Enzyme-Polyethylene Glycol Adducts: Modified Enzymes with Unique Properties

Davis, Frank F.; Abuchowski, Abraham; Es, Theodorus van; Palczuk, Nicholas C.; Chen, R.; Savoca, K.; Wieder, Kenneth J.

doi:10.1007/978-1-4684-6985-1_35

Cited by 52 publications

(31 citation statements)

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“…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.…”

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“…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).…”

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

Katre¹,

Knauf²,

Laird³

1987

Proc. Natl. Acad. Sci. U.S.A.

220

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

Katre¹,

Knauf²,

Laird³

1987

Proc. Natl. Acad. Sci. U.S.A.

220

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“…Il suo uso è approvato anche da solo sotto forma di idrogel per la cicatrizzazione delle ferite [2]. Sin dagli anni Settanta gli studi di Frank Davis [3] hanno dimostrato che la pegilazione di proteine è in grado di migliorarne la farmaco-…”

Section: La Pegilazioneunclassified

[Pegylation and interferons in multiple sclerosis]

Centonze

Puma

Saleri

et al. 2016

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cinetica e la farmacodinamica mediante l'aumento dell'idrosolubilità, la riduzione della clearance renale e la riduzione della tossicità. È anche in grado di conferire maggior stabilità rispetto alle variazioni di pH e di temperatura. Tali vantaggi risultano particolarmente rilevanti ai giorni nostri, dal momento che sono sempre di più i polipeptidi approvati e in studio per l'uso clinico e che spesso il loro principale difetto consiste nella suscettibilità alla distruzione da parte di enzimi proteolitici, nella bassa emivita e bassa durata di conservazione, nella bassa solubilità, nella rapida clearance renale e nella tendenza a causare la produzione di anticorpi neutralizzanti [2]. La pegiLazioneLa pegilazione è un processo chimico che si realizza legando covalentemente una molecola di polietilenglicole (PEG) a un farmaco (Figura 1). Il polietilenglicole è una molecola approvata per l'utilizzo come additivo in cibi, cosmetici e prodotti farmaceutici (vedi Tabella I per gli utilizzi di PEG approvati). Il suo metabolismo e la sua escrezione sono stati studiati approfonditamente e sono ben compresi negli animali e nell'uomo, nel quale non sono state riportate tossicità renali critiche [1]. Per le dosi generalmente utilizzate in campo farmaceutico l'indice terapeutico è piuttosto ampio, di circa 600 volte, pertanto non è considerata una molecola tossica né immunogenica per l'uomo [1]. Il suo uso è approvato anche da solo sotto forma di idrogel per la cicatrizzazione delle ferite [2]. Sin dagli anni Settanta gli studi di Frank Davis [3] hanno dimostrato che la pegilazione di proteine è in grado di migliorarne la farmaco- Figura 1. Una molecola di polietilenglicole (PEG) Corresponding author Diego Centonze centonze@uniroma2.it DisclosureIl presente supplemento è stato supportato da Biogen Italia. Gli autori hanno avuto il completo controllo editoriale del manoscritto e hanno approvato tutti i contenuti abstract Pegylation is a procedure used for drug development since the 1970s and consists of the conjugation of a polyethylene glycol molecule (PEG) to a drug. PEG has shown to be safe and effective in improving the pharmacokinetic and pharmacodynamic profile of drugs. Recently, a 20 kDa linear chain of PEG was conjugated to interferon beta-1a with the aim to offer a new treatment option to relapsing-remitting multiple sclerosis (RRMS) patients. Due to a prolonged bioavailability, this new drug can be administered less frequently (every two weeks) than the other interferons beta available, thus allowing to hypothesize a better adherence to the treatment, which, in turn, should result in better clinical and economic outcomes. A phase III clinical trial has proven its effectiveness compared to placebo in RRMS patients, as well as a safety profile comparable to that found in other interferon beta preparations. The immunogenicity of this new molecule is < 1%, thus minimizing the suppression or reduction of interferon beta biological activity that could come from the development of Neutralizing Antibodies (NAbs).

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“…It has been shown (4)(5)(6)(7)(8)(9)(10)24) conjugate induces immunosuppression through the regulatory mechanisms involving specific epitope recognition. One of the first steps in immune regulation takes place in the presentation of epitopes to T cells by antigen-presenting cells.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies have shown that conjugation ofmPEG with various protein antigens causes a loss of most of the antigenicity of the native antigens (4)(5)(6)(7)(8)(9)(10), and prior injection of animals with antigen-mPEG conjugates leads to the development of tolerance to subsequent immunization with the native antigen (5,7,9). In this paper, we present data showing that the mPEG-AcChoRa-(125-148) conjugate suppresses the development of EAMG by electrophysiological criteria and decreases the antibody response specific for the AcChoRa region from residue 125 to 148 without effect on the antibody responses to other AcChoR antigenic sites.…”

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confidence: 99%