A Cell Assay for Detecting Anti-PEG Immune Response against PEG-Modified Therapeutics

Abstract

Immunogenicity of PEGylated proteins and nanomedicines represents a potential impediment against their development and use in clinical settings. The purpose of this study is to develop a method for detecting anti-PEG immunity of PEGylated proteins and/or nanomedicines using flow cytometry. The binding of fluorescence-labeled mPEG-modified liposomes to HIK-G11 cells, PEG-specific hybridoma cells, or spleen cells was evaluated by flow cytometry for detecting immunogenicity of PEGylated therapeutics. The fluorescence-labeled methoxy PEG (mPEG)-modified liposomes were efficiently bound to HIK-G11 cells. Such staining with fluorescence-labeled mPEG-modified liposomes was significantly inhibited in the presence of either non-labeled mPEG-modified liposomes or mPEG-modified ovalbumin (OVA) but not polyglycerol-modified liposomes. In addition, we found that mPEG-modified liposomes, highly immunogenic, caused proliferation of PEG-specific cells, while hydroxyl PEG-modified liposomes, less immunogenic, scarcely caused. Furthermore, after intravenous injection of mPEG-modified liposomes, the percentage of PEG-specific cells in the splenocytes, as determined by flow cytometry, corresponded well with the production level of anti-PEG antibodies, as determined by ELISA. PEG-specific B cell assay we introduced may become a useful method to detect an anti-PEG immune response against PEGylated therapeutics and clarify the mechanism for anti-PEG immune responses.