ALBUMIN-BINDING FUSION PROTEINS IN THE DEVELOPMENT OF NOVEL LONG-ACTING THERAPEUTICS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 177

IL-1RA. These experiments demonstrated conclusively
thatAlbudAbscouldbeusedtogeneratetherapeu ic
molecules with improved pharmacokinetics and in vivo
efficacy due to binding of serum albumin in the circulation.

IFN- α/β/ω

IFNAR1 IFNAR2

Cell membrane

11.3 INTERFERON-a FUSED TO HUMAN SERUM
ALBUMIN OR AlbudAb—A DIRECT COMPARISON
OF HSA AND AlbudAb FUSION TECHNOLOGIES

Tyk2 JAK1

11.3.1 In Vitro Potency of HSA and AlbudAb Fusion
Proteins

Studies conducted by Holt and coworkers have demon-
strated the utility of AlbudAb technology in developing
antagonist molecules with improved pharmacokinetics as
potential therapeutics. Subsequently, we wished to deter-
mine whether AlbudAbs could be used to develop an agonist
molecule with improved half-life, while maintaining bio-
logical activity, which could potentially be used as a thera-
peutic in the treatment of chronic HCV infections. To this
end, we have developed human IFN- a 2b (IFN- a 2b) geneti-
cally fused to the N-terminus of an AlbudAb composed of a
single V k domain; clone DOM7h-14, via a short amino acid
linker (hereafter referred to as IFN- a 2b-DOM7h-14). In
order to determine the relative advantages of AlbudAb
technology in comparison to HSA fusion technology, we
compared the pharmacokinetics, in vitro potency and in vivo
efficacy of IFN- a 2b-DOM7h-14 with that of IFN- a 2b fused
to the C-terminus of HSA with no intervening linker
sequence generated in our laboratory (hereafter referred
to as HSA-IFN- a 2b), which is effectively the same molecule
as Albuferon [20]. Owing to the complex nature of these two
molecules, we decided to express them in a mammalian cell
culture system which uses transiently transfected human
embryonic kidney cells (HEK293E) to express proteins of
interest from the vector pTT5 into the cell culture super-
natant as soluble active protein [21]. This system has
successfully been used in the past to express soluble active
IFN- a molecules at high yield [22]. Following expression
for 5 days post transfection, IFN- a 2b-DOM7h-14 and HSA-
IFN- a 2b were purified from culture supernatants using
affinity ligands immobilized on a solid support. IFN- a 2b-
DOM7h-14 was bound to protein L, an affinity ligand that
specifically binds to immunoglobulin light chains [23], and
HSA-IFN- a 2b was bound to the affinity dye Cibacron Blue
F3GA, an affinity ligand with specificity for human IFNs
and serum albumin [24,25]. Following extensive washing,
the purified fusion proteins could then be eluted and their
activity determined using in vitro assays.
In order to compare the in vitro potency of purified IFN-
a 2b-DOM7h-14 and HSA-IFN- a 2b, activity of the two fusion
proteins was determined using the commercially available
HEK293 IFN- a / b cell line (a diagrammatic representation
of this assay is shown in Figure 11.1), in which HEK293 cells

P P IRF9

ISGF3
complex

IRF9

Nuclear membrane

ISRE SEAP

FIGURE 11.1 Schematic diagram of the HEK293 IFN- a / b
reporter cell assay. Type I interferon ( a , b ,or v ) binding to the
heterodimeric receptor composed of IFNAR1 and IFNAR2 results
in Tyk2 and JAK1 kinase activation. Tyk2 and JAK1 then phos-
phorylate STAT1 and STAT2, resulting in dimerization and inter-
action with IRF9, forming a complex named ISGF3. ISGF3 then
binds to interferon response elements (ISRE) in the promoters of
interferon-stimulated genes, in this case the reporter gene SEAP,
regulating their expression.

are stably transfected with a secreted alkaline phosphatase
reporter gene under the control of an IFN inducible promoter
element. Treatment of this cell line with type I IFNs (IFN- a ,
IFN- b ,andIFN- v ) results in induction of reporter gene
expression in a manner dependent upon the level of IFN
activity, the level of which can be determined by spectro-
photometric methods. Both fusion proteins were capable of
inducing reporter gene expression in this cell line, although
with reduced in vitro potency in comparison with a recombi-
nant IFN- a commercial standard (Figure 11.2). In order to
determine the likely effect of albumin binding on activity of
AlbudAbfusionproteins,theassaywascarriedoutinboththe
presence and absence of high concentrations of HSA. In the
absence of HSA, both IFN- a 2b-DOM7h-14 and HSA-IFN-
a 2b were able to induce reporter gene expression, though IFN-
a 2b-DOM7h-14 was able to do so with 55-fold greater

potency than HSA-IFN- a 2b. This result shows that genetic
fusion to the large HSA protein results in a significant loss of
IFN activity in vitro. In the presence of 100 m MHSA,the