Prolonging the half-life of human interferon-alpha 2 in circulation: Design, preparation, and analysis of (2-sulfo-9-fluorenylmethoxycarbonyl)7- interferon-alpha 2

Abstract

Polypeptide drugs are generally short-lived species in circulation. In this study, we have covalently linked seven moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the amino groups of human interferon-alpha2. The derivative thus obtained (FMS(7)-IFN-alpha2) has approximately 4% the biological potency and 33 +/- 4% the receptor binding capacity of the native cytokine. Upon incubation, FMS(7)-IFN-alpha2 undergoes time-dependent spontaneous hydrolysis, generating active interferon with t(1/2) values of 24 +/- 2 h at pH 8.5 and 98 +/- 10 h at pH 7.4. When native IFN-alpha2 is intravenously administered to mice, circulating antiviral activity is maintained for a short duration and then declines with t(1/2) = 4 +/- 0.5 h, reaching undetectable values at approximately 18 h after administration. With intravenously administered FMS(7)-IFN-alpha2, there is a lag period of 2 h, followed by a progressive elevation in circulating antiviral-active protein, which peaked at 20 h and declined with t(1/2) = 35 +/- 4 h. FMS(7)-IFN-alpha2 is resistant to alpha-chymotrypsin digest and to proteolytic inactivation by human serum proteases in vitro. We have thus introduced here an inactive IFN-alpha2 derivative, which is resistant to in situ inactivation and has the capability of slowly reverting to the native active protein at physiological conditions in vivo and in vitro. Having these attributes, FMS(7)-IFN-alpha2 maintains prolonged circulating antiviral activity in mice, exceeding 7-8 times the activity of intravenously administered native cytokine.

Figure 1

Progressive modification of the amino acid moieties of IFN-α2 with FMS-OSu; loss of receptor binding capacity as a function of FMS moieties incorporated into IFN-α2. Human IFN-α2 was modified at pH 8.5 with increasing concentrations of FMS-OSu, ranging from 1 equivalent up to 20 molar equivalents of FMS-OSu. For each treatment, receptor binding capacity (A) and moles FMS introduced covalently into IFN-α2 (B) were determined. Receptor binding capacity toward immobilized ifnar2-EC was assessed by the reflectometric interference spectroscopy procedure. The rising and declining curves represent, respectively, ligand association and ligand dissociation from ifnar2-EC. Moles of FMS/moles of IFN-α2 were determined by UV absorption at 301 nm after dialysis and by mass spectroscopy (see Materials and Methods).

Figure 2

Time course of reactivation of FMS₇–IFN-α2 upon incubation at 37°C, pH 7.4. FMS₇–IFN-α2 (1 mg/ml in PBS buffer, pH 7.4) was incubated at 37°C. Aliquots were withdrawn daily and analyzed for their antiviral potency to inhibit VSV-induced cytopathic effects in human WISH cells (see Materials and Methods). Results are expressed as percent of antiviral potency of the native cytokine. Half-maximal inhibition of cytopathic effect was obtained at 0.3 ± 0.03 pM IFN-α2. IFN-α2 derivative exhibiting half-maximal inhibition at a concentration of 30 ± 3 pM was considered as having 1% of the native antiviral potency.

Figure 3

Susceptibility of FMS₇–IFN-α2 toward enzymatic degradation by α-chymotrypsin. FMS₇–IFN-α2 and native IFN-α2 (1 mg/ml each in PBS buffer, pH 7.4) were incubated with α-chymotrypsin (1% wt/wt). At the indicated time points, aliquots were subjected to analytical HPLC. The quantity of the cytokine (peak area) at t = 0 was assigned 100%.

Figure 4

Resistance of FMS₇–IFN-α2 to inactivation in human serum. FMS₇–IFN-α2 and native IFN-α2 were incubated in human serum at 37°C. At the indicated time points, aliquots were tested for antiviral potency to inhibit VSV-induced cytopathic effects in human WISH cells (see Materials and Methods). (A) Antiviral activity of native IFN-α2. (B) Aliquots of FMS₇–IFN-α2 incubated in serum were either introduced directly to WISH cells (dashed columns) or pretreated for 18 h at pH 8.5, 37°C (open columns).

Figure 5

Intravenously administered FMS₇–IFN-α in mice facilitates prolonged circulating antiviral activity. Groups of mice (n = 5 for each group) received intravenously (10 μg/mouse) native-IFN-α2 (A) or FMS₇–IFN-α2 (B and C). Blood aliquots were withdrawn at the indicated time points. Circulatory antiviral activities in aliquots were determined in human WISH cells before (B) and after (C) an additional period of incubation for 18 h (pH 8.5, 37°C).

Figure 6

Intravenous coadministration of native IFN-α2 and FMS–IFN-α2 facilitates prolonged circulating antiviral activity from the time of administration. Groups of mice (n = 3 for each group) received intravenously native IFN-α2 (10 μg/mouse) or FMS₇–IFN-α2 (10 μg/mouse) or both (10 μg of each). Blood aliquots were withdrawn at the indicated time points and analyzed in human WISH cells for antiviral activity.