Imagine you’re suffering from severe chronic gout, a highly debilitating form of arthritis caused by uric acid crystals in the joint. Your body could not tolerate other gout treatments, so your doctor wanted you to try Krystexxa (PEGylated uricase), the last option available. After a couple of doses, your body developed antibodies to the PEG polymers in Krystexxa, which quickly clear the drug from the circulation and render this last line of therapy ineffective. Unfortunately, you just experienced what tens of thousands of Americans suffer from every year in dealing with severe chronic gout.^1,2

Our research group has always been fascinated by the adaptive immune system and its ability to orchestrate immune defense against a myriad of infectious agents. Building off our longstanding work in elucidating antibody function^3–9 and also engineering PEGylated drug carriers^10–12, we became very intrigued about how anti-PEG antibodies (APA), antibodies that specifically bind PEG, can render PEGylated therapies ineffective. A few years ago, we decided to initiate an active research program to understand the adaptive immune system’s response to synthetic polymers such as polyethylene glycol (PEG), a polymer regarded as very safe and widely used by the pharmaceutical industry to enhance the solubility, stability, pharmacokinetics, and biodistribution of protein therapeutics and drug delivery systems.^13–16

While there have been increasing insights into the mechanisms behind how APA can be induced and studies on their prevalence in the population and their impact on select therapeutics in both animal models and clinical trials,^17–19 we kept facing a puzzling question that sparked much speculation: how do APA specifically bind to a highly flexible polymer such as PEG that lacks any fixed 3-dimensional antigen conformation and is densely surrounded by water molecules?

Figure 1: Anti-PEG antibody (grey) binds tightly to PEG polymer chain backbone (blue and red) as antigen via an open ring substructure at the antibody paratope established primarily by virtue of an aromatic side chain from a tryptophan residue (magenta) in the hypervariable region of the antibody heavy chain.

To resolve this mystery, our group performed X-ray crystallography to first determine the structural properties and conformation of an APA in complex with PEG as antigen. We found, after several screens, two “hits” under different conditions, which revealed identical structures for our APA Fab bound with PEG. To our surprise, the PEG polymer chain appeared to thread through an open ring substructure at the APA Fab paratope (Fig. 1). We identified a critical tryptophan residue in the heavy chain complementarity-determining region 3 (HCDR3) primarily responsible for this interesting open ring motif. Through single amino acid point mutations to this tryptophan residue and other neighboring residues of the Fab paratope, we proved the importance of this ring substructure for stable binding to PEG. Finally, we developed a binding assay to suggest a potential molecular mechanism this APA Fab undergoes in solution to reach its final PEG-bound complex, which must rely on some degree of initial flexibility at the essential tryptophan residue before establishing the final continuous surface ring substructure (Fig. 2).

Figure 2: Step-by-step theoretical molecular mechanism depicting the dynamic binding processes of anti-PEG antibody (grey) binding to flexible PEG antigen (blue and red).

Our work not only answers a long-standing question from our research group and the more broad field of anti-polymer immunogenicity, but we believe these studies provide mechanistic insight into better understanding the humoral immune system and binding processes adopted by atypical antibody molecules.

To learn more about our recent work published in Communications Chemistry, please visit: https://www.nature.com/articles/s42004-020-00369-y

References:

1. Lipsky, P. E. et al. Pegloticase immunogenicity: The relationship between efficacy and antibody development in patients treated for refractory chronic gout. Arthritis Res. Ther. 16, 1–8 (2014).
2. Guttmann, A., Krasnokutsky, S., Pillinger, M. H. & Berhanu, A. Pegloticase in gout treatment - safety issues, latest evidence and clinical considerations. Ther. Adv. Drug Saf. 8, 379–388 (2017).
3. Newby, J. et al. A blueprint for robust crosslinking of mobile species in biogels with weakly adhesive molecular anchors. Nat. Commun. 8, (2017).
4. Schiller, J. L., Marvin, A., McCallen, J. D. & Lai, S. K. Robust antigen-specific tuning of the nanoscale barrier properties of biogels using matrix-associating IgG and IgM antibodies. Acta Biomater. 89, 95–103 (2019).
5. Schiller, J. L. et al. Antibody-mediated trapping in biological hydrogels is governed by sugar-sugar hydrogen bonds. Acta Biomater. 107, 91–101 (2020).
6. Schroeder, H. A. et al. Herpes simplex virus-binding IgG traps HSV in human cervicovaginal mucus across the menstrual cycle and diverse vaginal microbial composition. Mucosal Immunol. 11, 1477–1486 (2018).
7. McSweeney, M. D. et al. Overcoming anti-PEG antibody mediated accelerated blood clearance of PEGylated liposomes by pre-infusion with high molecular weight free PEG. J. Control. Release 311–312, 138–146 (2019).
8. Yang, Q. et al. Analysis of pre-existing IgG and IgM antibodies against polyethylene glycol (PEG) in the general population. Anal. Chem. 88, 11804–11812 (2016).
9. McCallen, J., Prybylski, J., Yang, Q. & Lai, S. K. Cross-Reactivity of Select PEG-Binding Antibodies to Other Polymers Containing a C-C-O Backbone. ACS Biomater. Sci. Eng. 3, 1605–1615 (2017).
10. Yang, Q. et al. Evading immune cell uptake and clearance requires PEG grafting at densities substantially exceeding the minimum for brush conformation. Mol. Pharm. 11, 1250–1258 (2014).
11. Parker, C. L. et al. Pretargeted delivery of PEG-coated drug carriers to breast tumors using multivalent, bispecific antibody against polyethylene glycol and HER2. Nanomedicine Nanotechnology, Biol. Med. 21, 102076 (2019).
12. Huckaby, J. T. et al. Engineering Polymer‐Binding Bispecific Antibodies for Enhanced Pretargeted Delivery of Nanoparticles to Mucus‐Covered Epithelium. Angew. Chemie 131, 5660–5664 (2019).
13. Veronese, F. M. & Pasut, G. PEGylation, successful approach to drug delivery. Drug Discov. Today 10, 1451–1458 (2005).
14. Milton Harris, J. & Chess, R. B. Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discov. 2, 214–221 (2003).
15. Zahr, A. S., Davis, C. A. & Pishko, M. V. Macrophage uptake of core-shell nanoparticles surface modified with poly(ethylene glycol). Langmuir 22, 8178–8185 (2006).
16. Jokerst, J. V, Lobovkina, T., Zare, R. N. & Gambhir, S. S. Nanoparticle PEGylation for imaging and therapy. Nanomedicine (Lond) 6, 715–728 (2011).
17. Yang, Q. & Lai, S. K. Anti-PEG immunity: Emergence, characteristics, and unaddressed questions. Wiley Interdiscip. Rev. Nanomedicine Nanobiotechnology 7, 655–677 (2015).
18. Zhang, P., Sun, F., Liu, S. & Jiang, S. Anti-PEG antibodies in the clinic: Current issues and beyond PEGylation. J. Control. Release 244, 184–193 (2016).
19. Verhoef, J. J. F., Carpenter, J. F., Anchordoquy, T. J. & Schellekens, H. Potential induction of anti-PEG antibodies and complement activation toward PEGylated therapeutics. Drug Discov. Today 19, 1945–1952 (2014).

Lai Research Group Website: https://pharmacy.unc.edu/research/faculty-labs/lai-research-group/