
13.3.3.3 Erythropoietin (EPO). Erythropoietin is a gly-
coprotein hormone produced primarily by cells of the
peritubular capillary endothelium of the kidney. EPO pro-
duction is stimulated by reduced oxygen content in the renal
arterial circulation. Circulating EPO binds to EPO receptors
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