Zwitterionic Polypeptide-protein Conjugation for the Safe and Efficient Delivery of Therapeutic Enzymes PROJECT SUMMARY One of the major obstacles that impede the wide application of therapeutic protein products is their potential immunological response, especially for those obtained from non-human sources. Currently, the most successful strategy to mitigate immune response induced by foreign proteins is ?PEGylation?, i.e., to shield the protein surface epitopes with polyethylene glycol (PEG). This surface conjugation strategy has been shown to decrease to some extent immune responses to the underlying protein and more than ten PEGylated protein products have been approved by the Food and Drug Administration (FDA). However, recent studies both in animal models and clinical trials have demonstrated the presence of induced anti-PEG antibodies after repeated administrations and pre-existing anti-PEG antibodies, which directly challenge the future of this PEGylation technology. We believe that there are two shortcomings for the current PEGylation technology: 1) the haptenic character of amphiphilic PEG leading to anti-PEG antibodies and 2) non- degradable character of amphiphilic PEG leading to vacuolation of kidney and liver. Previously, we developed a zwitterionic poly(carboxybetaine) (PCB) protected uricase, which was shown to be capable of maintaining protein bioactivity, reducing the immunogenicity of encased proteins and extending their circulation time without induced anti-polymer Abs. Extended from the zwitterionic concept, poly(EK) (PEK), a zwitterionic polypeptide comprising of alternative lysine (K) and glutamic acid (E), has all properties of zwitterionic PCB and is also degradable to promote its elimination from the body while PEG or PCB is not. Thus, PEK is a great alternative beyond PEGylation, but has never been studied for its application to drug delivery. The purpose of this R21 proposal is to explore the potential of degradable zwitterionic poly(EK) (PEK)-protein conjugates. As a degradable version of PCB, PEK is anticipated to provide similar benefits as PCB to its modified protein drugs while possessing excellent biodegradability. Our preliminary results showed that PEK did not induce any anti-PEK antibodies even when it was conjugated onto highly-immunogenic keyhole limpet hemocyanin (KLH) commonly used to boost the immunogenicity of haptens. However, significant anti-PEG antibodies were elicited by PEG-conjugated KLH in the parallel study. In this proposal, uricase, a non-human enzyme with strong immunogenicity, will be used as a protein target. We hypothesize and will confirm though this study that PEK-uricase conjugates will provide a safe and efficient strategy for enzyme delivery by greatly reducing immunogenicity, improving pharmacokinetics (PK), and enhancing pharmacodynamics (PD). Since PEGylation has been widely used in protein therapeutics, successful completion of this project will culminate in a new approach as an alternative to or a replacement over the PEGylation technology, producing safer and more effective enzyme therapeutics.
The objective of the proposed work is to study zwitterionic poly(peptide)-enzyme conjugates beyond the existing PEGlylation technology. Success of this work will provide a safe and effective enzyme delivery platform to eliminate immunological response from both polymer and protein and to increase enzyme circulation half- life, activity and efficacy. The immediate application is the use of peptide-uricase conjugates for the treatment of chronic refractory gout.
