On the origin of the low immunogenicity and biosafety of a neutral α-helical polypeptide as an alternative to polyethylene glycol

Poly(ethylene glycol) (PEG) is a prominent synthetic polymer widely used in biomedicine. Despite its notable success, recent clinical evidence highlights concerns regarding the immunogenicity and adverse effects associated with PEG in PEGylated proteins and lipid nanoparticles. Previous studies have found a neutral helical polypeptide poly(γ-(2-(2-(2-methoxyethoxy)ethoxy)ethyl l-glutamate), namely L-P(EG3Glu), as a potential alternative to PEG, displaying lower immunogenicity. To comprehensively assess the immunogenicity, distribution, degradation, and biosafety of L-P(EG3Glu), herein, we employ assays including enzyme-linked immunosorbent assay, positron emission tomography-computed tomography, and fluorescent resonance energy transfer. Our investigations involve in vivo immune responses, biodistribution, and macrophage activation of interferon (IFN) conjugates tethered with helical L-P(EG3Glu) (L20k-IFN), random-coiled DL-P(EG3Glu) (DL20k-IFN), and PEG (PEG20k-IFN). Key findings encompass: minimal anti-IFN and anti-polymer antibodies elicited by L20k-IFN; length-dependent affinity of PEG to anti-PEG antibodies; accelerated clearance of DL20k-IFN and PEG20k-IFN linked to anti-IFN and anti-polymer IgG; complement activation for DL20k-IFN and PEG20k-IFN but not L20k-IFN; differential clearance with L20k-IFN kidney-based, and DL20k-IFN/PEG20k-IFN accumulation mainly in liver/spleen; enhanced macrophage activation by DL20k-IFN and PEG20k-IFN; L-P(EG3Glu) resistance to proteolysis; and safer repeated administrations of L-P(EG3Glu) in rats. Overall, this study offers comprehensive insights into the lower immunogenicity of L-P(EG3Glu) compared to DL-P(EG3Glu) and PEG, supporting its potential clinical use in protein conjugation and nanomedicines.