Therapeutic Protein PEPylation: The Helix of Nonfouling Synthetic Polypeptides Minimizes Antidrug Antibody Generation

[Image: see text] Polymer conjugation is a clinically proven approach to generate long acting protein drugs with decreased immune responses. Although poly(ethylene glycol) (PEG) is one of the most commonly used conjugation partners due to its unstructured conformation, its therapeutic application is limited by its poor biodegradability, propensity to induce an anti-PEG immune response, and the resultant accelerated blood clearance (ABC) effect. Moreover, the prevailing preference of unstructured polymers for protein conjugation still lacks strong animal data support with appropriate control reagents. By using two biodegradable synthetic polypeptides with similar structural compositions (l-P(EG(3)Glu) and dl-P(EG(3)Glu)) for site-specific protein modification, in the current study, we systematically investigate the effect of the polymer conformation on the in vivo pharmacological performances of the resulting conjugates. Our results reveal that the conjugate l(20K)-IFN, interferon (IFN) modified with the helical polypeptide l-P(EG(3)Glu) shows improved binding affinity, in vitro antiproliferative activity, and in vivo efficacy compared to those modified with the unstructured polypeptide analogue dl-P(EG(3)Glu) or PEG. Moreover, l(20K)-IFN triggered significantly less antidrug and antipolymer antibodies than the other two. Importantly, the unusual findings observed in the IFN series are reproduced in a human growth hormone (GH) conjugate series. Subcutaneously infused l(20K)-GH, GH modified with l-P(EG(3)Glu), evokes considerably less anti-GH and antipolymer antibodies compared to those modified with dl-P(EG(3)Glu) or PEG (dl(20K)-GH or PEG(20K)–GH). As a result, repeated injections of dl(20K)-GH or PEG(20K)-GH, but not l(20K)-GH, result in a clear ABC effect and significantly diminished drug availability in the blood. Meanwhile, immature mouse bone marrow cells incubated with the helical l(20K)-GH exhibit decreased drug uptake and secretion of proinflammatory cytokines compared to those treated with one of the other two GH conjugates bearing unstructured polymers. Taken together, the current study highlights an urgent necessity to systematically reassess the pros and cons of choosing unstructured polymers for protein conjugation. Furthermore, our results also lay the foundation for the development of next-generation biohybrid drugs based on helical synthetic polypeptides.