Biomedical Engineering Reference
In-Depth Information
Fc-fusion proteins are preferable for dimer ic molecu les
since the intera ction of the antibody heavy chain and the
disulfide bride at the hinge region automa tically forces the
formati on of homod imers. However, thes e large molecu les
might not always be favorable. There fore, attempt s have
been under taken to establ ish also Fc-fusi on prot eins with
monome ric effector molecu les. This means only one heavy
chain is fused to the effector molecu le, thus creating a
heterodim er that is fully symme tric only for the Fc part.
Often Fc-fusi on protei ns prepared for pulmona ry delivery
and using the FcRn expresse d alveolar epithel ial cells have a
monome ric conform ation. For instanc e, mono meric eryth -
ropoietin (EPO ) Fc fusion had a twofold highe r affinity to
the FcRn and a 30% better half-life than the dimeric variant.
Compa red to the origina l EPO, mono meric EPO- Fc reached
a muc h highe r system ic conce ntration when administ ered at
equimolar conce ntrations [23]. Th e improved distribution of
the mono mer is a result of smaller size, better transpor t, and
less steric hindranc e. Th e EPO- Fc dimer achieved 70%
bioavailabil ity in the central lung regions during a Ph ase
I clini cal study [24] . Re cently it was shown with pri mates
that an inha led interfer on-
b
(IFN-
b
) mono meric Fc fusion
was fourfold more active than the corr espond ing dimer, but
still 2.3-f old less poten t than the origina l IFN-
b _
s However,
the pres ence of the Fc-part extended the half-life threef old
[25]. Be sides IFN -
b
, also interf eron
a
(IFN-
a
) was succe ss-
fully tested as mono meric Fc-f usion protein for pu lmonary
delivery [26]. Monom eric Fc-fusion prot eins are disc ussed
in detail in Chapte r 7.
The attractiveness of monomeric Fc fusion has initiated the
search for novel solutions. One approach takes advantage
from the ability of antibodies to assemble their CH
3
domains
asymmetrically. By alternating the sequences of IgA and IgG
complementary Fc domains can be generated. They effi-
ciently form heterodimers, while disfavoring the generation
of homodimers. This process is much more efficient than
other approaches to produce monomeric Fc-fusions, which
suffer from low yields. These findings can establish the basis
to generate on one hand bispecific antibodies or on the other
hand monomeric Fc-fusions. This technique is called SEED
(strand exchange engineered domain) platform and was used
to generate a monomeric interleukin 2 (IL-2) Fc-fusion. This
molecule exhibited extended serum half-life [27]. In addition,
SEED was used to evaluate the ability to form bispecific
antibodies [28]. More details can be found in Chapter 37.
Fc-based hybrid molecules can also be built symmetri-
cally. One example is a hybrid Fc domain that combines the
properties of IgG4 with those of IgD. This results in a highly
flexible hinge region and the lack of secondary toxic func-
tions while maintaining the long half-life of
Therefore, it is worthwhile trying to optimize it. Mutagene-
sis toward higher affinity to FcRn at pH 6.0 while leaving the
binding properties at pH 7.4 unchanged can lead to a 2.5-fold
longer half-life in primates. This means less antibody is
released from the FcRn during intracellular passage, thus
avoiding lysosomal degradation of weakly bound antibody
[29]. Mutagenesis at the Fc part always has to take other
functionalities into account. This includes effector functions
such as antibody-derived cellular cytotoxicity (ADCC) or
complement-dependent toxicity (CDC) [30]. The best way
to avoid any unwanted activities would be to switch to the Fc
part of IgG4, which does not provoke CDC at all and has low
ADCC stimulation ability. So far most of the mutagenesis
studies were directed to improve ADCC and CDC [31].
However, the Fc region can be engineered for high affinity to
FcRn independently of the pH, too. This then blocks the
FcRn salvage function for all other antibodies or Fc-fusion
proteins and causes much shorter half-life for Fc-containing
proteins. This was described as an approach to lower IgG
concentration in antibody-mediated diseases or to promote
the rapid clearance of immunotoxins or antibody drug
conjugates [32].
Despite the well-known behavior of Fc domains, the
construction of fusion protein containing additional domains
can negatively influence stability. Often conformational
stability is pH dependent. In the case of Orencia, consisting
of an IgG1 Fc part and the soluble part of T-cell receptor
CTLA-4, the instability of the CH
2
domain can cause
aggregation [33].
One group of therapeutic molecules that could particu-
larly benefit from half-life extension is peptides. Peptides
have important functions in the organism but because of
their small size they are quickly removed from circulation by
excretion. Their typical half-life is in the range of minutes,
therefore, any improvement would have a huge impact. The
trusted and proven platform of Fc fusion is certainly one of
the first choices. And consequently a number of examples
exist. One already approved molecule is Nplate
1
to treat
chronic idiopathic (immune) thrombocytopenic purpura. It
consists of thrombopoietin fused to Fc. This leads to an
improved half-life of 3.5 days that makes chronic treatment
feasible [34]. Another therapeutic peptide utilized in the so-
called Mimetibody
TM
platform is angiopoietin, currently
being in clinical Phase III. Beyond the relatively straightfor-
ward fusion to Fc further optimization that includes intro-
duction of disulfide bridges or mutagenesis to reduce
immunogenicity was performed. The whole Chapter 8
addresses Mimetibodies
TM
[35].
A bit larger than typical peptides bit still suffering from
short circulation times are cytokines. They play a role in
regulating immune response and inflammation but vanish
from the blood stream within minutes. But not only size-
dependent rapid clearance contributes to the short half-life
but also degradation and receptor-mediated endocytosis. A
20 days. This
technol ogy was designed by Gen exin e in Korea (http://
www.genexine.com/20 070801_h tml_e /rnd/rnd_03. asp) .
The contribution of recycling to half-life extension is a
function of the affinity between the Fc part and FcRn.
Search WWH ::
Custom Search