Biomedical Engineering Reference
In-Depth Information
are already suffic ient. However, max imum activity was
obtaine d with a helical 12 aa linker which was 1.7 times
or 2.9 times better than a short rigid or the standar d five aa
long G 4 S linker [39]. Th e knowledge about linker s was
continuo usly expanded [40]. Recent ly, a database collec ting
natural linker sequences and their propert ies coul d be est ab-
lished. 1 Int er domain linkers have to be different iated from
intradoma in loops based on their functi on, but also becau se
of their amino acid preference. Interestingl y proline is the
most frequent amino acid in both case s. Prol ine dest roys
a -helix and b -sheet structu res as well and cannot form
hydrog en bonds [41].
But linker peptide s can also be engi neered to cont ain
additional functional ities. To optimise the pharmac okinetic
profile of a granulocyte-colony-stimulating factor (G-CSF )
fused to transferrin, a linker with an intramol ecular disu lfide
bridge was introdu ced. Th e peptide sequence was thromb in
sensitive, whi ch allowed an in vitr o cleavage by adding this
proteas e, leaving the two doma ins only connect through the
disulfide bridge. This labile bond could then easily be
cleaved in vivo , releasing free G- CSF that was more active
than the fused molecu le [42] . Previous ly, a similar molecu le
was engineer ed to improve oral efficacy. Here, a long rig id
helical li nker composed of 50 aa betwee n G-CSF and
transferr in led to a 10 times lower EC 50 than the initial
fusion protein with a two aa space r [43] . Th is particula r
linker also increas ed the expression level more than 10-f old
compare d to the direct fusion with only two aa in b etween.
However, this time the construct was in an opposite ori en-
tation having transferrin at the N- and G- CSF at the
C-terminu s. Int erestingly an unst ructured 50 aa linker inhi b-
ited expression totally [44] .
The orientation of doma ins and the impact of space rs
were also studied whe n a scFv against huma n transferr in
recepto r was combine d with the fungal ribonucl eolytic toxin
restrictoci n. Indep endent of the ori entation in both case s, the
introdu ction of a proteas e-sensi tive linker drastical ly
improved the EC 50 2- to 3 0-fold [45]. The influence o f
linkers on fusi on protei ns is disc ussed in Chapte r 4.
with two molecules of a truncated Pseudomonas exotoxin A
resulted in an almost 40-fold more active fusion protein than
with only one toxin molecule [47]. Oligomerization can also
help to improve valency of recombinant proteins. Sometimes
it is required to induce cross-linking of receptors to execute
specific functions such as activation or internalization. It has
been demonstrated that by the right choice of linkers between
V H and V L in scFv, aggregates can spontaneously form having
a higher avidity [48].
Using an Fc part auto matical ly delivers dimer ization,
however there are also different appro aches possib le to
enforce dimer s. Freque ntly leucine zipp ers are used for
that purpos e. An early example is the gener ation of bivalent
scFv antibodi es with Fos or Jun leucine zipp ers. A covalent
bond could be introdu ced by posi tioning cysteine in prox-
imity to the zipp er. These molecu les spont aneously formed
dimer s whe n secr eted to Escheri chia coli perip lasm [49].
The leuc ine zipper GCN 4 of Sacchar omyc es cerevi siae has
been used to dimer ize the solu ble insulin recepto r. This
resulted in an improved binding const ant, very similar to the
origina l membrane bound native rece ptor [50].
As in the case of OX40 ligand (OX40L) even a trime-
rization can b e require d for full functional ity. Here an act ive
construc t in the right confo rmation coul d be obta ined by
fusing OX40L to a GCN4 zipp er d omain connecte d to an Fc
part. The final hexameric molecu le consist ed of three
Fc-induce d disulfide link ed dimer s that allowed the forma-
tion of two trimers [51] . Another mol ecule having a trimer as
active natural confo rmation is tumo r necr osis factor-related
apopto sis-inducing ligand (TRAIL ). As before, the GCN4
zipper-induced self-a ssembled trim ers of TRAIL based on
three- strande d coiled coils [52]. However, TRAIL can also
be trim erized as linear fusion in a head to tail configur ation
interrupt ed by short flexible pept ide loops. To improve
targeting, even a scFv coul d b e include d at the N-ter minus
[53]. Trimeriz ation through tand em repe ats in a sing le chain
molecu le coul d also be shown with TNF [54]. CD95 ligand
(CD9 5L) as anot her membe r o f the TNF homo logy do main
(THD) family could be forced into covalent trimerization by
fusion to a tenascin-C (TNC) oligomerization domain, again
resulting in improved bioactivity [55]. Higher oligomeriza-
tion of multiple trimers was tested with the CD40 ligand
(CD40L). First a di-trimer conformation was achieved by
incorporating the N-terminal part of adiponectin (Acrp30).
A tetra-trimer construct could be obtained by combining the
N-terminal part of mouse surfactant protein-D (SP-D) with
the extracellular part of CD40L. Additional leucine zippers
were not required because both Acrp30 and SP-D sponta-
neously self-assemble into the desired multimers. These
constructs have been successfully used as adjuvant in
DNA vaccines [56]. A similar approach was also initiated
with soluble Fas ligand (FasL). The bioactivity of a single
FasL trimer was much lower than di-trimer (hexameric)
constructs that either relied on the oligomerization effects of
1.4.3 Oligo meriza tion of Fusion Prote ins
The next level of design involves the correct oligomerization
of the protein. In many cases, proteins are dependent on
multimerization or have a higher bioactivity as a multimer.
One example is the combination of stem cell factor (SCF) and
macrophage-colony-stimulating factor (M-CSF) with a 12 aa
flexible peptide linker. The fusion protein forms dimers that
have a 10- to 20-fold higher potency than the individual
monomeric proteins, also benefitting from a synergistic effect
[46]. A higher cytoxicity could also be demonstrated for
divalent antibody toxin fusions. Combining a Fab fragment
1 http://www.ibi.vu.nl/programs/linkerdbwww/
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