Biomedical Engineering Reference
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(A)
potency of the IFN- a standard and HSA-IFN- a 2b was largely
unaffected, whereas the potency of IFN- a 2b-DOM7h-14 was
reduced approximately threefold, presumably due to steric
effects following direct binding of the AlbudAb to HSA.
However, when comparing the in vitro potency of the two
fusion proteins directly in the presence of serum albumin, it
appears that IFN- a 2b-DOM7h-14 is
110
90
70
50
18-foldmore active than
HSA-IFN- a 2b. While the concentration of HSA used in this
assay is lower than the 600- m M concentration found in vivo,no
differences were observed in this assay when the two concen-
trations of HSA were compared directly; therefore, we con-
clude that even in the presence of physiologically relevant
concentrations of HSA, IFN fused to the AlbudAb DOM7h-14
has greater potency than IFN fused directly to HSA.
In order to determine whether potency in the HEK293
IFN- a / b assay correlates directly with in vitro antiviral
efficacy, we studied the activity of IFN- a 2b-DOM7h-14,
HSA-IFN- a 2b, and unfused IFN- a in a cell-based assay in
which the A549 human lung carcinoma cell line is infected
with encephalomyocarditis virus, hereafter referred to as the
EMCV assay (a diagram of the EMCV assay is shown in
Figure 11.3). In this assay, the level of IFN-mediated
protection against EMCV infection was determined for
each fusion protein and the activity, measured in interna-
tional units per milligram of protein (IU/mg), calculated by
comparison with international reference standard material.
Since the in vitro potency of IFN- a 2b-DOM7h-14 in the
HEK293 IFN- a / b assay appears to be slightly reduced in the
presence of HSA, we also carried out the EMCV antiviral
assay in the absence or presence of 100 m M HSA. When
HSA was present in the assay, the activity of both human
IFN- a 2b fusion proteins and the unfused IFN- a were
slightly reduced (Figure 11.4), indicating some nonspecific
inhibition of EMCV infection by HSA in this cell line. IFN-
a 2b-DOM7h-14 and HSA-IFN- a 2b activity were calculated
at 3.8
30
10
-10
Concentration (pM)
(B)
110
90
70
50
30
10
-10
Concentration (pM)
FIGURE 11.2 Activity of interferon fusion proteins in HEK293
IFN- a / b reporter cell assay. To assay for IFN activity in vitro IFN-
a 2b-DOM7h-14 (- - - & - -), HSA-IFN- a 2b, (- ~ -), and unfused
IFN- a (- -) were tested for their ability to induce SEAP expression
in the HEK293 IFN- a / b reporter cell line in the absence (A) or
presence (B) of 100 m M HSA. Source: Reproduced from Refer-
ence [20] by permission of Oxford University Press.
10 5 IU/mg, respectively. In compari-
son with the unfused IFN- a , this represents a fold reduction
in activity of 17.4 for IFN- a 2b-DOM7h-14 and 275 for
10 6 and 2.4
FIGURE 11.3 Schematic diagram of the EMCV assay. A549 cells are preincubated with human
IFN- a 2b or human IFN- a 2b fusion proteins. Following wash off of interferon cells, these cells are
then infected with virus, after which protection against virus infection is measured by determining
cell viability using dye exclusion methods.
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