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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Complement activation induced by PEG enhances humoral immune responses against antigens encapsulated in PEG-modified liposomes.

Splenic marginal zone B (MZ-B) cells have attracted attention as alternative antigen-presenting cells. We recently developed an original delivery system, using PEGylated liposomes (PEG-Lip) to deliver antigens to MZ-B cells. In this system, to induce antigen-specific immunity, empty PEG-Lip and antigen-containing PEG-Lip were intravenously (i.v.) injected sequentially at 3 day intervals. Since complement activation by the second dose is required for the delivery of antigen-containing PEG-Lip to splenic MZ-B cells, we investigated the ability of liposomes, modified with various PEG derivatives having different functional terminal groups (methoxy PEG (CH3O-PEG), hydroxy PEG (HO-PEG) or polyglycerol (PG), to activate the complement system and deliver a model antigen, ovalbumin (OVA), to splenic MZ-B cells in vitro and in vivo. Hydroxy PEG-modified liposomes (HO-PEG-Lip) both activated the complement system in vitro, and facilitated the preferential association of HO-PEG-lip with MZ-B cells in vitro. Manipulating HO-PEG density, in particular a density of 2 mol% in total lipids, significantly enhanced the association of HO-PEG-Lip with splenic MZ-B cells in vivo. Consequently, a single i.v. injection of HO-PEG-Lip (2 mol%) containing OVA induced OVA-specific IgG response. Our immunization system with HO-PEG-Lip, achieved efficient antigen delivery to MZ-B cells after a single i.v. injection, improving on our previous immunization system. This new delivery technique may be an improved, simple, antigen delivery system to MZ-B cells that induces meaningful levels of humoral immune response.

A hydroxyl PEG version of PEGylated liposomes and its impact on anti-PEG IgM induction and on the accelerated clearance of PEGylated liposomes.

Surface decoration of liposomes with polyethylene glycol (PEG), PEGylation, is recognized as a method to bestow liposomes with a prolonged circulation time following intravenous administration. However, many reports have emphasized that a first dose of PEGylated liposomes (PL) elicits an anti-PEG IgM antibody response that can trigger a rapid systemic clearance of a second dose of PL via a phenomenon that is referred to as "accelerated blood clearance (ABC)." Such a phenomenon is usually observed with PL that has been modified with methoxy-PEG. In the current study, we introduced various functional groups, methoxy (OCH3), amino (NH2), carboxyl (COOH), and hydroxyl (OH), at the chain ends of PEG to investigate the effect on anti-PEG IgM induction. Among different PEG-modified liposomes, hydroxyl PEG-modified liposomes (PL-OH) efficiently attenuated the anti-PEG IgM response in vitro. In addition, PL-OH was less recognizable by anti-PEG IgM compared with other PLs. These findings raised the possibility that PL-OH could attenuate/abrogate elicitation of the ABC phenomenon. Nonetheless, upon repeated intravenous injection, PL-OH triggered the enhanced clearance of a subsequently injected second dose. Furthermore, in vitro studies have demonstrated that, as a complement activator, PL-OH is stronger than PL-OCH3 and induces further complement activation in the presence of anti-PEG IgM, which was the predominant contributor to the rapid clearance of a second dose of PL-OH. Our results suggest that the screening of complement activation by polymer-modified products in tandem with anti-polymer antibody production should be a prerequisite in the development of polymers that might enhance the therapeutic efficacy of nanocarriers.