Biomedical Engineering Reference
In-Depth Information
tumor necrosis factor-
a
(TNF-
a
), thus generating a miniature
bispecific molecule. This novel dual binder combines a high
affinity to albumin and the resulting half-lifewith a nanomolar
affinity against TNF-
a
, making it an interesting therapeutic
molecule [54]. The minimal albumin binding domain (ABD)
derived from streptococcal protein G does not interfere with
the FcRn binding of HSA and extends the half-life of a HER2-
affibody molecule to values similar to HSA alone [55].
Bispecific molecules incorporating HSA binding can also
be designed on the basis of camelid heavy-chain-only anti-
bodies, the so-called nanobodies. As expected also this
approach resulted in prolonged circulation. Even more
important was the improved tumor accumulation and pene-
tration compared to a full-length antibody with the same
specificity against epidermal growth factor receptor (EGFR)
[56]. A summary of albumin binding peptides and proteins is
given in Chapter 11.
The concept of albumin as carrier should also be trans-
ferable to the second most abundant molecule class in
human serum, the immune globulins. The simplest case
would be a bispecific molecule of the same type, binding
to antibodies and having specificity for a second target. Such
a diabody had a fivefold longer half-life and could also
recruit complement and cellular toxicity through the Fc part
of the antibody it bound to. A further advantage is the low
manufacturing cost by expression in Escherichia coli [57].
Alternatively bacterial immunoglobulin binding proteins
can be used for the same purpose as fusion partner. In an
experimental study, the half-life prolonging abilities of
Staphylococcus protein A (SpA), Streptococcus protein G
(SpG), and Peptostreptococcus protein L (PpL) were com-
pared when fused to a single-chain Fv fragment (scFv) or a
related bispecific single-chain diabody (scDb). It was found
that the C3 domain of SpG had the best effect, which was
also supported by the lowest affinity to IgG [58].
A more extreme example combines the strengths of
antibodies with the functionality of peptides. The so-called
CovX bodies consist of a catalytic antibody with aldolase
activity and a small pharmacophore. To this pharmacophore
peptides can be chemically grafted. Mixing antibody and the
peptide-pharmacophore molecule, creates a covalent bond
between a specific lysine residue of the antibody and the
azetidinone moiety of the pharmacophore,
FIGURE 6.4
Kidney filtration. Molecules below a diameter of
60 A
passage from the glomerular lumen to the Bowman capsule
through small pores in the endothelium. After crossing the nega-
tively charged network of the basal membrane, they reach the
filtration slit between the podocytes. Larger molecules such as
some fusion proteins remain in the capillaries.
hydrodynamic radius. Large molecules cannot be excreted
by kidney filtration (Figure 6.4). Therefore, a number of
fusion protein concepts have evolved over the last years
utilizing this phenomenon. Most of them rely on the attach-
ment of peptide (repeats) that are attached to one or both of
the termini. To achieve the desired effect, the peptide should
be hydrophilic, ideally uncharged to avoid aggregation, and
without ordered structure. The absence of structure together
with Brownian motion supports a cloud-like random con-
formation of the peptide chain that causes a huge hydro-
dynamic radius. The size of this “cloud” is only dependent
on the number of amino acids if no other intramolecular
forces are present.
The phenomenon of a protein cloud can be observed even
in nature. Some protozoan parasites seem to utilize this
mechanism to prolong the half-life of virulence factors such
as trans-sialidase. This enzyme is secreted and remains in
circulation for up to 35 h because of the presence of a long
hydrophilic repetitive carboxy terminal peptide. The same
enzyme without this tail has a fivefold decreased half-life.
This effect could also be repeated with heterologous proteins
and a different natural sequence containing 13 repeats of
EPSKA [60]. An engineered less immunogenic variant with
the repeat sequence PSTAD still was able to extend the half-
life approximately fivefold [61].
linking both
molecules
irreversibly together
[59]. This concept
is
explained in more detail in Chapter 38.
6.3 HALF-LIFE EXTENSION THROUGH
INCREASE OF HYDRODYNAMIC RADIUS
6.3.1 Repetitive Peptide Fusions
Neglecting the contribution of receptor-mediated recycling;
half-life
can be
extended by solely increasing the
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