FUSION PROTEINS: APPLICATIONS AND CHALLENGES - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 6

FIGURE 1.3 Fusion protein design. The typical variations of fusion proteins comprise orientation,
linkers, and oligomerization. Sometimes proteins require a free N- or C-terminus for their activity.
The linker can vary in length, flexibility, and susceptibility to protease cleavage. Dimeric forms of
fusion proteins can be generated by including leucine zippers in the linker region. Two examples
of higher oligomerization are depicted as well; first, a trimer built from the trimerization domain of
human endostatin and second, a hexamer constructed through the combination of three Fc dimers
connected via an ILZ coiled coil to two trimers ( 1 see Morris et al. [51]).

disulfide bridge formation, aggregates formed that drasti-
cally reduced the recovery. Interestingly, this phenomenon
could be completely abolished by positioning the IFN- a 2b
on the N-terminus instead [30]. In another case the order of
two angiostatic proteins, human angiostatin (hAS) and endo-
statin (hES), combined in a fusion protein had to be in a
specific orientation to obtain maximum activity. The fusion
hES-hAS was 28% more potent than hAS-hES or 7% better
than hAS and hES when administered separately [31].
Sometimes a free N-terminus is required. The second partner
is then connected at the C-terminus as in the case of receptor
traps such as Etanercept. Here, the extracellular receptor
domain is combined with an Fc part at the C-terminus to
maintain the natural conformation [6]. Another striking
example for the positioning effect of fusion partners is
the case of elastin-like peptides (ELP). It was observed
that C-terminal fusions of ELP resulted in a higher expres-
sion level, better yield, and bioactivity. The underlying
reason could be increased misfolding, induced by ELPs at
the N-terminus; thus reducing the amount of active proteins
and increasing their susceptibility to proteolysis [32]. Ori-
entation has a high impact on functionality particularly when
fusion proteins contain enzymes that require either a free N-
or C-terminus. This has been demonstratedwith the Immuno-
RNAse consisting of angiogenin (ANG) and a single-chain
variable domain (scFv) against CD22. Only constructs in the
scFv-ANG orientation did not aggregate and were fully
functional [33].

1.4.2 Linker Engineering

Again starting with Fc fusions as example, the hinge region
fulfils the function of a linker, allowing some spatial flexi-
bility [34]. Besides this exception where a part of a fusion
protein ends with a flexible peptide chain, in most cases
specific linkers between protein molecules have to be artifi-
cially introduced. The multiple aspects of linker design have
recently been reviewed [35]. Many researchers use a simple
glycine and serine (G 4 S)-containing linkers as proposed by a
large study of natural domain separating linkers [36]. Spacer
peptides that connect both modules of a fusion protein in a
spatial conformation are frequently needed to maintain
functionality. For instance, the highest potency could be
observed when a spacer was introduced between a single
chain variable domain (scFv) and ANG [37]. Since fusion
proteins ideally consist only of a single polypeptide chain, a
reformatting of Fab fragments to scFv is required. This is
done with the help of a linker sequence that frequently
consists of repeats of glycine and serine, as for example
the popular (G 4 S) 3 linker. The basis of linker design is the
rational engineering of both length and conformation. A
controlled distance between domains can be achieved by
defined repeats of a -helical peptides A(EAAAK) n A that
maintains the separation of domains in contrast to flexible
linkers [38].
When evaluating the optimal linker between IFN- a 2b
and HSA it could be demonstrated that five amino acids (aa)