IGF-I and IGF-II are essential regulators of mammalian growth, development and metabolism, whose actions are modified by six high-affinity IGF binding proteins (IGFBPs). New lines of knockout (KO) mice lacking either IGFBP-3, -4, or -5 had no apparent deficiencies in growth or metabolism beyond a modest growth impairment (approximately 85-90% of wild type) when IGFBP-4 was eliminated. To continue to address the roles of these proteins in whole animal physiology, we generated combinational IGFBP KO mice. Mice homozygous for targeted defects in IGFBP-3, -4, and -5 remain viable and at birth were the same size as IGFBP-4 KO mice. Unlike IGFBP-4 KO mice, however, the triple KO mice became significantly smaller by adulthood (78% wild type) and had significant reductions in fat pad accumulation (P < 0.05), circulating levels of total IGF-I (45% of wild type; P < 0.05) and IGF-I bioactivity (37% of wild type; P < 0.05). Metabolically, triple KO mice showed normal insulin tolerance, but a 37% expansion (P < 0.05) of beta-cell number and significantly increased insulin secretion after glucose challenge, which leads to enhanced glucose disposal. Finally, triple KO mice demonstrated a tissue-specific decline in activation of the Erk signaling pathway as well as weight of the quadriceps muscle. Taken together, these data provide direct evidence for combinatorial effects of IGFBP-3, -4, and -5 in both metabolism and at least some soft tissues and strongly suggest overlapping roles for IGFBP-3 and -5 in maintaining IGF-I-mediated postnatal growth in mice.

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.

Type I interferon-α(IFN-α) has proven to be a clinically effective antiviral and antineoplastic drug for 20 yr (1). Recently, pegylated forms of IFN-α have been commercially produced and have shown superior clinical efficacy to unpegylated IFN-α for reducing HCV viral load with less frequent dosing required for the patient (2,3). The superior clinical efficacy is probably derived from the enhanced serum half-life of the pegylated IFN-α in patients. However, pegylation also reduces the in vitro activity of the core IFN-α protein (4,5). Understanding the structural implications of pegylation on IFN-α activity is critical for biologically characterizing the commercial drug product. In vitro characterization provides a basis for establishing consistency in the manufacturing process for the precursor IFN-α and the final pegylated product. The better characterized the product is, the higher the confidence is for assessing and demonstrating comparability between pegylated products when comparing changes in process of manufacture, site of manufacture, or like-product from different sources.