We have modified recombinant interleukin-2 (rIL-2) to facilitate site-directed covalent attachment of monomethoxy polyethylene glycol (PEG). The site chosen for modification and subsequent covalent attachment with PEG (PEGylation) was the single glycosylation position found in the native interleukin-2 (IL-2). The mutant protein was expressed in E. coli, purified, and PEGylated with a PEG-maleimide reagent to obtain PEG-cys3-rIL-2. The PEG-cys3-rIL-2 had full bioactivity relative to the unmodified molecule and had an increase in hydrodynamic size sufficient to increase its systemic exposure by approximately 4 fold. This method has general applicability for modifying any therapeutic protein at a specific site and thereby alter its potency. In particular, it can be used to attach PEG to prokaryotically expressed recombinant proteins at their glycosylation sites.

Recombinant human interleukin 2 purified from Escherichia coli has limited solubility at neutral pH and a short circulatory half-life. This recombinant interleukin 2 was chemically modified by an active ester of polyethylene glycol. The modified interleukin 2 was purified by hydrophobic interaction chromatography and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and isoelectric focusing. This conjugate was compared to unmodified recombinant interleukin 2 in vitro and in vivo. Covalent attachment of the hydrophilic polymer polyethylene glycol enhanced the solubility of interleukin 2, decreased its plasma clearance, and increased its antitumor potency in the Meth A murine sarcoma model.Interleukin 2 (IL-2), a glycosylated lymphokine with approximate molecular weight 15,000 (1), has therapeutic potential in treating cancers and infectious diseases (2-4). Human IL-2 has been obtained from genetically engineered Escherichia coli as an unglycosylated protein, rIL-2 (5-7), with biological activities equivalent to those of native glycosylated IL-2 (6, 8). However, the IL-2 expressed in E. coli is contained in insoluble refractile bodies within the bacteria and therefore denaturants are required during its purification. Both native IL-2 and rIL-2 are rapidly cleared from the circulation of mice (9-12) and rats (13), resulting in limited bioavailability of the protein. Nevertheless, Rosenberg and co-workers have shown that systemic administration of rIL-2 in high doses causes regression of established metastases in mice (14) and, in conjunction with lymphokine-activated killer cells, in humans (15). This suggests that increasing the bioavailability of rIL-2 may increase its potency, thus facilitating more effective use of this protein as a drug.We have modified rIL-2 to increase its solubility and plasma half-life. This has been accomplished by chemically attaching the polymer monomethoxy polyethylene glycol (PEG) to the protein. This straight-chain amphiphilic polymer has been used to covalently modify several proteins (16, 17). Abuchowski, Davis, and co-workers have modified several enzymes with PEG, including asparaginase and uricase, resulting in partially active conjugates with longer half-lives in vivo (18,19).We report here the preparation, purification, and characterization of rIL-2 covalently modified by monomethoxy polyethylene glycol. This PEG-rIL-2 conjugate is soluble in aqueous medium at neutral pH, is as active as IL-2 in three in vitro bioassays, exhibits an increased circulatory half-life in vivo, and is efficacious in vivo in a mouse tumor model. MATERIALS AND METHODSPreparation of an Active Ester of PEG. Monomethoxy PEG of average molecular weight 5000 (Aldrich) was used without further purification. PEG was converted to the active ester in two steps. In the first step, PEG-glutarate was prepared from PEG and glutaric anhydride as described by Zalipsky et al. (20). The second step was the preparation of the Nhydroxysuccinimide ester of 21). This ester reacts predomin...

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