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Drug delivery: Brushing off antigenicity

Conjugation of a diabetes drug with a brush polymer reduces the reactivity of the drug conjugate towards pre-existing polymer antibodies in human plasma and improves the drug's performance in diabetic mice.

Therapeutic peptides and proteins can make safe and effective drugs for humans. Over 100 such drugs have so far been approved for clinical use1,2, and interest in developing more is high given the increased rate of discovery of new biological targets and of new peptides and proteins with untapped therapeutic potential. However, naturally occurring peptides and proteins are often unstable, or have poor physicochemical properties or short half-lives in blood. These limitations lead to short efficacy time windows and frequent dosing, which reduce patient compliance and clinical appeal. Half-life extension techniques, such as drug depots or recombinant fusions with the Fc region of an antibody or of albumin, have however decreased the dosage of biologic drugs from twice daily to once a week or once a month3. One of the most established and commercially successful ways of synthetically extending a biologic's half-life is PEGylation — the covalent attachment of bioactive molecules to a poly(ethylene glycol) (PEG) chain. Since the early 1990s, over 10 PEGylated biological drugs have been approved by the US Food and Drug Administration (FDA), with many more currently enlisted in both early and late clinical trials3. Although PEG has been regarded as a non-immunogenic material with a proven safety record in humans, antibodies against it have recently been found in patients taking PEG–drug conjugates and in healthy individuals4. Reporting in Nature Biomedical Engineering, Ashutosh Chilkoti and colleagues now show that a brush-like, PEG-mimic polymer overcomes obstacles associated with current PEGylation techniques5. In particular, they demonstrate that a conjugate of exendin-4 (exendin) — a clinical drug for type 2 diabetes — and the PEG mimic has a markedly extended half-life, improved pharmacodynamics and, most notably, eliminates PEG antigenicity in human plasma without compromising biological efficacy in mouse models.

PEG is a polymer made of ethylene glycol (EG) subunits that bind to water molecules. Hence, hydrated peptides or proteins conjugated with PEG have improved solubility and stability. However, PEGylation reduces the drug's potency by restricting the interaction between the drug and its target6. For clinical applications, the rational design of a biologic with an optimized PEGylation process needs to balance the dose/efficacy ratio; for peptides, this is particularly challenging because of their relatively small size (typically <5,000 Da) compared with that of a PEG molecule (20,000–50,000 Da). Although significant progress has been made in the synthesis of PEG molecules carrying site-specific functional groups or having specific shapes, developing an optimum form of PEGylated biologics, suitable to be scaled-up for clinical testing, is not trivial, and is further limited by low production yield, complicated purification and lack of site-specificity.

Humans develop antibodies towards PEG because of the chronic exposure to the polymer, which is present in many consumer products, foods and pharmaceuticals. In patients, anti-PEG antibodies can reduce the efficacy of PEGylated drugs and increase the risk of hypersensitivity reactions. In fact, anti-PEG antibodies have abrogated the clinical efficacy of pegaspargase (l-asparaginase) and pegloticase (porcine-like uricase) — two PEGylated non-human proteins. This suggests that the formation of anti-PEG antibodies may depend on the immunogenicity, triggered by T-cell recognition of foreign (that is, non-human) epitopes, of the conjugated peptides or proteins. Because of this, the FDA currently requests anti-PEG antigenicity analyses in patients treated with PEGylated compounds that are under clinical development.

Chilkoti and co-authors covalently attached poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) to exendin (currently marketed as Byetta) — a peptide agonist of the glucagon-like peptide-1 receptor that facilitates insulin release in type 2 diabetes — by means of sortase-A-catalysed polymer conjugation (Fig. 1). Sortase A is a transpeptidase derived from the bacterium Staphylococcus aureus that recognizes the peptide sequence lysine–proline–glutamic-acid–threonine–glycine (LPETG) fused to the C-terminus of exendin. Sortase A selectively cleaves the T–G amide bond and generates a reactive thioacyl intermediate that can be attached to the reactive atom transfer radical polymerization (ATRP) initiator AEBMP by forming a stable peptide bond. Once the ATRP initiator is installed on the C-terminus of the peptide, the peptide is grafted to the brush-shaped POEGMA through controlled in situ ATRP in aqueous buffer. By using this approach, the authors synthesized exendin–POEGMA conjugates with molecular weights in the 20,000–200,000 Da range and with precisely controlled polydispersity. They also controlled the shape of the POEGMA brush by using OEGMA monomers with varying side-chains.

Figure 1: A PEGylation approach for improving drug half-life and eliminating PEG antigenicity.
Figure 1

Sortase-A-catalysed installation of the brominated, atom transfer radical polymerization (ATRP) initiator AEBMP on the C-terminus of a peptide (or protein) enables a controlled in situ ATRP in aqueous buffer that generates the brush-shaped PEG-mimic POEGMA. Peptide–POEGMA conjugates show increased solubility and stability, extended half-life and improved pharmacodynamics, and absence of PEG antigenicity5. Ab, antibody; LPETG, lysine–proline–glutamic-acid–threonine–glycine, a peptide sequence fused to the C-terminus of the peptide that is recognized by sortase A.

Unlike conventional polymer-conjugation strategies involving multiple purification steps and low yields, Chilkoti and co-authors’ approach leads to high-purity peptide– polymer conjugates at a high production yield (>80%) by using easy purification steps. In a type 2 diabetes mouse model, the exendin– POEGMA conjugate showed extended half-life and improved pharmacodynamics, comparable to what had been obtained by using a site-specific PEGylated peptide7. Remarkably, the authors also demonstrate that the PEG antigenicity of exendin–POEGMA can be eliminated without compromising exendin efficacy by optimizing the side-chain length of the polymer brushes.

Although the safety of PEGylated drugs is not in dispute and patients with pre-existing anti-PEG antibodies treated with PEGylated interferons did not experience any reduction in the half-life or efficacy of their treatment4, the potential negative impact of PEG antigenicity should not be underestimated, and ought to be monitored during the early stages of PEGylated drug development. Chilkoti and co-authors’ POEGMA-conjugation approach provides a possible solution for the elimination of PEG antigenicity triggered by PEGylated drugs. However, to establish POEGMA conjugates as clinically suitable biologics, it will be necessary to demonstrate that the approach works for other types of therapeutic peptides and proteins such as (l-asparaginase and uricase). Also, as the authors recognized, any immunogenic reactions to POEGMA will need to be carefully investigated in multiple species, including humans5. All things considered, Chilkoti and colleagues have provided a strategy for overcoming the many limitations of traditional PEGylation techniques.


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Author information


  1. Kang Choon Lee is at the School of Pharmacy, Sungkyunkwan University, Suwon 16469, Korea.

    • Kang Choon Lee
  2. Seulki Lee is at The Russell H. Morgan Department of Radiology and Radiological Sciences and The Center for Nanomedicine at the Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.

    • Seulki Lee


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Corresponding authors

Correspondence to Kang Choon Lee or Seulki Lee.