Peritoneal B Cells Play a Role in the Production of Anti-polyethylene Glycol (PEG) IgM against Intravenously Injected siRNA-PEGylated Liposome Complexes.

Polyethylene glycol (PEG)-modified (PEGylated) cationic liposomes are frequently used as delivery vehicles for small interfering RNA (siRNA)-based drugs because of their ability to encapsulate/complex with siRNA and prolong the circulation half-life in vivo. Nevertheless, we have reported that subsequent intravenous (IV) injections of siRNA complexed with PEGylated cationic liposomes (PLpx) induces the production of anti-PEG immunoglobulin M (IgM), which accelerates the blood clearance of subsequent doses of PLpx and other PEGylated products. In this study, it is interesting that splenectomy (removal of spleen) did not prevent anti-PEG IgM induction by IV injection of PLpx. This indicates that B cells other than the splenic version are involved in anti-PEG IgM production under these conditions. In vitro and in vivo studies have shown that peritoneal cells also secrete anti-PEG IgM in response to the administration of PLpx. Interleukin-6 (IL-6) is a glycoprotein that is secreted by peritoneal immune cells and has been detected in response to the in vivo administration of PLpx. These observations indicate that IV injection of PLpx stimulates the proliferation/differentiation of peritoneal PEG-specific B cells into plasma cells via IL-6 induction, which results in the production of anti-PEG IgM from the peritoneal cavity of mice. Our results suggest the mutual contribution of peritoneal B cells as a potent anti-PEG immune response against PLpx.

Impact of Anti-PEG IgM Induced via the Topical Application of a Cosmetic Product Containing PEG Derivatives on the Antitumor Effects of PEGylated Liposomal Antitumor Drug Formulations in Mice.

Poly(ethylene glycol) (PEG) is used in many common products, such as cosmetics. PEG, however, is also used to covalently conjugate drug molecules, proteins, or nanocarriers, which is termed PEGylation, to serve as a shield against the natural immune system of the human body. Repeated administration of some PEGylated products, however, is known to induce anti-PEG antibodies. In addition, preexisting anti-PEG antibodies are now being detected in healthy individuals who have never received PEGylated therapeutics. Both treatment-induced and preexisting anti-PEG antibodies alter the pharmacokinetic properties, which can result in a subsequent reduction in the therapeutic efficacy of administered PEGylated therapeutics through the so-called accelerated blood clearance (ABC) phenomenon. Moreover, these anti-PEG antibodies are widely reported to be related to severe hypersensitivity reactions following the administration of PEGylated therapeutics, including COVID-19 vaccines. We recently reported that the topical application of a cosmetic product containing PEG derivatives induced anti-PEG immunoglobulin M (IgM) in a mouse model. Our finding indicates that the PEG derivatives in cosmetic products could be a major cause of the preexistence of anti-PEG antibodies in healthy individuals. In this study, therefore, the pharmacokinetics and therapeutic effects of Doxil (doxorubicin hydrochloride-loaded PEGylated liposomes) and oxaliplatin-loaded PEGylated liposomes (Liposomal l-OHP) were studied in mice. The anti-PEG IgM antibodies induced by the topical application of cosmetic products obviously accelerated the blood clearance of both PEGylated liposomal formulations. Moreover, in C26 tumor-bearing mice, the tumor growth suppressive effects of both Doxil and Liposomal l-OHP were significantly attenuated in the presence of anti-PEG IgM antibodies induced by the topical application of cosmetic products. These results confirm that the topical application of a cosmetic product containing PEG derivatives could produce preexisting anti-PEG antibodies that then affect the therapeutic efficacy of subsequent doses of PEGylated therapeutics.

Anti-PEG IgM production and accelerated blood clearance phenomenon after the administration of PEGylated exosomes in mice.

Recently, there is an increasing interest in exosomes or extracellular vesicles as potential candidates for delivering RNAs, proteins, genes, and anticancer agents. Engineering of exosome properties is rapidly evolving as a means of expanding exosome applications. PEGylation of exosomes is a technique used to improve their in vivo stability, circulation half-lives, and sometimes to allow the binding targeting ligands to the exosome exterior. According to FDA guidelines for the development of PEGylated proteins, immunological responses to PEGylated molecules and particles should be examined. In this study, we prepared PEGylated exosomes and investigated the production of anti-PEG IgM antibodies after single i.v. injections in mice. In addition, we monitored blood concentrations and tumor accumulation of a second dose of PEGylated exosomes administered after the initial dose. Single injections of PEGylated exosomes in mice induced anti-PEG IgM production in a T cell-dependent manner. The anti-PEG IgM production decreased when the injection dose of PEGylated exosomes was further increased. Anti-PEG IgM induced by injection of PEGylated exosomes decreased blood concentrations of a second dose of PEGylated exosomes and suppressed their tumor accumulation in a C26 murine colorectal cancer model. Initial injection doses of either PEGylated liposomes or PEGylated ovalbumin (PEG-OVA), both of them induced anti-PEG IgM production, also decreased the blood concentration of PEGylated exosomes. Interestingly, anti-PEG IgM induced by injection of PEGylated exosomes did not affect the blood concentration of PEG-OVA. These results imply the importance of monitoring anti-PEG IgM when repeat PEGylated exosome doses are required and/or when PEGylated exosomes are used together with other PEGylated therapeutics.

Pegfilgrastim (PEG-G-CSF) induces anti-PEG IgM in a dose dependent manner and causes the accelerated blood clearance (ABC) phenomenon upon repeated administration in mice.

Pegfilgrastimis a recombinant PEGylated human granulocyte colony-stimulating factor (G-CSF) analog filgrastim (trade names Neulasta® or G-Lasta®) that stimulates the production of white blood cells (neutrophils). It is employed as an alternative to filgrastim (G-CSF) for chemotherapy-induced neutropenia in patients due to its longer half-life. In clinical settings, PEG-G-CSF is administered to cancer patients via both the s.c. and i.v. routes. In a murine study, we showed that, regardless of administration route, initial doses of PEG-G-CSF above 0.06 mg/kg elicited anti-PEG immune response in a dose-dependent manner. I.v. administration elicited higher levels of anti-PEG IgM than the s.c. route. Initial doses of PEG-G-CSF (6 mg/kg) that were high enough to trigger production of anti-PEG IgM, did not trigger the accelerated clearance of a lower subsequent dose (0.06 mg/kg) that was similar to i.v. clinical doses of PEG-G-CSF, but when the subsequent dose of PEG-G-CSF was raised to (6 mg/kg), the initial dose triggered the accelerated clearance of the second dose via an anti-PEG IgM-mediated complement activation. Similar observations were noted when an increased PEG-OVA dose was given as the second dose, indicating that pre-existing and/or treatment-induced anti-PEG antibodies might compromise the therapeutic activity and/or reduce tolerance of other PEGylated formulations. To the best of our knowledge, this is the first report to suggest the induction of the ABC phenomenon upon repeated injections of pegfilgrastim. In the clinic, cancer patients, receiving multiple cycles of chemotherapy, receive multiple cycles of pegfilgrastim to avoid infections and substantial morbidity. The ABC phenomenon to pegfilgrastim appears to be the cause of loss of clinical benefit of sequential treatments with pegfilgrastim in patients.