POEGMAlation – A Next-Generation PEGylation Technology (2016)

&lt;p&gt;The delivery of therapeutic peptides and proteins is often challenged by a short circulation half-life, necessitating frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. The covalent conjugation of therapeutic peptides and proteins, and more recently oligonucleotide-based drugs, with the “stealth” polymer poly(ethylene glycol) (PEG), termed PEGylation, is one of the most commonly used approaches to increase the in vivo half-life and reduce the immunogenicity of these therapeutic biomolecules. However, after several decades of research and clinical use, the limitations of PEGylation have begun to emerge.&lt;/p&gt;&lt;p&gt;Conventional methods for synthesizing peptide/protein-polymer conjugates have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of control over site and stoichiometry of conjugation, which results in heterogeneous products with significantly compromised biological activity. Additionally, anti-PEG antibodies have been induced in patients treated with PEGylated drugs and have been shown to correlate with rapid clearance of these drugs. High levels of pre-existing anti-PEG antibodies have also been found in individuals naïve to PEGylated agents, which are associated with serious first-exposure allergic reactions.&lt;/p&gt;&lt;p&gt;To address the synthetic limitations of PEGylation, a general approach for the high-yield synthesis of site-specific (C-terminal) and stoichiometric (1:1) peptide/protein-polymer conjugates, named sortase-catalyzed polymer conjugation, was developed. Demonstrating proof-of-concept of the approach with green fluorescent protein (GFP) as a model protein, sortase A from Staphylococcus aureus was used to site-specifically attach an initiator solely at the C-terminus of GFP, followed by in situ growth of the PEG-based brush polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) from the protein macroinitiator by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeded with high specificity and near-complete (~ 95%) product conversion. Subsequent in situ ATRP in aqueous buffer produced 1:1 stoichiometric conjugates with &gt; 90% yield, tunable MW, low dispersity, and no denaturation of the protein. The extraordinarily high yield compares favorably to order of magnitude losses typically seen in conventional PEGylation processes.&lt;/p&gt;&lt;p&gt;Next, the therapeutic potential of POEGMAlation, or the conjugation of POEGMA to a peptide or protein, was demonstrated by implementing the developed sortase-catalyzed polymer conjugation strategy with exendin-4 (exendin), a therapeutic peptide for treating type 2 diabetes, to synthesize exendin-C-POEGMA conjugates with a wide and tunable range of molecular weights (MWs) and low dispersity. A single subcutaneous injection of exendin-C-POEGMA conjugates lowered blood glucose for up to 120 h in a diabetic mouse model. Most intriguingly, we showed that appending PEG as oligomeric side-chains on the conjugated POEGMA and tuning the side-chain length completely eliminated the reactivity of exendin-C-POEGMA conjugates toward patient-derived anti-PEG antibodies without compromising in vivo efficacy. Clinically, the lack of anti-PEG antigenicity of POEGMA conjugates is expected to completely eliminate serious first-exposure allergic reactions and the accelerated blood clearance of POEGMA-drug conjugates due to pre-existing anti-PEG antibodies in patients.&lt;/p&gt;&lt;p&gt;Collectively, these results establish POEGMAlation as a next-generation PEGylation technology that is highly useful for improving the pharmacological performance of therapeutic biomolecules while providing a timely solution to the increasing levels of pre-existing anti-PEG antibodies in patients that are seriously hindering the safety and efficacy of traditional PEGylated drugs.&lt;/p&gt;