Site of Pegylation and Polyethylene Glycol Molecule Size Attenuate Interferon-α Antiviral and Antiproliferative Activities through the JAK/STAT Signaling Pathway*

Therapeutic pegylated interferon-αs (IFN-α) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-α molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-α molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-α2b that were monopegylated using 5–30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys1, His34, Lys31, Lys83, Lys121, Lys131, and Lys134. The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-α2b control. The highest residual activity was observed with the His34 positional isomers, and the lowest was observed with the Cys1 positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys134 > Lys83 ∼ Lys131 ∼ Lys121 > Lys31. The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-α to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His34 positional isomer suggests that this site may be favorable for pegylating IFN-α2b molecules.

IFNs), which have provided a dramatic increase in virological response, especially in combination with ribavirin. Standard IFN-␣ therapy has a short (Ͻ12-h) half-life that requires subcutaneous injection three times weekly to maintain effective levels in the blood (2). The short half-life of IFN-␣ has led to the development of longer lasting preparations achieved by the attachment of a large polyethylene glycol (PEG) molecule directly to IFN-␣. Two different commercial preparations of PEG-IFN-␣ have been developed for clinical use, PEG-IFN-␣2b (PEG-INTRON®) and PEG-IFN-␣2a (Pegasys®); both have long half-lives (40 and 80 h, respectively) that permit once weekly administration (3). Both of these preparations have been demonstrated to be effective for the treatment of patients with hepatitis C (4), and clinical trial results have shown further that both of the pegylated molecules produce sustained viral response rates superior to those achieved with their respective standard IFN-␣s (5)(6)(7).
Whereas pegylation has proven to be highly effective for slowing the clearance of biological molecules, including IFN-␣, and thus increasing serum half-life, it has been shown to also modify in vitro biological activity (8). For instance, we have reported that pegylation of IFN-␣2b with a 12-kDa linear PEG molecule results in a preparation that has a specific activity of 28% relative to IFN-␣2b; the loss in activity was not due to structural perturbation of the core IFN-␣2b core protein (9). Other groups have reported that pegylation of IFN-␣2a with a 40-kDa branched PEG molecule results in a preparation that contains from 1 to 7% relative specific activity compared with IFN-␣2a (10,11). These two pegylated interferon-␣s (PEG-IFN-␣s) differ substantially in their postpegylation constituent properties. PEG-IFN-␣2b has a 12-kDa linear PEG molecule attached using succinimidyl carbonate polyethylene glycol (SC-PEG) chemistry via a covalent urethane-like bond to the IFN-␣2b protein (12). The pegylation linkage process results in a heterogeneous mixture of pegylation positional isomers that occur predominantly (ϳ50%) at the His 34 amino acid residue, with the remaining positional isomers pegylated at various lysines, the N-terminal cysteine, a serine, tyrosine, and an alternate histidine residue (12). PEG-IFN-␣2a has a 40-kDa branched PEG molecule attached using N-hydroxysuccinimide-PEG chemistry via a covalent amide bond to the IFN-␣2a protein (11). PEG-IFN-␣2a is also a heterogeneous mixture of pegylated positional isomers consisting of four major positional isomers at Lys 31 , Lys 121 , Lys 131 , and Lys 134 (10).
The different relative specific activities reported for the IFN-␣ preparations suggested that the significant differences in the size of the PEG molecule or the distribution of pegylation positional isomers or both might be accountable. Fractionation of specific pegylation positional isomers for 12-kDa PEG-IFN-␣2b have demonstrated that positional isomers had differential relative specific activities, with the His 34 positional isomer retaining higher relative activity (37%) than the mixture of positional isomers (9). Thus, changes in the activity of pegylated proteins appear to be influenced by the site of pegylation and, in some cases, the molecular weight of the PEG moiety (13).
Our study was undertaken to directly determine the effects of size and site of pegylation on in vitro activity for IFN-␣2b. This information is important for understanding how specific characteristics of PEG-IFN-␣s influence their in vitro activity that may be translated to their in vivo efficacy.
Production and Isolation of Pegylated Interferon Isomers for Activity Studies-The primary amino acid residues of IFN-␣ reactive to pegylation are Cys 1 , Lys 31 , His 34 , Lys 83 , Lys 121 , Lys 131 , and Lys 134 . His 34 and Cys 1 are most reactive at a neutral pH, whereas the ⑀-amine groups of lysine residues are most reactive at basic pH (15). Therefore, pegylation reactions utilizing SC-PEG 5 kDa, SC-PEG 12 kDa, SC-PEG 20 kDa, and SC-PEG 30 kDa were performed at room temperature at pH 6.5 to produce His 34 -and Cys 1 -modified positional isomers and pH 10 to produce the ⑀-amine-modified lysine positional isomers.
Reactions at pH 6.5 were performed in 50 mM sodium phosphate at 22°C, with SC-PEG 5 kDa, SC-PEG 12 kDa, and SC-PEG 20 kDa present in a 4.2-fold molar excess relative to IFN-␣2b (5 mg/ml). For the reactions involving SC-PEG 30 kDa, the linker-to-protein molar ratio was limited by linker solubility to 2:1. After 70 min, reactions were quenched with 1 M glycine to minimize the formation of multipegylated species. Reactions at pH 10 were performed in 30 mM sodium tetraborate at 22°C. Due to the increased rate of reaction at this pH, the molar ratio of linker to protein was reduced to 1.0 for SC-PEG 5 kDa, SC-PEG 12 kDa, and SC-PEG 20 kDa. To adjust for the reduced reactivity of higher M r PEG polymers, the ratio was increased to 1.5 for SC-PEG 30 kDa. Reactions were again quenched with 1 M glycine.
Single PEG-IFN-␣2b positional isomers were isolated from the mono-PEG-IFN-␣2b pools by high performance ion exchange chromatography (HPIEX) on a preparative scale sulfopropyl (SP) column (21.5 mm ϫ 15.0 cm, 13-m particle size; TosoHaas, Philadelphia, PA) using a Waters HPLC system (717ϩ Autosampler with a 2.5-ml syringe, 486 detector, two 510 HPLC pumps with high pressure gradient mixing, 6 ml/min flow rate) with monitoring by UV light at 280 nm. This procedure, previously utilized to resolve positional isomers of 12-kDa PEG-IFN-␣2b (15), was optimized to improve the resolution of positional isomers with varying PEG molecular weights. The purified mono-PEG-IFN-␣2b size exclusion pools were dialyzed overnight at 5°C against HPIEX buffer A (1.8 mM citric acid monohydrate, 3.3 mM sodium phosphate dibasic heptahydrate, pH 5.3). The dialysates were concentrated to 2.2 A 280 prior to loading onto the SP column, preequilibrated with buffer A. Loading was adjusted over a range of 19 -65 g of protein/ml of resin. Bound positional isomers were eluted using pH gradients formed by buffer B (30 mM trisodium citrate dihydrate, 70 mM sodium phosphate monobasic monohydrate, pH 6.0). To optimize resolution of positional isomers, the pH gradient was adjusted according to both attached PEG length and site of pegylation. As can be seen in Fig. 1A, the His 34 and Cys 1 positional isomers are well resolved from other positional isomers generated at a reaction pH of 6.5 when eluted with a gradient of 0 -10% B in 100 min, followed by 10 -30% B over 80 min. The separation of lysine-modified 12-kDa PEG-IFN-␣2b positional isomers also involved two sequential linear gradients; from 0 to 14% buffer B in 180 min followed by 14 -20% buffer B in 40 min. Due to weaker binding to the SP column, shallower gradients (0 -12% buffer B in 180 min followed by 12-15% buffer B in 40 min) were employed to elute higher M r PEG-IFN-␣2b lysine-modified positional isomers (Fig. 1B). Peak fractions were manually collected in order to generate pools consisting of single positional isomers. The purity and identity of the positional isomers were assessed by HPSEC, analytical HPIEX (SP-5PW, 7.5 mm ϫ 7.5 cm, 10-m particle size; TosoHaas), and peptide mapping followed by amino acid sequencing. There was no free IFN-␣ in any of the purified positional isomer pools detectable by HPSEC.
Stability of Positional Isomer Pools-Evaluation of the pools by HPSEC following five freeze/thaw cycles to simulate handling conditions demonstrated that the positional isomers were stable and the pools remained free of IFN-␣ contamination.
Peptide Mapping-Characterization of purified positional isomer peaks was carried out as described previously (15). Peak fractions were concentrated to 0.5-1 mg/ml using a centrifugal spin filter (Ultrafree 15 ml, BioMaxx 50; Millipore Corp.). After adding a 10% (v/v) aliquot of 50 mM ammonium bicarbonate, the concentrate was digested with trypsin (1:10 mol/mol) overnight at 37°C. Dithiothreitol (100 mM) was added to a final concentration of 5 mM, and incubation was carried out for 1 h at ambient temperature. The reduced digest was resolved by HPSEC with manual collection of pegylated peptides. The entire pegylated peptide peak was pooled and characterized by N-terminal sequence analysis. All peptide sequencing was performed by Commonwealth Biotechnologies (Richmond, VA) using Agilent hardware.
Production and Isolation of Branched 20-kDa PEG-IFN-␣2b and di-20-kDa PEG-IFN-␣2b for Estimation of Stokes Radius-Two additional preparations of pegylated IFN-␣2b were made solely for the study of the effect of pegylation on Stokes radius. Branched 20-kDa PEG-IFN-␣2b was produced by using PEG2-N-hydroxysuccinimide 20 kDa at a 2:1 molar ratio. Pegylation was performed at pH 8.8 at 22°C in 40 mM sodium tetraborate with quenching at 60 min using 1 M glycine. The reaction mixture was isolated and purified as described for the positional isomers used for activity measurements.
Di-20-kDa PEG-IFN-␣2b was produced as a secondary product from the SC-PEG reactions at pH 10. The mixture was purified from the pegylation reaction by collecting the unbound fraction obtained from anion exchange chromatography on Q HyperD (BioSepra). The reaction products were dialyzed against 10 mM sodium phosphate, pH 8.2. The dialyzed product was applied at 1.4 mg of protein/ml of resin to a 20-cm column with a flow of 1.66 cm/min. Dipegylated IFN-␣ was collected from the unbound fraction. This fraction was further purified by size exclusion chromatography on Superdex 200 HiLoad under conditions identical to those used for the purification of monopegylated interferon ␣. The purity of dipegylated IFN-␣ purified by these columns was Ͼ99% by size exclusion HPLC.
STAT1 Translocation Assay-Human hepatoma (Huh-7) cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 2 mM GlutaMax-1, 100 units/ml penicillin/streptomycin, and nonessential amino acids. Cells (10,000 cells/well) were seeded overnight in 96-well Packard black view plates (Packard Instrument Co.). 3-Fold serial dilutions of the test IFN-␣s were prepared in Dulbecco's modified Eagle's medium and incubated with cells at 37°C in 5-6% CO 2 for 30 min. The cells were washed with phosphate-buffered saline, fixed with 3.7% formaldehyde, and permeabilized with 0.5% Triton X-100. The cells were then incubated with either a polyclonal anti-STAT1 p84/p91 (clone E-23; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a polyclonal anti-STAT2 (clone C-20; Santa Cruz Biotechnology) primary antibody for 1 h at room temperature. The cells were sequentially washed with phosphate-buffered saline and 0.01% Tween 20. They were then incubated for 1 h with a mixture of Alexa Fluor® 488-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR) and Hoechst 33342 (Molecular Probes). The cells were again washed with 0.01% Tween 20 and phosphate-buffered saline. The plates were imaged on the ArrayScan® II High Content Screening System (Cellomics, Pittsburgh, PA) with a ϫ10 objective using the ArrayScan version 2.1 soft-ware (Cellomics). The resulting images were processed using the Cytoplasm to Nucleus Translocation Application software (Cellomics). The cytonuclear difference, defined as the difference in fluorescence intensity of the target STAT in the nuclear region minus the cytoplasmic region, was used as a measure of STAT translocation. Cytoplasmic and nuclear STAT1 levels were also examined for each cytonuclear difference determination. We confirmed that the cytonuclear difference was primarily caused by an increase in nuclear STAT1, whereas cytoplasmic STAT1 levels remained constant or only decreased slightly. This observation was consistent with data reported for TNF-␣-mediated NF-B cytonuclear translocation measurement using this instrumentation and software (16).
Antiviral and Antiproliferation Assays-The antiviral assay was performed by titrating serial 2-fold dilutions in 96-well microtiter assay plates seeded with either human foreskin fibroblast cells (FS-71) or non-small cell lung carcinoma cells (A549) infected with encephalomyocarditis virus as described previously (17). The relative potency of IFN-␣2b and all of the PEG-IFN-␣s tested was determined by comparing the dose of the test preparation, which affords 50% protection to infected cells, with the dose of control IFN-␣2b reference standard. The IFN-␣2b standard was calibrated against the NIBSC IFN-␣2b (95/566) standard. The materials for the PEG-IFN-␣s were dose-ranged in preliminary experiments to assure that potency would be determined across a suitable response range. Specific activities for 12-kDa PEG-IFN-␣2b and 40-kDa PEG-IFN-␣2a were calculated from the empirically determined titer at 50% protection divided by the calculated IFN-␣2b protein weight at the 50% point.
The antiproliferation assay was performed by titrating serial 2-fold dilutions in 96-well microtiter assay plates seeded with Daudi cells. Daudi cells (ATCC), a human lymphoblastoid cell line, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. After a 72-h incubation, cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide/SDS and analyzed spectrophotometrically for cellular mitochondrial activity associated with viability. The relative potencies of IFN-␣2b and all of the PEG-IFN-␣s tested were determined by comparing the dose of the test preparation, which induced a 50% decrease in formazon-determined proliferation, with the dose of control IFN-␣2b reference standard. The IFN-␣2b standard was calibrated against the NIBSC IFN-␣2b (95/566) standard. For the PEG-IFN␣s, materials were dose-ranged in preliminary experiments to assure that potency would be determined across a suitable response range. Specific activities for 12-kDa PEG-IFN-␣2b and 40-kDa PEG-IFN-␣2a were calculated from the empirically determined titer at 50% proliferation divided by the calculated IFN-␣2b protein weight at the 50% point.
Statistical Analysis of STAT Translocation and Antiviral Data-The 50% inflection point for each of the dose-response curves of the STAT translocation responses and the antiviral responses were determined using iterative curve fitting software. SoftMaxPro version 4.3 (Molecular Devices) software was used for reducing the STAT translocation data. A proprietary version of AllFit, Allwin 2.03, was used for reducing the antiviral data. The descriptive statistics were generated using SAS JMP version 5.0.1 (SAS). An analysis of variance was carried out using Student's, Tukey-Kramer, Dunnett's, and Hsu MCB. All comparisons were first carried out against the respective PEG size isomer of His 34 . Additional comparisons were also conducted looking at the effect of PEG size change within each isomer. Other analyses for lysine comparisons in the antiviral assay were also performed. In selective cases where the variances were flagged as unequal, a Welch ANOVA test was also carried out to confirm results. Because the STAT translocation assay was more variable than the antiviral assay, the Student's t test was a more rugged comparator of variance.
Determination of Stokes Radius of PEG-IFN-␣2b-The apparent molecular masses and Stokes radii of 5-kDa, 12-kDa, and 20-kDa PEG-IFN-␣2b, branched 20-kDa IFN-␣2b (containing a PEG moiety with 2 ϫ 10-kDa PEG branches for a total molecular mass of 20 kDa), and a di-PEG 20-kDa IFN-␣2b (containing two linear 20-kDa PEG molecules attached to the protein at two different sites, for a total molecular mass of 40 kDa) were determined. The 5-kDa, 12-kDa, 20-kDa, branched 20-kDa, and di-PEG 20-kDa IFN-␣2b were produced using SC-PEG chemistry and purified as preparations of mixed positional isomers. Apparent M r values were obtained from a standard curve (log M r versus retention time by HPSEC analysis) constructed using 10 different marker proteins. The Stokes radius (R S ) for the PEG-IFN-␣2bs were then calculated based on a standard curve of log R S versus log M r also obtained using these markers. HPSEC analysis of the PEG-IFN-␣2bs was performed using a Sigma Chrom TM GFC-1300 column (12-15-m particle size; Sigma) in 0.1 M sodium phosphate and 0.1 M NaCl, pH 7.0, at a flow rate of 0.5 ml/min.

Confirmation of Differential Activity between 12-kDa Monopegylated IFN-␣2b and 40-kDa Monopegylated IFN-␣2a-Be-
cause the reported activity for 12-kDa monopegylated IFN-␣2b and 40-kDa monopegylated IFN-␣2a was generated from different laboratories using different antiviral assays, we first measured the differential activity directly. 12-kDa PEG-IFN-␣2b was consistently more than 25-fold more active than 40-kDa PEG-IFN-␣2a in an antiviral protection assay using either FS-71 or A549 cells (Table I). The activity of 12-kDa PEG-IFN-␣2b ranged between 25 and 35% of that for IFN-␣2b control, values comparable with those previously reported (9). The activity of 40-kDa PEG-IFN-␣2a was ϳ1% of that for IFN-␣2b. This value is lower than that from one previous report (10), but consistent with those from another recent evaluation of the activity of PEG-IFN-␣2a in the Madin-Darby bovine kidney antiviral assay (11). IFN-␣2a, the core interferon protein for 40-kDa PEG-IFN-␣2a, was titrated in the FS-71 antiviral assay with IFN-␣2b and was found to be equivalent in activity within the variation of the assay. Thus, the lower activity seen with the 40-kDa PEG-IFN-␣2a versus 12-kDa PEG-IFN-␣2b cannot be accounted for by differences in the core proteins in the assay. In addition, circular dichroism spectroscopy revealed no perturbation in the near or far UV for the core protein of 40-kDa PEG-IFN-␣2a (data not shown).
Results obtained in a Daudi cell antiproliferation assay were similar to those for the antiviral assay (Table I). The specific activity of 12-kDa PEG-IFN-␣2b was 28.9% of that for IFN-␣2b as compared with a value of 0.7% obtained for 40-kDa PEG-IFN-␣2a.

TABLE I
Comparative antiviral and antiproliferation activities for 12-kDa PEG-IFN-␣2b and 40-kDa PEG-IFN-␣2a 12-kDa PEG-IFN-␣2b or 40-kDa PEG-IFN-␣2a were diluted in serial 2-fold fashion by IFN-␣ protein weight. Antiviral protection was determined using an FS-71/EMCV CPE assay, and antiproliferative activity was determined using a Daudi cell assay. All titrations were performed in triplicate on plates with a calibrated IFN-␣2b reference standard. The mean titer (IU/ml) was determined at the 50% point of the titration curve and was an average of curves from three different experimental days (n ϭ 9 curves). Specific activity was calculated using the concentration of the IFN-␣2 protein, independent of weight contribution from PEG. The relative percentage of IFN-␣2b activity was calculated using the specific activity of IFN-␣2b at 2.6 ϫ 10 8 IU/mg. Effect of PEG Molecule Size on PEG-IFN-␣2b Stokes Radius and Apparent Molecular Weight-Because of the hydrophilic nature of PEG polymers, increased polymer length can significantly increase the relative Stokes radius of a pegylated protein (18). In addition, due to the linear nature of the PEG moiety, the increase in the apparent molecular weight of a globular protein such as IFN-␣2b is much greater than the sum of the molecular weights of the protein and the attached PEG group. Using SC-PEG chemistry, we purified mixed positional isomer preparations of 5-kDa, 12-kDa, linear 20-kDa, branched 20-kDa, and di-20-kDa PEG-IFN-␣2b and measured the apparent molecular weight (aM r ) and R S using HPSEC analysis. The addition of a 5-kDa PEG group to IFN-␣2b increased the aM r to 73,300, a value of more than 3 times the additive molecular mass of the 23.1-kDa protein and the 5-kDa PEG group and the R S was increased by 58% (Table II). With the addition of the 12-kDa PEG, the R S increased by 119% relative to IFN-␣2b, and the aM r increased to 165,600. The branched and linear 20-kDa PEGs increased R S by 166 and 184% relative to IFN-␣2b, and aM r increased to 272,300 and 320,600, respectively. For the di-20-kDa PEG (with a total molecular mass of 63.1 kDa), the R S was 275% higher than IFN-␣2b, and the aM r was 639,700. This increase in R S as pegylated moieties increase in size or position might have implications for receptor binding, activation, and clinical efficacy.
Production and Isolation of Pegylated Positional Isomers of IFN-␣2b-Pegylated positional isomers at Cys 1 , His 34 , Lys 31 , Lys 83 , Lys 121 , Lys 131 , and Lys 134 were chosen for study because they have been reported as being major positional isomers for 12-kDa PEG-IFN-␣2b or 40-kDa PEG-IFN-␣2a (10, 12). All reaction products were resolved by size exclusion chromatography to generate mono-PEG-IFN-␣ that was Ͼ99% pure by HPSEC. These monopegylated reaction products were then resolved into individual positional isomers by HPIEX (Fig. 1, A and B) to 98% purity by HPSEC and 91-99% purity by analytical HPIEX (15). Peptide mapping analysis can usually detect Ͼ5% impurities. However, one exception is the inability to resolve by HPIEX the neighboring peaks of 5-kDa Lys 131 and 5-kDa Lys 164 , hence the lower purity estimation by analytical HPIEX relative to HPSEC. The HPIEX chromatography profiles and positional isomer elution order in this study matched those reported previously (12,15,19). In addition, the identity and purity of the purified positional isomers were confirmed by peptide mapping analysis.
STAT1 Translocation Studies-To study the effect of pegylation size and site on JAK/STAT IFN-␣ signal transduction, an assay was developed to assess STAT1 translocation from the cytoplasm to the nucleus in Huh-7 cells. The STAT1 translocation activity for the titration curves of each of the seven positional isomers at each respective PEG molecule size is shown in Fig. 2. The ED 50 (inflection points) for the positive control curves of 12-kDa PEG-IFN-␣2b and 40-kDa PEG-IFN-␣2a ranged from 2.0 to 4.8 and from 33.0 to 76.0 ng/ml, respectively, across the experiments in Fig. 2; a statistical analysis (USP 27 ͗111͘) determined that none of the control infection points were outliers. This variation is consistent with newly developed cell-based bioassays. Assay results showed that the 12-kDa PEG-IFN-␣2b was more active than the 40-kDa PEG-IFN-␣2a. The STAT1 translocation activity for the His 34 positional isomer was consistently similar to that for 12-kDa PEG-IFN-␣2b. There was also little significant reduction in STAT1 translocation activity for the His 34 positional isomers from 5 through 30 kDa (Fig. 2A). The Lys 31 positional isomers exhibited less STAT1 translocation activity, demonstrated by a rightward shift in the titration curve, as the size of the PEG molecule increased from 5 to 30 kDa (Fig. 2B). The 5-kDa Lys 31 PEG-IFN-␣2b isomer had less translocation activity than the 12-kDa PEG-IFN-␣2b control curve (Fig. 2B). Similar trends of decreased translocation activity associated with increasing PEG molecule size were observed with the other Lys and Cys positional isomers (Fig. 2, C-G).
The ED 50 was calculated for each of the site-pegylated IFN-␣2b isomers at PEG molecule sizes from 5 through 30 kDa PEG (Fig. 3). The His 34 positional isomers at 5-30 kDa were the most active for STAT1 translocation, and increasing PEG molecule size at His 34 had only a small effect on STAT translocation activity. Cys 1 and Lys 31 had the lowest translocation activity across all PEG molecule sizes, and both were very sensitive to increasing PEG molecule size above 5 kDa. The STAT1 translocation activity for each differently sized PEG positional isomer for both Cys 1 and Lys 31 was significantly different (p Ͻ 0.05, Student's t test) compared with the respective His 34 positional isomer. The Lys 134 positional isomer trended to be the most active for STAT translocation of the lysine isomers studied, whereas the Lys 83 , Lys 121 , and Lys 131 isomers were roughly equivalent in their respective STAT translocation activities. These four lysine positional isomers also appeared to trend to lower STAT1 translocation activity compared with the His 34 positional isomer, although statistical significance was observed only with the 5-kDa Lys 83 and 20-kDa Lys 121 . For all of these positional isomers, there was a consistent decrease in translocation activity associated with increased PEG molecule size to 20 or 30 kDa. Antiviral Protection Activity of Pegylated Positional Isomers-The same site and size pegylation isomers were also studied using an antiviral protection assay in FS-71 cells. Overall, the results from the antiviral assay (Fig. 4) were consistent with the STAT1 translocation results. The most active positional isomer was His 34 , and the least active were Cys 1 and Lys 31 . The most active lysine positional isomer was Lys 134 , whereas Lys 83 , Lys 121 , and Lys 131 were roughly comparable in activity. Of note, all differently sized PEG positional isomers, compared against the respective His 34 positional isomer, were significantly lower (p Ͻ 0.05) in antiviral activity. However, the Lys 134 positional isomer, although lower in activity than His 34 , was statistically the most active of the lysine positional isomers studied. In addition, significant decreases occurred (p Ͻ 0.05) in the antiviral protection activity associated with increasing PEG molecule size for each of the positional isomers studied. These results confirm the significant differences and trends that were observed in the STAT1 translocation assay.

DISCUSSION
Our initial studies were performed using unfractionated PEG-IFN-␣2a and PEG-IFN-␣2b, which are known to be mixtures of positional isomers (10, 12). These results confirmed that there was a consistent 25-35-fold difference between the in vitro antiviral and antiproliferative activities of the two different PEG-IFN-␣ mixtures. The differences in relative activities between 12-kDa PEG-IFN-␣2b and 40-kDa PEG-IFN- ␣2a may result from the different sizes of the PEG moieties, the different distribution of site-pegylated isomers, differences in the core IFN-␣ proteins, and/or differences in the bonds linking the PEG molecules to their respective core protein (4,20). It is unlikely that differences between the activities of the core proteins, IFN-␣2a and IFN-␣2b, contribute to the observed differences between the two pegylated preparations, since their activities have been reported to be similar. In addition, prior studies have shown that pegylation does not detectably alter the secondary or tertiary conformation of the IFN-␣2 core protein as well as for both the 12-kDa PEG-IFN-␣2b and 40-kDa PEG-IFN-␣2a (9). Thus, the site of pegylation and size of PEG moiety would appear to be the critical elements influencing the relative activities of 12-kDa PEG-IFN-␣2b and 40-kDa PEG-IFN-␣2a.
The size of the PEG moiety increased the aM r of the pegylated molecule in excess of the additive empirical molecular weight of the PEG to IFN-␣2 core protein. This is due, in part, to the effect of the hydrophilic nature of PEG polymers on the Stokes radius of the pegylated molecule. Increases in linear PEG molecules from 5 to 20 kDa resulted in increased aM r from 23,100 for unpegylated IFN-␣2b to 320,600 for 20-kDa PEG-IFN-␣2b. Modification in PEG structures also impacted aM r .
Branching two 10-kDa PEG molecules to make 20-kDa PEG decreased the aM r from 320,600 to 272,300, whereas conjugating two 20-kDa PEG molecules as separate dipegylated moieties onto a single IFN-␣2b core protein increased the aM r to 639,700. Thus, increasing PEG moiety size could potentially impact the IFN-␣2 core protein when the PEG IFN-␣2 is presented to the interferon ␣ receptor.
The relative difference in STAT1 translocation activity that we observed between the 12-kDa PEG-IFN-␣2b and the 40-kDa PEG-IFN-␣2a was ϳ30:1 and consistent with their relative antiviral and antiproliferative activities. Several laboratories have reported that IFN-␣2 antiviral and antiproliferative activity in vitro is dependent upon JAK/STAT signaling through a potentially rate-limited interaction with the IFNAR1-IF-NAR2 heterodimeric receptor complex (21)(22)(23). IFN-␣2 binds to the IFNAR1-IFNAR2 heterodimeric complex, activating the JAK1 and Tyk2 kinases. This leads to phosphorylation and dimerization of STAT1 and STAT2 and subsequent translocation of the dimer to the nucleus with IRF-9 (24 -27). Translocation is required for the dimer to form the ISGF-3 complex and bind to the ISRE element that in turn initiates the transcription of IFN-␣-inducible genes (28). The advantage of using a STAT1 translocation assay for signaling assessment is that the translocation event is linked to both the receptor-ligand interaction and the ultimate expression of IFN-␣ gene transcription, which is necessary for antiviral and antiproliferative activity.
It seems likely that the very different distributions of positional isomers for 12-kDa PEG-IFN-␣2b and 40-kDa PEG-IFN-␣2a may contribute substantially to their differences in in vitro activity. The His 34 positional isomer is the major isomer in 12-kDa PEG-IFN-␣2b due to the chemical conditions used for the SC-PEG conjugation (29). The remaining positional isomers for 12-kDa PEG-IFN-␣2b are predominantly lysine conjugates (9). In contrast, 40-kDa PEG-IFN-␣2a comprises almost completely lysine positional isomers and includes no histidine positional isomers (11). The 12-kDa PEG-IFN-␣2b and 40-kDa PEG-IFN-␣2a share positional isomers at Lys 31 , Lys 83 , Lys 121 , Lys 131 , and Lys 134 , although in different distributions. Significantly, the His 34 positional isomer demonstrated a consistently lower ED 50 for induction of STAT1 translocation than any of the other positional isomers studied. Conversely, the Lys 31 positional isomer had one of the highest ED 50 values for induction of STAT1 translocation. The difference in activity for these two positional isomers may seem surprising, since they are located in close proximity on the AB1 loop (amino acids 22-51) of IFN-␣2, which appears to be involved in one of two putative binding domains for IFNAR2 (30 -33).
There are several potential explanations for difference in ac-tivity of the His 34  isomer had higher STAT1 translocation and antiviral activity. This site is located in the DE loop of IFN-␣2b, suggesting that steric hindrance from a PEG molecule at the Lys 134 site is less than at either Lys 121 or Lys 131 . Lys 83 is reported to be located at an interaction point between IFN-␣2b and IFNAR1, so it is reasonable to observe diminished activity with this positional isomer (34). Finally, the Cys 1 site was found to be particularly sensitive to pegylation. The structural relevance of this site is important, since it is absolutely conserved; in the NH 2 terminus portion of IFN-␣, this has been shown to be important in mediating antiviral activity (33,35).
Another possible explanation for diminished activity based on the site of pegylation is differential influence on IFN-␣2b and IFNAR1/IFNAR2 interaction kinetics (21,36). For example, it has been postulated that the charge loss associated with His 34 pegylation may perturb the electrostatic potential at or near the receptor binding site less than with ⑀-amine pegylation (15). However, it should be noted that Piehler and Schreiber have observed that the IFN-␣ binding site on IFNAR2 is not a highly negatively charged area on the protein surface, and this limits the potential role for electrostatic forces in determining the rate of association (37). Nevertheless, a cooperative interaction, first through electrostatic steering, followed by docking of the IFN-␣2b and IFNAR2 and IFNAR1, might be impacted by the pegylation.
It is also possible that the site of pegylation in conjunction with size-mediated steric hindrance might cause the core IFN-␣ protein to be presented in a different contextual interaction with the heterodimeric receptor such that unique recognition domains are not engaged. There have been many reports of differential activity conferred by different IFN-␣ species with the intact receptor (35). Hybrid IFN-␣ recombinants have been shown to have specific domains for antiviral activity (35,38), antiproliferative activity (39), and cytotoxic activity (40 -42). Studies using human IFN-␣2a/2c hybrids have shown that the N terminus region and the hydrophobic residues on the C-helix region are particularly important for antiproliferative activity (43). Splice variants of the human IFNAR1 have been shown to differentially recognize different IFN-␣s through unique receptor subdomains for major histocompatibility complex class I antigen expression (44). PEG-IFN-␣2b has been shown to have roughly equivalent reductions in the antiviral, antiproliferative, cytotoxic, and major histocompatibility complex class I expression activities, suggesting that the sites of pegylation and the 12-kDa PEG molecule impact these potential recognition domains on the heterodimeric receptor equivalently (9). In these studies, we confirmed that PEG-IFN-␣2a lost approximately equivalent activity in the antiviral and antiproliferative assays, although significantly more activity was lost than that observed for PEG-IFN-␣2b. This suggests that similar domain recognition may exist even with the differences in site of pegylation and size of the PEG molecule for PEG-IFN-␣2a and PEG-IFN-␣2b.
In light of our results, it is interesting to note that both antiviral and antiproliferative activity in vitro is transduced by a relatively small receptor occupancy that appears to be independent of mass action but dependent on continuity of signal induction (22,37). Site of pegylation and PEG molecule size both significantly affect in vitro signaling of IFN-␣2b, suggesting that a discontinuity in signaling may be occurring as a result of a decreased K on rate. This may have important clinical implications, since the K on rate is critical for receptor-mediated signaling. Another clinically important component is the serum residency time of the molecule. Pegylation of IFN-␣ significantly increases the serum residency and thus the number of potential interactions with the receptor. However, at the same time, pegylation decreases the ability of IFN-␣ to interact with its receptor once it reaches the cell surface. These two consequences of pegylation are likely to have opposing effects on the clinical utility of the resulting preparations, and they must be carefully balanced in the design of PEG-IFN-␣s.
Conclusion-The higher in vitro specific activity of 12-kDa PEG-IFN-␣2b relative to 40-kDa PEG-IFN-␣2a can be attributed to differences in the respective size of the PEG moiety and the distribution of positional isomers. This study provides evidence that the overall in vitro activity of PEG-IFN-␣s is governed both by PEG moiety size and by specific positional isomers. In particular, the 12-kDa PEG-His 34 positional isomer, a major constituent of 12-kDa PEG-IFN-␣2b, retained the highest postpegylation specific activity. This activity was significantly higher for in vitro antiviral activity compared with all other isomers studied. The higher activity for 12-kDa PEG-IFN-␣2b was observed to occur as early as the STAT1 translocation step within the IFN-␣-mediated JAK/STAT signaling pathway. However, increasing the PEG moiety size significantly attenuated the in vitro antiviral activity of all pegylation sites studied. The correlative effects of site and size of pegylation observed with the antiviral, antiproliferation, and STAT1 translocation activity point to a receptor-mediated mechanism. Unfortunately, extant reported clinical studies have not been appropriately balanced by protein weight to help clearly elucidate the impact of any receptor-mediated activity on in vivo efficacy. Further study of the in vitro effects of pegylation and the subsequent impact on in vivo efficacy are needed to improve our understanding of the optimal balance of receptor-mediated activity against extended serum half-life.