The delivery of therapeutic peptides and proteins is often challenged by a short half-life, and thus the need for frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. Here, we demonstrate that a single subcutaneous injection of site-specific (C-terminal) conjugates of exendin-4 (exendin) — a therapeutic peptide that is clinically used to treat type 2 diabetes — and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) with precisely controlled molecular weights lowered blood glucose for up to 120 h in fed mice. Most notably, we show that an exendin-C-POEGMA conjugate with an average of 9 side-chain ethylene glycol (EG) repeats exhibits significantly lower reactivity towards patient-derived anti-poly(ethylene glycol) (PEG) antibodies than two FDA-approved PEGylated drugs, and that reducing the side-chain length to 3 EG repeats completely eliminates PEG antigenicity without compromising in vivo efficacy. Our findings establish the site-specific conjugation of POEGMA as a next-generation PEGylation technology for improving the pharmacological performance of traditional PEGylated drugs, whose safety and efficacy are hindered by pre-existing anti-PEG antibodies in patients.

In this review, we summarize —from a materials science perspective— the current state of the field of polymer conjugates of peptide and protein drugs, with a focus on polymers that have been developed as alternatives to the current gold standard, poly(ethylene glycol) (PEG). PEGylation, or the covalent conjugation of PEG to biological therapeutics to improve their therapeutic efficacy by increasing their circulation half-lives and stability, has been the gold standard in the pharmaceutical industry for several decades. After years of research and development, the limitations of PEG, specifically its non-degradability and immunogenicity have become increasingly apparent. While PEG is still currently the best polymer available with the longest clinical track record, extensive research is underway to develop alternative materials in an effort to address these limitations of PEG. Many of these alternative materials have shown promise, though most of them are still in an early stage of development and their in vivo distribution, mechanism of degradation, route of elimination and immunogenicity have not been investigated to a similar extent as for PEG. Thus, further in-depth in vivo testing is essential to validate whether any of the alternative materials discussed in this review qualify as a replacement for PEG.

We report a new methodology for the synthesis of polymer-drug conjugates from “compound”—all in one—prodrug monomers that consist of a cyclic polymerizable group that is appended to a drug through a cleavable linker. We show that organocatalyzed ring-opening polymerization can polymerize these monomers into well-defined polymer prodrugs that are designed to self-assemble into nanoparticles and release drug in response to a physiologically relevant stimulus. This method is compatible with structurally diverse drugs and allows different drugs to be copolymerized with quantitative conversion of the monomers. The drug loading can be controlled by adjusting the monomer(s) to initiator feed ratio and drug release can be encoded into the polymer by the choice of linker. Initiating these monomers from a polyethylene glycol macroinitiator yields amphiphilic diblock copolymers that spontaneously self-assemble into micelles with a long plasma circulation, which is useful for systemic therapy.

Conventional methods for synthesizing protein/peptide–polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site-specificity, which results in heterogeneous products with significantly compromised bio activity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with > 90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide–polymer conjugates.

We report a new methodology for the synthesis of polymer-drug conjugates from "compound"-all in one-prodrug monomers that consist of a cyclic polymerizable group that is appended to a drug through a cleavable linker. We show that organocatalyzed ring-opening polymerization can polymerize these monomers into well-defined polymer prodrugs that are designed to self-assemble into nanoparticles and release drug in response to a physiologically relevant stimulus. This method is compatible with structurally diverse drugs and allows different drugs to be copolymerized with quantitative conversion of the monomers. The drug loading can be controlled by adjusting the monomer(s) to initiator feed ratio and drug release can be encoded into the polymer by the choice of linker. Initiating these monomers from a polyethylene glycol macroinitiator yields amphiphilic diblock copolymers that spontaneously self-assemble into micelles with a long plasma circulation, which is useful for systemic therapy. KeywordsPolymerizable prodrug; Ring-opening polymerization; Polymer-drug conjugate; Nanoparticle; Cancer therapy Most small-molecule drugs utilized in the clinic have poor bioavailability and suboptimal pharmacokinetics because of their hydrophobicity and low molecular weight. Polymeric drug delivery systems can improve the efficacy of these drugs by increasing their water solubility, prolonging their circulation time, increasing the amount of drug deposited in the target tissue, and decreasing their exposure to normal tissues. [1] Conjugation of hydrophobic drugs to hydrophilic polymers can address these problems, [2] and is typically carried out by separate synthesis of the polymer, drug and linker, and sequential conjugation of the three entities to create the polymer-drug conjugate. This conventional strategy requires multiple reaction steps with limited yield, and has limited control of the site and degree of drug loading. New methods are hence needed to synthesize polymer-drug conjugates that have the following attributes: (1) are compatible with a structurally diverse set of drugs; (2) enable more than one drug to be conjugated to the same polymer with tunable control of the * chilkoti@duke.edu. Supporting information for this article is given via a link at the end of the document. Motivated by this rationale, we report herein a new method to synthesize polymer-drug conjugates by living ring-opening polymerization (ROP) of prodrugs (Scheme 1). This scheme inverts the conventional approach of conjugating a drug to a polymer post-synthesis, and instead directly incorporates the drug during synthesis of the polymer. These polymer prodrugs are composed of a "compound" monomer that consists of three covalently linked moieties: (1) a cyclic group that can undergo ROP to yield a biodegradable main chain, that is attached to (2) a cleavable linker, and which is attached to (3) a drug of interest. Living ROP of the compound monomer leads to the synthesis of a polymer with a biodegradable main chain with pendant drug molecules th...

Many proteins suffer from sub-optimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom transfer radical polymerization (ATRP) at the N-terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by reaction of the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methylacrylate) with a 1 carbon spacer (C1) (PCBMA), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of 1 carbon (C1) and 2 carbon (C2) spacer. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar MW. The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.

A new and versatile method is described to engineer precisely defined protein/peptide–polymer therapeutics by a modular approach that consists of three steps: (1) fuse a protein/peptide of interest with an elastin–like–polypeptide that enables facile purification and high yield; followed by (2) installation of a clickable group at the C–terminus of the recombinant protein/peptide with close to complete conversion by enzyme–mediated ligation; and subsequently (3) attachment of a polymer by a click reaction with near quantitative conversion. We demonstrate that this modular approach is applicable to various protein/peptide drugs and used it to conjugate structurally diverse water-soluble polymers that prolong the plasma circulation duration of proteins. These protein/peptide–polymer conjugates exhibit significantly improved pharmacokinetics and improved therapeutic effects over the native protein/peptide after administration to mice. The studies reported here provide a facile methodology for synthesis of protein/peptide-polymer conjugates for therapeutic use and other applications.

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