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.
Microbial transglutaminase (mTGase) is an enzyme that catalyzes site-specific protein derivatization at specific glutamines. mTGase-mediated conjugation with PEG-NH2 to granulocyte colony stimulating factor (G-CSF) yields a site selective mono-derivative conjugate involving Gln135. The same enzymatic reaction of mTGase, i.e. the transfer of the Gln acyl group to an amino donor, was investigated by reversing the substrates. A specific acyl donor PEG derivative was synthesized by coupling the Z-QG mTGase substrate to PEG. The mTGase-mediated conjugation of this PEG-ZQG in the presence of G-CSF generated a high-yield PEG-G-CSF conjugate in which the polymer was selectively coupled to Lys41 of the protein. The PEG-K41-G-CSF conjugate was compared with the PEG-Q135-G-CSF one obtained through mTGase conjugation of PEG-NH2 to Gln135. Biophysical characterization showed that the two positional isomers have similar behaviors, and pharmacokinetic studies in rats demonstrated that they have comparable half-life extensions. Overall, the study demonstrates that mTGase protein derivatization is linked to inherent advantages because it carries with it the possibility of targeting lysines or glutamines, in both cases with a high site-selective specificity
The use of therapeutic proteins is often impaired by their short in vivo half-lives. PEGylation has been exploited to enhance protein stability and to prolong the pharmacokinetic. The biophysical characterization of two site-specific mono-PEGylated forms of human growth hormone (hGH)--chemically N-terminal PEGylated hGH (PEG-Nter-hGH) and enzymatically Gln141 PEGylated hGH (PEG-Gln141-hGH) via transglutaminase--is outlined here and their pharmacodynamics are compared. The thermal stability of PEG-Nter-hGH was increased with respect to that of hGH and PEG-Gln141-hGH. Pharmacodynamic studies in rats showed that a single injection of the conjugates had a better or comparable potency with respect to a daily hGH on a week schedule in terms of weight gain, femoral length, and tibial diaphysis width.
One of the main goals of vaccine research is the development of adjuvants that can enhance immune responses and are both safe and biocompatible. We explored the application of the natural polymer hyaluronan (HA) as a promising immunological adjuvant for protein-based vaccines. Chemical conjugation of HA to antigens strongly increased their immunogenicity, reduced booster requirements, and allowed antigen dose sparing. HA-based bioconjugates stimulated robust and long-lasting humoral responses without the addition of other immunostimulatory compounds and proved highly efficient when compared to other adjuvants. Due to its intrinsic biocompatibility, HA allowed the exploitation of different injection routes and did not induce inflammation at the inoculation site. This polymer promoted rapid translocation of the antigen to draining lymph nodes, thus facilitating encounters with antigen-presenting cells. Overall, HA can be regarded as an effective and biocompatible adjuvant to be exploited for the design of a wide variety of vaccines.
While interferon alpha (IFNα) is used in several viral and cancer contexts, its efficacy against ovarian cancer (OC) is far from being incontrovertibly demonstrated and, more importantly, is hindered by heavy systemic side effects. To overcome these issues, here we propose a strategy that allows a targeted delivery of the cytokine, by conjugating IFNα2a with an aldehyde-modified form of hyaluronic acid (HA). The resulting HA-IFNα2a bioconjugate was biochemically and biologically characterized. The conjugation with HA did not substantially modified both the antiviral function and the anti-proliferative activity of the cytokine. Moreover, the induction of STAT1 phosphorylation and of a specific gene expression signature in different targets was retained. In vivo optical imaging biodistribution showed that the i.p.-injected HA-IFNα2a persisted into the peritoneal cavity longer than IFNα2a without being toxic for intraperitoneal organs, thus potentially enhancing the loco-regional therapeutic effect. Indeed, in OC xenograft mouse models bioconjugate significantly improved survival as compared to the free cytokine. Overall, HA-IFNα2a bioconjugate disclosed an improved anticancer efficacy, and can be envisaged as a promising loco-regional treatment for OC.
Microbial transglutaminase enzyme (mTGase) is an extremely useful enzyme that is increasingly employed in the food and pharmaceutical industries and as a tool for protein modification and tagging. The current study describes how we immobilised mTGase (iTGase) on a solid support to improve its stability during the PEGylation process by which polyethylene glycol chains are attached to protein and peptide drugs. When the enzyme was immobilised at the N-terminal sequence on agarose beads, it retained more than 53% of its starting activity. Kinetic studies on the immobilised and free mTGase disclosed a 1.7 and 1.5 fold decrease of K and V, respectively. Protein PEGylation was carried out using α-lactalbumin (α-LA) and granulocyte colony stimulating factor (G-CSF). In the former case, the iTGase showed a selective conjugation towards only one Gln residue of α-LA, avoiding formation of a mono- and bi-conjugate mixture that is achieved using the free enzyme. In the latter case, the immobilised enzyme still remained selective towards only one Gln, but avoided the undesired formation of deamidated G-CSF that took place when free mTGase was used. Overall, the results of the current study highlight the suitability of iTGase in preparing site-selective protein-polymer conjugates.
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