Therapeutic Protein PEPylation: The Helix of Nonfouling SyntheticPolypeptides Minimizes AntidrugAntibody Generation

摘要

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(EG3Glu) and dl-P(EG3Glu)) 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 l20K-IFN, interferon (IFN) modified with the helical polypeptide l-P(EG3Glu) shows improved binding affinity, in vitro antiproliferative activity, and in vivo efficacy compared to those modified with the unstructured polypeptide analogue dl-P(EG3Glu)or PEG. Moreover, l20K-IFN triggered significantlyless antidrug and antipolymer antibodies than the other two. Importantly,the unusual findings observed in the IFN series are reproduced ina human growth hormone (GH) conjugate series. Subcutaneously infused l20K-GH, GH modified with l-P(EG3Glu), evokes considerably less anti-GH and antipolymer antibodiescompared to those modified with dl-P(EG3Glu) orPEG (dl20K-GH or PEG20K–GH).As a result, repeated injections of dl20K-GH orPEG20K-GH, but not l20K-GH, resultin a clear ABC effect and significantly diminished drug availabilityin the blood. Meanwhile, immature mouse bone marrow cells incubatedwith the helical l20K-GH exhibit decreased druguptake and secretion of proinflammatory cytokines compared to thosetreated with one of the other two GH conjugates bearing unstructuredpolymers. Taken together, the current study highlights an urgent necessityto systematically reassess the pros and cons of choosing unstructuredpolymers for protein conjugation. Furthermore, our results also laythe foundation for the development of next-generation biohybrid drugsbased on helical synthetic polypeptides.