Biomedical Engineering Reference
In-Depth Information
this new N-terminus [14]. The recombinant fusion protein,
called KGLP-1/HSA, bound and activated GLP-1 receptors;
the activity of which was less potent than that of either
KGLP-1 or wild-type GLP-1, possibly due to increased
spatial blockade caused by the albumin moiety. Neverthe-
less, KGLP-1/HSA retained much of the biological activity
of GLP-1, and its duration of effect was prolonged; up to
eightfold longer for its glucose-lowering effect and up to
twofold longer for its insulin-releasing effect [14].
Albumin has also been fused to the C-terminal end of
exendin-4, a potent GLP-1 agonist [15]. Exendin-4 is a 39-
amino-acid peptide isolated from the salivary gland of the
Heloderma suspectum lizard [59] and has 53% homology to
mammalian GLP-1. Synthetic exendin-4 (exenatide) has
greater efficacy compared to GLP-1, but its half-life of
2.4 h necessitates twice-daily injections. Extended-release
formulations of exenatide have also been developed that can
be given on a weekly basis [60,61]. As an alternative
approach, the gene encoding exendin-4 was fused to the
rHA N-terminus, linked by a short peptide (GGGGS), to
create the novel fusion protein rEx-4/rHA [15]. The rEx-
4/rHA fusion protein had improved and prolonged glucose-
lowering effects in diabetic mice compared to a GLP-1-
albumin conjugated analog (36 vs. 12 h) [62]. In monkeys,
the half-life of rEx-4/rHAwas 70-80 h, which was 33 times
longer than that of exenatide (2.4 h) [63] and much longer
than values reported for other GLP-1 modifications that
employed fatty acid chain modification (11-15 h) [64] or
PEGylation (33.4min) [65]. These results indicate that
albumin fusion prolongs the half-life of exendin-4 while
retaining its glucose-lowering effects, potentially allowing
for weekly dosing. Indeed, this is currently being evaluated
in Phase III clinical trials [61].
than with rhG-CSF (up to 4 days vs. less than 3 days). Thus,
Albugranin may provide similar efficacy as rhG-CSF while
allowing for weekly administration, and may therefore
improve convenience and, possibly, treatment effects.
While a PEGylated form of rhG-CSF (Neulasta
1
) is availa-
ble that has extended activity compared to rhG-CSF [66],
Albugranin has the added advantages of being simpler to
manufacture and easier to produce a uniform product.
Albugranin is currently in Phase III clinical
trials for
chemotherapy-induced
neutropenia
in
breast
cancer
patients.
10.3.2 Albumin Fusion Proteins in Early-Stage
Development
10.3.2.1 Interleukin-2
Albuleukin
1
is an 81.8-kDa
fusion protein derived from albumin and the cytokine inter-
leukin-2 (IL-2) and is produced using an S. cerevisiae
expression system [30]. A comparison of the biodistribution
of radiolabeled albumin, IL-2, and Albuleukin in mice using
whole-body autoradiography revealed striking differences.
IL-2 was cleared rapidly from the blood: less than 1% of the
injected dose (ID) per gram of tissue was detectable 20min
after administration, while levels in the kidney (120% ID/g)
suggested rapid uptake that persisted for up to 6 h. In
contrast, Albuleukin remained in circulation longer
(14% ID/g at 6 h) and had less uptake in the kidneys (less
than 6% ID/g) but increased localization in the lymphocyte-
rich tissues, such as the liver, spleen, and lymph nodes
(maximal uptake
22% for all three tissues). Albumin
also remained in circulation longer than did IL-2, but was
found in low levels in the liver, spleen, and lymph nodes,
suggesting that uptake of Albuleukin in these tissues is
mediated in part by the IL-2 portion of the fusion protein.
This targeting of tissues, where lymphocytes reside, indicates
that albumin fusion technology may improve the efficacy and
reduce the toxicity of IL-2 therapy [30]. Preclinical studies
have evaluated the pharmacokinetics of Albuleukin [31].
In vitro experiments showed that Albuleukin increased the
proliferation of primary human and mouse T cells and B cells
as well as primary human natural killer (NK) cells. The fusion
protein competed with recombinant IL-2 for binding to IL-2
receptors, and induced the production of IFN-
g
in primary
human NK cells. In mice, Albuleukin had a prolonged
half-life of 6-8 h, compared to only 19-57min for recombi-
nant IL-2. Administration of Albuleukin inhibited tumor
growth in mice with hepatic metastases and reduced the
incidence of residual microscopic hepatic tumors, leading
to prolonged survival compared to controls and mice treated
with recombinant IL-2 [31].
10.3.1.3 Granulocyte Colony-Stimulating Factor
Re-
combinant human granulocyte colony-stimulating factor
(rhG-CSF; filgrastim) is expressed in bacteria and used in
the treatment of neutropenia, but its short circulating
half-life necessitates daily or twice-daily injections [21].
Albugranin
TM
is a fusion protein derived from albumin and
rhG-CSF, in which the N-terminus of rhG-CSF is fused to
the C-terminus of rHA [21]. In mice and monkeys, Albu-
granin had improved effects compared to rhG-CSF, includ-
ing sustained neutrophilic leukocytosis in both species [21].
In mice, clearance was eightfold slower with Albugranin
than with rhG-CSF, and the half-life was more than
twofold greater with either intravenous or subcutaneous
administration. The number of peripheral granulocytes
and hematopoietic progenitors was increased for up to 4
days after a single dose, compared to 1 day with rhG-CSF. In
monkeys, administration of Albugranin every 4 or 7 days
over a 14-day period was well tolerated and increased
absolute granulocyte and white blood cell counts. The dura-
tion of hematopoietic effects was longer with Albugranin
10.3.2.2 Insulin
An albumin-insulin fusion protein
(albulin) was developed to improve upon the performance
of currently available long-acting insulin analogs, such as
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