FUSION PROTEINS FOR HALF-LIFE EXTENSION - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 93

when blood clotting was required [17]. Interestingly, a
similar pharmacodynamic (PD) effect was observed in an
IFN- a 2b and HSA fusion protein. The insertion of a cleav-
able linker increased the potency of the molecule. Basic
amino acids in the spacers lead to a factor Xa susceptibility.
The effect was even tunable by the number of inserted
amino acids. Choosing linkers with intermediate release
rate had the most balanced pharmacokinetic/-dynamic
effect [18].
However, despite the documented success with albumin
fusions, in some cases it was found that the bulky partner
HSA reduced the activity. One of the examples for this effect
is the fusion to soluble human tumor necrosis factor recep-
tor. Independent of an N- or C-terminal fusion, both variants
had drastically reduced activity [19]. Albumin fusions are
discussed in Chapter 10.

diseases. Chapter 9 deals exclusively with the topic of
soluble receptors as traps.
The clinical success of Fc fusion proteins is primarily
based on utilizing the high and specific affinity of extrac-
ellular receptor domains and the well-known principle of
receptor-mediated antibody recycling through the Fc part.
Antibodies and albumin share the FcRn, although they
bind to different epitopes of this receptor. But the general
process is identical; binding at acidic and release at
neutral pH. The understanding of this phenomenon paved
the way to combine the Fc part with other protein mol-
ecules. Of course it should not be neglected that already
the addition of a 50-kDa moiety aids escaping fast kidney
filtration.
The recycling through FcRn does not only protect
Fc-containing molecules from lysosomal degradation, but
also strips bound molecules from the antibody at low pH in
the endosome, thus freeing the binding domain to become
available again after release from the receptor.
The contribution of the FcRn-mediated recycling on half-
life was evaluated in a study that correlated the affinity of
antibodies and Fc-fusion proteins to the FcRn with their
half-life. It was clearly demonstrated, that low affinity
corresponds to short half-life and vice versa. To some
respect the observed shorter half-lives for Fc-fusions could
be attributed to a sterical or conformational effect of the
molecules fused to the N-terminus. Some antibodies with
exceptionally short half-life despite high affinity are proba-
bly eliminated by other antibodies or through internalizing
cell-surface antigens [21]. Therefore, engineered Fc
domains with higher affinity to FcRn, particularly at low
pH should have longer half-lives. This was elegantly proven
by optimizing Avastin 1 to have 11-fold higher affinity,
resulting in a threefold longer half-life. An identical effect
could be shown by modifying Erbitux 1 that addresses an
internalizing antigen [22].

6.2.1.2 Fc Fusions Looking at the currently marketed
fusion proteins, it is obvious that Fc fusions dominate the
field. The fragment crystallizable (Fc) region of an antibody
consists of the second and third constant domain of the
heavy chain (CH 2 ,CH 3 ), forming a dimer. In the hinge
region at the N-terminal end, two disulfide bridges connect
both chains. Enbrel 1 , the first ever approved fusion protein
can be found in the group of Fc fusion proteins. This
molecule belongs to the family of receptor traps that com-
bine the extracellular domain of a receptor with the Fc part
of an antibody [20]. Other members are Amevive 1 or
Orencia 1 . A complete overview of approved Fc fusion
proteins can be seen in Table 6.3. Comparing the first
approved fusion protein Enbrel with its antibody competi-
tors it shows that all antibodies have a better half-life than
Enbrel. Nevertheless, the fusion protein is still quite suc-
cessful on the market. Only the immunotoxin Ontak 1 has a
relatively short half-life around 1 h. But this is not neces-
sarily a disadvantage since Ontak is not used for chronic

TABLE 6.3 Half-Life of Fusion Proteins Compared to Antibodies

Type Drug Brand kDa t 1/2 range Average t 1/2 (h)

Nplate 1 Fusion protein Romiplostim 60 1-34 days 408

Orencia 1 Abatacept 92 8-25 days 400

Amevive 1

Alefacept 92 11 days 264

Nulojix 1 Belatacept 90 8-10 days 220

Arcalyst 1

Rilonacept 251 9 days 216

Eylea 1 Aflibercept 115 5-6 days 130

Enbrel 1 Etanercept 150 3-6 days 120

Ontak 1

Denileukin difitox 58 70-80 min 1.2

Remicade 1 Antibody Infliximab 150 9.5 days 228

Humira 1

Adalimumab 150 10-20 days 360

Cimzia 1 Certolizumab pegol 91 14 days 334

Simponi 1 Golimumab 150 14 days 334