title: Transglutaminase-mediated dual and site-specific incorporation of poly(ethylene glycol) derivatives into a chimeric interleukin-2.
abstract: To expand the applications of poly(ethylene glycol) (PEG)-protein conjugates for clinical use, we have developed a novel method for dual and site-specific incorporations of PEG derivatives into proteins using a substrate peptide (AQQIVM, named TG2) and transglutaminase (TGase). In our previous studies, TG2 was shown to be a special peptide with two adjacent Gln substrates for guinea pig liver transglutaminase (G-TGase). We have now constructed a chimeric protein (named rTG2-IL-2) of human interleukin-2 (IL-2), in which TG2 was fused to the N-terminus of IL-2. For the G-TGase-catalyzed reaction, rTG2-IL-2 was dually and site-specifically modified with alkylamine derivatives of PEG (PEG10, average M(r) 10 kDa) at both the Gln2 and Gln3 residues in the appended tag. To demonstrate the effectiveness of the G-TGase-catalyzed PEG-incorporation, we have compared the characteristics and the biological properties of PEG10-rTG2-IL-2 species with two PEG10 molecules attached to rTG2-IL-2 [(PEG10)(2)-rTG2-IL-2] with that of (PEG10)(2)-rhIL-2(R), in which PEG10 was randomly incorporated into rhIL-2 by a general procedure using a N-hydroxysuccinimidyl ester of PEG (PEG10-COOSu) (M(r) 10 kDa). (PEG10)(2)-rTG2-IL-2 was found to be superior in its in vitro bioactivities and equivalent in its pharmacokinetic profiles to (PEG10)(2)-rhIL-2(R). Unlike most previous methods, this approach can place dual PEG chains at designed sites on chimeric proteins without decreasing their bioactivities. Thus, TGase-catalyzed PEG-incorporation would improve the therapeutic utility of PEG-protein conjugates.
authors.affiliation: Ajinomoto Company Inc., Central Research Laboratories, 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, 210-8681, Japan. haruya_satou@ajinomoto.com
authors.forename: H
authors.initials: H
authors.lastname: Sato
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
authors.forename: E
authors.initials: E
authors.lastname: Hayashi
authors.forename: Y
authors.initials: Y
authors.lastname: Takahara
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D008297:Male; D011092:Polyethylene Glycols / Q000378:metabolism*; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; C000717609:Tgm2 protein, rat; D011092:Polyethylene Glycols; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases