Biomedical Engineering Reference
In-Depth Information
with Fc resulted in marked improvement of the half-life for
several therapeutic proteins, it was hypothesized and con-
firmed that the Fc construct could be improved to further
enhance the biological properties of the therapeutic moiety.
This improved construct is referred to as a monomeric Fc
fusion molecule and is comprised of a single therapeutic
protein coupled to dimeric Fc. The applications and chal-
lenges of this novel Fc fusion configuration are discussed in
the current review.
found [24,28]. Initial uptake of IgG is reported to occur by
fluid-phase pinocytosis, the process bywhich other proteins in
the blood enter endothelial cells. Binding of IgG to FcRn takes
place in the sorting endosomes at acidic pH [29,30]. IgG is
then routed along with FcRn back to the cell surface and
released at neutral pH in some cell types (e.g., endothelial cell)
or transcytosed across the cell in other cell types such as the
mucosal epithelial cells lining the intestine. In contrast, pro-
teins that do not bind FcRn are targeted for degradation in
lysosomal compartments. It is through this specific interaction
with FcRn that IgG or Fc-containing proteins are protected
from degradation and conferred a relatively longer half-life.
7.2 FcRn AND MONOMERIC Fc-FUSION
PROTEINS
7.2.2 FcRn for the Delivery of Fc-Fusion Proteins
7.2.1 FcRn Expression and Function
The FcRn pathway was explored as a means to deliver
large therapeutic proteins noninvasively through the lung
[12,31-33]. Because FcRn expression was demonstrated in
the lungs of adult humans, as was the transcytosis of IgG
across human lung epithelial cells, it was hypothesized that
this natural pathway could enable delivery of aerosolized Fc-
fusion proteins through the lung. The prototype fusion
proteins were dimeric with respect to both the therapeutic
protein and Fc (Figure 7.1). Erythropoietin (Epo) was
selected as the fusion partner because it is a potent cytokine
that elicits a readily measured biological response [34].
Proof-of-principle studies were conducted with the EpoFc
dimer in cynomolgus monkeys. Monkeys were selected
because the expression of FcRn was determined to be similar
to human, and also because the characteristics of a monkey
lung were reported to be similar to human [12,18]. EpoFc
was administered to cynomolgus monkeys targeting the
central airways. Absorption of the EpoFc dimer was
achieved, while a mutant EpoFc which contained three
mutations at Fc domain (I253A, H310A, and H435A)
(EpoFc/IHH) that differed from the wild-type EpoFc in
three amino acids in the Fc domain known to be necessary
for binding to FcRn was taken up very poorly [12]. Pulmo-
nary delivery of EpoFc dimer was also demonstrated in
healthy human volunteers and the resulting pharmacokinetic
(PK) parameters were consistent with what was expected
based on the nonhuman primate studies [35]. While these
data were encouraging, it was postulated that the Fc fusion
construct could be modified to improve the PK parameters,
including bioavailability. EpoFc is
Passive immunity is accomplished through the uptake of
maternal IgG from ingested colostrum and milk across the
epithelial cell layer of the small intestine via FcRn in neonatal
rodents during the suckling period. Expression of the receptor
is high during the first few weeks of life, but at the time of
weaning, it is markedly reduced (
1000-fold) in rodents [22].
FcRn transport of IgG also takes place in the yolk sac of
rodents during gestation [23]. Therefore, the importance of
FcRn is clear during both the gestational and neonatal time
periods in rodents. In contrast, passive immunity in humans
occurs through the transport of IgG across the syncytiotro-
phoblast of the placenta where FcRn mediates the maternal
transfer of IgG to the fetus during gestation [17]. Over the
years, it has been demonstrated that FcRn expression is not
limited to the gestational and neonatal periods in humans, but
rather is found in many cell and tissue types throughout life.
The major site of IgG homeostasis is thought to be in the
vascular endothelium, primarily in skin and muscle, as well as
in liver and adipose tissue [24]. These sites provide a large
contact area for FcRn with the blood. Elegant experiments in
mice inwhichFcRnwas conditionallydeleted inendothelial and
hematopoietic cells demonstrated that these tissues are indeed
the primary sites for the regulation of IgG homeostasis [25].
FcRn expression remains relatively high throughout life
in the epithelial cells lining the intestine, as well as in the
lung [12,14,18]. Moreover, the human receptor is functional
in adult life in the intestinal epithelium when studied in a
humanized mouse model [26]. Further examination of
expression in the lung showed that FcRn was most promi-
nent in the central airways, with much less receptor located
in the peripheral lung regions [12]. FcRn is also expressed in
the kidney where the receptor is thought to contribute to the
resorption of IgG [11,15], as well as in hepatocytes in the
liver where it may contribute to maintaining circulating IgG
levels and protection from catabolism [11,27].
There appears to be limited expression of FcRn at the
surface of cells, with expression occurring mostly in intra-
cellular compartments in most cell types in which FcRn is
100 kDa in size, so it is
possible that having two effector molecules may pose steric
hindrance between the Fc and Epo and on the binding to
their respective receptors. In addition, Epo is a highly
glycosylated protein. It is known that the charge on the
molecule may affect the penetration of mucus that bathes the
epithelial cells in the lung, as well as affect endocytosis into
cells [36,37]. Therefore, it was hypothesized that removal of
one therapeutic moiety may reduce size and charge and
possibly relieve some steric hindrance between the
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