Site-Specific Transglutaminase-Mediated Conjugation of Interferon α-2b at Glutamine or Lysine Residues

Abstract

Interferon α (IFN α) subtypes are important protein drugs that have been used to treat infectious diseases and cancers. Here, we studied the reactivity of IFN α-2b to microbial transglutaminase (TGase) with the aim of obtaining a site-specific conjugation of this protein drug. Interestingly, TGase allowed the production of two monoderivatized isomers of IFN with high yields. Characterization by mass spectrometry of the two conjugates indicated that they are exclusively modified at the level of Gln101 if the protein is reacted in the presence of an amino-containing ligand (i.e., dansylcadaverine) or at the level of Lys164 if a glutamine-containing molecule is used (i.e., carbobenzoxy-l-glutaminyl-glycine, ZQG). We explained the extraordinary specificity of the TGase-mediated reaction on the basis of the conformational features of IFN. Indeed, among the 10 Lys and 12 Gln residues of the protein, only Gln101 and Lys164 are located in highly flexible protein regions. The TGase-mediated derivatization of IFN was then applied to the production of IFN derivatives conjugated to a 20 kDa polyethylene glycol (PEG), using PEG-NH for Gln101 derivatization and PEG modified with ZQG for Lys164 derivatization. The two mono-PEGylated isomers of IFN were obtained in good yields, purified, and characterized in terms of protein conformation, antiviral activity, and pharmacokinetics. Both conjugates maintained a native-like secondary structure, as indicated by far-UV circular dichroism spectra. Importantly, they disclosed good in vitro antiviral activity retention (about only 1.6- to 1.8-fold lower than that of IFN) and half-lives longer (about 5-fold) than that of IFN after intravenous administration to rats. Overall, these results provide evidence that TGase can be used for the development of site-specific derivatives of IFN α-2b possessing interesting antiviral and pharmacokinetic properties.

Dual, Site‐Specific Modification of Antibodies by Using Solid‐Phase Immobilized Microbial Transglutaminase

“…Fontana et al. recently described MTG‐mediated lysine modifications of interferon α subtypes and avidin at multiple sites with the nonstoichiometric use of glutamine‐containing ZQG derivatives (ZQG, N ‐benzyloxycarbonyl‐ l ‐glutaminylglycine). As immobilization of enzymes can increase site selectivity, as, for example, reported for lipase, we speculated whether this would also apply to immobilized MTG.…”

From Synthesis to Characterization of Site-Selective PEGylated Proteins

“…Spolaore et al studied the reactivity of IFN α-2b to microbial mTGase to obtain a site-specific conjugation of this biopharmaceutical. Characterization by mass spectrometry of the conjugates indicated that among the 10 Lys and 12 Gln residues of the protein only Gln101 and Lys164 were selectively conjugated with a PEG-NH 2 for Gln101 and a PEG modified with carbobenzoxy--glutaminyl-glycine for Lys164 derivatization, with activity retention and improvements at pharmacokinetic levels (Spolaore et al, 2016). A mono-PEGylated derivative of filgrastim (granulocyte colonystimulating factor) was also prepared using mTGase.…”

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

“…The most well used partner for bioactive peptide drugs is human serum albumin. The choice of this protein has been justified for a variety of reasons [13,14] and its use is exemplified by human serum albumin-interferon fusion proteins [6,7,[15][16][17], which are typically expressed in yeast [15]. The expression of such proteins in E. coli is problematic since serum albumin contains 35 cysteine residues engaged in 17 disulphide bonds.…”

“…In general, pegylation occurs randomly on the ε-amino group of surface lysine residues [4], though, site-specific pegylation has also been reported [5,6]. For more specific targeting, however, fusion technology to graft a protein specifically to the N-or C-terminal of a drug has been developed.…”

“…Albumin fusion to interferon is one such example [6,7]. Most commonly, E. coli is the host of choice for the heterologous expression of recombinant proteins.…”