Biomedical Engineering Reference
In-Depth Information
prolonging the half-life of IgG through the same mechanism,
although albumin and IgG bind to distinct domains on FcRn.
Owing to its size, wide distribution within the body,
relative lack of immunological and enzymatic function,
and innate protection from intracellular degradation, conju-
gation or fusion to HSA presents an attractive mechanism for
prolonging the half-life of therapeutic proteins and peptides
while addressing some of the development challenges asso-
ciated with PEGylated molecules. Genetic fusion of thera-
peutic proteins to HSA results in significant improvements in
serum half-life while maintaining biological activity of the
therapeutic fusion partner [9]. These HSA fusion proteins can
be expressed to high level in yeast systems and purified to
homogeneity [10]. Examples of therapeutic proteins and
peptides genetically fused to HSA can also be found in the
clinic, for example, HSA fused to a glucagon-like peptide 1
(GLP-1) mimetic, currently being developed for the treatment
of type 2 diabetes [11]. HSA fused to IFN-
a
(Albuferon
1
) has
also been developed for potential use in the treatment of
chronic HCV infection. Albuferon has similar efficacy and
superior pharmacokinetics compared with PEGylated IFN
[12,13]. Indeed, because of its increased half-life (7 days in
man), once fortnightly or once monthly dosing schedules may
be possible with Albuferon, although further clinical devel-
opment of this molecule has been halted following Phase III
clinical trials. However, as with PEGylated molecules, devel-
opment challenges are also associated with HSA fusion
proteins. Owing to its large size and the number of disulfide
bonds present in the molecule, HSA expression in bacterial
systems such as Escherichia coli is challenging; therefore,
production of HSA fusion proteins generally requires yeast-
based expression systems. A study carried out by Zhao and
coworkers also revealed that HSA-IFN fusion proteins
expressed in Pichia pastoris were unstable during long-
term storage and were heterogeneous following purification
[14]. In summary, the development of HSA fusion proteins
such as those described earlier has resulted in a number of
therapeutics in clinical development. HSA fusion proteins can
be expressed to high yield and purity and may allow greater
improvement in pharmacokinetics compared to those
achieved with PEGylation. However, additional process chal-
lenges are associated with production of HSA fusion proteins,
with expression limited inmany cases to yeast-based systems.
This has led to the development of alternative half-life
extension technologies based on albumin-binding antibody
fragments (AlbudAbs), which are described in more detail in
Section 2.3.
variable domain (V
H
or V
L
). dAbs with antigen-binding
activity can be expressed in E. coli [15], and using phage
display methods, dAbs with high affinity and desirable
biophysical properties, such as high solubility, resistance
to aggregation following thermal denaturation and mono-
meric state in solution, have been isolated [16,17] although
these desirable properties appear to be dependent on certain
critical CDR residues affecting the hydrophobicity of the
former V
L
-V
H
interface [18]. Terminal half-lives of these
small proteins are typically
25min in mice; however, dAbs
with antigen-binding activity toward serum albumin have
been isolated, and these “AlbudAbs” have circulating half-
lives approaching that of serum albumin [19]. Many of these
AlbudAbs demonstrated a high degree of cross-reactivity
with serum albumin from discovery relevant species such as
mouse, rat, and cynomolgus monkey meaning they can
easily be assayed for in vivo activity in preclinical animal
models. Owing to their small size, ease of expression in
multiple systems (including yeast, bacteria, and mammalian
cell culture), desirable biophysical properties, and high degree
of species cross-reactivity, the potential for AlbudAbs in the
generation of novel therapeutics with improved pharmaco-
kinetics was investigated.
Holt and coworkers have generatedAlbudAb fusions to the
interleukin-1 receptor antagonist IL1-RA [19], a therapeutic
with short in vivo half-life approved for use in the treatment of
rheumatoid arthritis. The resulting AlbudAb/IL1-RA fusion
was expressed in E. coli using the pETexpression system and
the activity of the purified fusion protein determined. Affinity
for serum albumin, determined by surface plasmon resonance
assays, was largely unaffected by fusion to IL1-RA. Similarly,
when the AlbudAb/IL1-RA fusion protein was assayed for
biological activity, measured by its ability to neutralize the
secretion of IL-8 from MRC-5 cells, the activity was compa-
rable to that of unfused IL1-RA. These experiments demon-
strated conclusively that AlbudAbs could potentially be used
to generate fusion proteins of potential therapeutic benefit
without affecting the activity of either the AlbudAb or its
fusion partner. In order to determine whether the in vitro
activity of the AlbudAb/IL1-RA fusion protein would be
likely to translate into clinical efficacy, the activity of
the molecule was determined in a collagen-induced mouse
model of arthritis in which animals were injected intra-
peritoneally with IL-1RA or AlbudAb/IL1-RA fusion pro-
tein 21 days after collagen injection to induce arthritis in
these animals. Using clinical scores for the severity of
arthritis, the authors demonstrated that AlbudAb/IL1-RA
was significantly more efficacious in lowering the arthritis
score compared to IL-1RA when the two were adminis-
tered at the same dose. Biodistribution of
3
H labeled
AlbudAb/IL1-RA, IL1-RA, and mouse serum albumin
(MSA) was also determined by quantitative whole body
autoradioagraphy and the distribution of AlbudAb/IL1-RA
shown to be more similar to that of MSA than that of
<
11.2.3 Anti-Serum Albumin Domain Antibodies in the
Development of Novel Half-Life Extension Technologies
Domain antibodies (dAbs) are the smallest binding unit of an
immunoglobulin. They are
12-14 kDa in size and are
composed solely of a single human immunoglobulin
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