Antonella Grigoletto

Chemical and Enzymatic Site Specific PEGylation of hGH: The Stability and in vivo Activity of PEG‐N‐Terminal‐hGH and PEG‐Gln141‐hGH Conjugates

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.

Site-selective enzymatic chemistry for polymer conjugation to protein lysine residues: PEGylation of G-CSF at lysine-41

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.

Covalent immobilisation of transglutaminase: stability and applications in protein PEGylation

Abstract 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 Km and Vmax, 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.

Transgultaminase-Mediated Nanoarmoring of Enzymes by PEGylation

PEGylation, the covalent attachment of polyethylene glycol to bioactive molecules, is one of the leading approaches used to prolong pharmacokinetics, to improve the stability, and to reduce the immunogenicity of therapeutic proteins. PEG-conjugated products are associated with better therapy outcomes and improved patient compliance. Widely applied in clinical practice, the technology is mainly used to modify proteins, peptides, and oligonucleotides but also other drug delivery systems such as the liposomal one. Undergoing continuous attempts to optimize therapeutic efficacy and to tune the formation of conjugates, a number of different PEGylation processes are now available to researchers for protein conjugation. Although the possibility of obtaining highly homogeneous conjugate mixtures, preferably formed by a single monoconjugate, from a chemical conjugation reaction continues to be limited, several enzymatic conjugation approaches have recently been investigated to address this need. PEGylation mediated by microbial transglutaminase and its many advantages and modifications are outlined in detail in the current work permitting interested readers to perform site-specific protein derivatization to glutamines or lysines.

A site-selective hyaluronan-interferonα2a conjugate for the treatment of ovarian cancer

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.

Transglutaminase and Sialyltransferase Enzymatic Approaches for Polymer Conjugation to Proteins

Proteins hold a central role in medicine and biology, also confirmed by the several therapeutic applications based on biologic drugs. Such therapies are of great relevance thanks to high potency and safety of proteins. Nevertheless, many proteins as therapeutics might present issues like fast kidney clearance, rapid enzymatic degradation, or immunogenicity. Such defects implicate frequent administrations or administrations at high doses of the therapeutics, thus yielding or exacerbating potential side effects. A successful technology for improving the clinical profiles of proteins is the conjugation of polymers to the protein surface. The design of a protein-polymer conjugate presents critical aspects that determine the efficacy and safety of the final product. The control over stoichiometry and conjugation site is a strict criterion on which researchers have been intensively focused during the years, in order to obtain homogeneous and batch-to-batch reproducible products. An innovative site-specific conjugation strategy relies on the use of enzymes as tools to mediate polymer conjugation. Enzymatic approaches are attractive because they allow site-selective polymer conjugation at specific protein amino acids. In these reactions, the polymer is a substrate analog that replaces the native substrate. Furthermore, enzymes can count other advantages such as high yields of conversion and physiological conditions of reaction. This chapter provides a meaningful description of protein-polymer conjugation through transglutaminase-mediated and sialyltransferase-mediated enzymatic strategies, reporting the mechanism of action and some relevant examples.