title: Development of copper-catalyzed azide-alkyne cycloaddition for increased in vivo efficacy of interferon β-1b by site-specific PEGylation.
abstract: The development of protein conjugate therapeutics requires control over the site of modification to allow for reproducible generation of a product with the desired potency, pharmacokinetic, and safety profile. Placement of a single nonnatural amino acid at the desired modification site of a recombinant protein, followed by a bioorthogonal reaction, can provide complete control. To this end, we describe the development of copper-catalyzed azide-alkyne cycloaddition (CuAAC, a click chemistry reaction) for site-specific PEGylation of interferon β-1b (IFNb) containing azidohomoalanine (Aha) at the N-terminus. Reaction conditions were optimized using various propargyl-activated PEGs, tris(benzyltriazolylmethyl)amine (TBTA), copper sulfate, and dithiothreitol (DTT) in the presence of SDS. The requirement for air in order to advance the redox potential of the reaction was investigated. The addition of unreactive PEG diol reduced the required molar ratio to 2:1 PEG-alkyne to IFNb. The resultant method produced high conversion of Aha-containing IFNb to the single desired product. PEG-IFNbs with 10, 20, 30, and 40 kDa linear or 40 kDa branched PEGs were produced with these methods and compared. Increasing PEG size yielded decreasing in vitro antiviral activities along with concomitant increases in elimination half-life, AUC, and bioavailability when administered in rats or monkeys. A Daudi tumor xenograft model provided comparative evaluation of these combined effects, wherein a 40 kDa branched PEG-IFNb was much more effective than conjugates with smaller PEGs or unPEGylated IFNb at preventing tumor growth in spite of dosing with fewer units and lesser frequency. The results demonstrate the capability of site-specific nonnatural amino acid incorporation to generate novel biomolecule conjugates with increased in vivo efficacy.
authors.affiliation: Allozyne, 1600 Fairview Avenue E, Suite 300, Seattle, WA 98102, USA. nnairn@allozyne.com
authors.forename: Natalie W
authors.initials: NW
authors.lastname: Nairn
authors.forename: Kurt D
authors.initials: KD
authors.lastname: Shanebeck
authors.forename: Aijun
authors.initials: A
authors.lastname: Wang
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Graddis
authors.forename: Michael Pete
authors.initials: MP
authors.lastname: VanBrunt
authors.forename: Kenneth C
authors.initials: KC
authors.lastname: Thornton
authors.forename: Kenneth
authors.initials: K
authors.lastname: Grabstein
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D000998:Antiviral Agents / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D001386:Azides / Q000737:chemistry*; D001665:Binding Sites; D001682:Biological Availability; D002384:Catalysis; D045744:Cell Line, Tumor; D003300:Copper / Q000737:chemistry*; D061565:Cycloaddition Reaction; D006801:Humans; D000068576:Interferon beta-1b; D016899:Interferon-beta / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008715:Methionine / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D023041:Xenograft Model Antitumor Assays
chemical_list: D000480:Alkynes; D000970:Antineoplastic Agents; D000998:Antiviral Agents; D001386:Azides; D000068576:Interferon beta-1b; D011092:Polyethylene Glycols; D016899:Interferon-beta; D003300:Copper; D008715:Methionine