MONOMERIC Fc-FUSION PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 111

12.5 IU/kg
25 IU/kg
50 IU/kg
100 IU/kg

which is markedly longer than reported for the recombi-
nant protein (11.4 h) [74]. The longer half-life of FSHFc
heterodimer likely explains the significantly increased
bioactivity of the fusion protein in rats compared to
recombinant FSH.
Recombinant FSH and FSHFc heterodimer were also
tested in a testis weight gain assay in neonatal rats after
oral administration [75]. FcRnexpressionandIgGtrans-
port is relatively high during the first few weeks of life in
rodents, while secreted acid in the stomach and the level of
digestive enzymes are low. As a result, this model is useful
in studying the transport and PKs of Fc-fusion proteins
from the epithelial cells in the intestine into the circulation
[76-77]. Recombinant FSH had no effect on testis weight
compared to vehicle control treated animals while FSHFc
heterodimer administrationresutedina ignificant
increase in testis weight [33] indicating the FSHFc heter-
odimer is more stable than the recombinant protein after
oral administration in rats, and that the Fc fusion in
combination with FcRn expression has the potential for
alternate routes of administration for molecules that are
otherwise restricted to subcutaneous and intramuscular
injections.

100

10

1

0.1

0 100 200

Time post dosing (h)

300 400

FIGURE 7.5 Dose-dependent pharmacokinetic profiles of
rFIXFc after a single intravenous dose of in subjects with severe
hemophilia B. Plasma FIX activity levels were measured over time
after a single intravenous infusion of 12.5 (n ¼ 1), 25 (n ¼ 1), 50
(n ¼ 5), or 100 IU/kg (n ¼ 5) rFIXFc. Results presented are mean
SEM. Source: This research was originally published in
Reference [65].

monomeric Fc fusion technology is also useful for proteins
that require a heterodimeric partner for optimal activity.

7.4.2 Cytokine-Fc-Fusion Proteins

Cytokines have an impact on many critical biological pro-
cesses including regulation of gene expression, cell prolif-
eration, and promotion of chronic inflammation. Cytokines
are also known to be involved in the initiation and perpetua-
tion of human diseases and these effects can be dampened by
the use of soluble receptors that act as competitors with
endogenous receptors for cytokine binding. For example,
Etanercept is a homodimer of the ectodomain of TNF- a
receptor fused to human Fc of IgG1. Etanercept thus inhibits
the inflammatory function of TNF- a and is used in the
treatment of rheumatoid, juvenile rheumatoid and psoriatic
arthritis, plaque psoriasis, and ankylosing spondylitis. In
many cases however, cytokines act as multicomponent
systems and the use of soluble receptors is limited since
cytokine affinity for monomeric receptors is often low
suggesting that Fc monomer technology would have limited
utility for improving cytokine function. However, it has been
demonstrated that heterodimeric Fc-fusion proteins of inter-
leukin (IL)-2/IL-12 and IL-4/granulocyte-macrophage col-
ony stimulating factor results in synergistic biological
activity similar to that seen with the combination of indi-
vidual cytokines [78]. In addition, cytokine traps consisting
of the extracellular domains of two distinct cytokine recep-
tors (e.g., IL-6 receptor a and gp-130) each fused to human
Fc and expressed in mammalian cells as IL-6 receptor
a Fc/gp-130Fc heterodimer fusions, have been shown to
constitute one of the most potent inhibitors of cytokine
action reported [79].

7.4.1 Follicle Stimulating Hormone-Fc-Fusion Proteins

Follicle stimulating hormone (FSH) is commonly used in the
treatment of infertility in both men and women and is
administered daily by subcutaneous or intramuscular injec-
tion for several days [68,69]. FSH binds to FSH receptors in
the granulosa cells of the ovary resulting in the selection and
growth of ovarian follicles. This mechanism of action has
commonly been used in rats as a bioactivity assay in which
ovarian weight is measured in response to FSH treatment
[70]. FSH is a noncovalently linked heterodimer of a and b
subunits [71]. Subunit assembly is essential for activity of
the heterodimer [72] as well as protection of the b subunit
which is unstable when not in complex with the a subunit
[73]. Thus, fusion of FSH a and b subunits with Fc in a
heterodimeric configuration that maintained full activity of
the effector moiety could be useful in stabilizing FSH,
reducing the frequency of administration and providing
an alternate, less invasive route of administration that could
improve the tolerability of current formulations.
FSHFc heterodimers were generated by co-transfecting
mammalian cells with FSH a Fc and FSH b Fc fusion plas-
mids [33]. Purified FSHFc heterodimer was injected into
juvenile female rats subcutaneously resulting in significantly
increased ovarian weights compared to vehicle control
animals as well as animals administered recombinant
FSH [33]. PK studies with FSHFc heterodimer revealed
that the protein had a terminal half-life of 69 h in rats,