Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in the murine Meth A sarcoma model.

Abstract

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...

Polyion complex micelles as vectors in gene therapy – pharmacokinetics and in vivo gene transfer

“…23,24 Modifications of peptides or proteins with PEG increase their stability and solubility in the blood and can increase their potency. 25,26 It has already been reported that PIC micelles of DNA with PEG-PLL are highly water soluble, and form nuclease resistant nanoparticles. 18 Moreover, addition of PEG was reported to have reduced cytotoxicity compared with polycation homopolymer.…”

Molecular design of hybrid tumour necrosis factor-alpha. II: The molecular size of polyethylene glycol-modified tumour necrosis factor-alpha affects its anti-tumour potency

“…Chemical modification of bioactive proteins with polyethylene glycol (PEG) increases their molecular size and steric hindrance, both of which are derived from PEG attached to bioactive proteins, resulting in augmented plasma half-lives and stability (Katre et al, 1987;Hershfield et al, 1991). These PEG-modified bioactive proteins have increased therapeutic potency, so PEGylation enables the therapeutic dose and frequency to be decreased.…”

Site-specific chemical modification with polyethylene glycol of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves antitumor activity and reduces animal toxicity and immunogenicity

“…PEGylation of proteins increases their plasma half-lives by reducing proteolysis and restricting tissue-distribution such as glomerular filtration by the kidney and hepatic uptake (12)(13)(14)(15)(16)(17)(18)(19)(20). This effect is attributed to the increased molecular size and steric hindrance that result from attaching PEG to proteins.…”

“…This results in an improvement of plasma half-lives and in proteolytic-stability, and a decrease in immunogenicity and hepatic uptake (13,15). PEGylation of interleukin-2 has been reported to increase its antitumor potency in vivo (16), and PEGylation of an F(abЈ)2 derived from the monoclonal antibody A7 has improved its tumor localization (17). We previously reported that a PEGylated chimeric toxin composed of transforming growth factor-␣ and PE showed an improvement in its blood-residency time and Abbreviation: PE, Pseudomonas exotoxin; PEG, polyethylene glycol.…”