anti-PEG IgM Antibody Research Articles

Anti-PEG IgM production induced by PEGylated liposomes as a function of administration route.

Modifying the surface of nanoparticles with polyethylene glycol (PEG) is a commonly used approach for improving the in vitro stability of nanoparticles such as liposomes and increasing their circulation half-lives. We have demonstrated that, in certain conditions, an intravenous (i.v.) injection of PEGylated liposomes (PEG-Lip) induced anti-PEG IgM antibodies, which led to rapid clearance of second doses in mice. SARS-CoV-2 vaccines, composed of mRNA-containing PEGylated lipid nanoparticles, have been widely administered as intramuscular (i.m.) injections, so it is important to determine if PEGylated formulations can induce anti-PEG antibodies. If the favorable properties that PEGylation imparts to therapeutic nanoparticles are to be widely applicable this should apply to various routes of administration. However, there are few reports on the effect of different administration routes on the in vivo production of anti-PEG IgM. In this study, we investigated anti-PEG IgM production in mice following i.m., intraperitoneal (i.p.) and subcutaneous (s.c.) administration of PEG-Lip. PEG-Lip appeared to induce anti-PEG IgM by all the tested routes of administration, although the lipid dose causing maximum responses varied. Splenectomy attenuated the anti-PEG IgM production for all routes of administration, suggesting that splenic immune cells may have contributed to anti-PEG IgM production. Interestingly, in vitro experiments indicated that not only splenic cells but also cells in the peritoneal cavity induced anti-PEG IgM following incubation with PEG-Lip. These observations confirm previous experiments that have shown that measurable amounts of PEG-Lip administered i.p., i.m. or s.c. are absorbed to some extent into the blood circulation, where they can be distributed to the spleen and/or peritoneal cavity, and are recognized by B cells, triggering anti-PEG IgM production. The results obtained in this study have important implications for developing efficient PEGylated nanoparticular delivery system.

Impact of spontaneous liposome modification with phospholipid polymer-lipid conjugates on protein interactions

ABSTRACT Liposome surface coating has been studied to avoid the immunological responses caused by the complement system, and alternative materials to poly(ethylene glycol) (PEG) have been explored recently since the production of anti-PEG IgM antibodies has been found in humans. We previously reported a liposome coating with poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC))-conjugated lipids (PMPC-lipids) and demonstrated its protective effect on blood protein interactions. Here, we attempted to modify the liposome surface by exogenously adding PMPC-lipids, which were spontaneously incorporated into the outer membrane via hydrophobic interactions. The polymerization degree of the PMPC segment was regulated from 10 to 100. The incorporated ratio of PMPC-lipid increased with a decrease in the degree of PMPC polymerization. Due to surface modification with PMPC-lipids, increase in the length of the PMPC-chain increased the size of the liposomes. The modified liposomes were kept stable for 14 d in terms of their size, polydispersity, and surface properties, where approximately 70% of PMPC-lipids were incorporated into the liposome surface. We demonstrated that liposome surface modification with PMPC-lipids can inhibit protein adsorption when exposed to serum, regardless of the degree of polymerization of PMPC. In addition, the PMPC-lipid modified surface was not recognized by the anti-PEG IgM antibody, whereas PEG-lipid was recognized by the antibody. Thus, we successfully fabricated an inert liposome surface via spontaneous modification with PMPC-lipids, where only the outer bilayer surface was modified. This technique can be available for full loading of water-soluble active pharmaceutical ingredient inside the modified liposome.

Toxicity of high-molecular-weight polyethylene glycols in Sprague Dawley rats

Polyethylene glycol (PEG) is present in a variety of products. Little is known regarding the accumulation of high-molecular-weight PEGs or the long-term effects resulting from PEG accumulation in certain tissues, especially the choroid plexus. We evaluated the toxicity of high-molecular-weight PEGs administered to Sprague Dawley rats. Groups of 12 rats per sex were administered subcutaneous injections of 20, 40, or 60 kDa PEG or intravenous injections of 60 kDa PEG at 100 mg PEG/kg body weight/injection once a week for 24 weeks. A significant decrease in triglycerides occurred in the 60 kDa PEG groups. PEG treatment led to a molecular-weight-related increase in PEG in plasma and a low level of PEG in cerebrospinal fluid. PEG was excreted in urine and feces, with a molecular-weight-related decrease in the urinary excretion. A higher prevalence of anti-PEG IgM was observed in PEG groups; anti-PEG IgG was not detected. PEG treatment produced a molecular-weight-related increase in vacuolation in the spleen, lymph nodes, lungs, and ovaries/testes, without an inflammatory response. Mast cell infiltration at the application site was noted in all PEG-treated groups. These data indicate that subcutaneous and intravenous exposure to high-molecular-weight PEGs produces anti-PEG IgM antibody responses and tissue vacuolation without inflammation.

Accelerated blood clearance and hypersensitivity by PEGylated liposomes containing TLR agonists

Systemic administration of toll-like receptor (TLR) agonists have demonstrated impressive preclinical results as an anti-cancer therapy due to their potent innate immune-stimulatory properties. The clinical advancement has, however, been hindered by severe adverse effects due to systemic activation of the immune system. Liposomal drug delivery systems may modify biodistribution, cellular uptake, and extend blood circulation, and thus, potentially enable systemic administration of TLR agonists at therapeutic doses.In this study, we investigated potential barriers for the administration of TLR agonists formulated in polyethylene glycosylated (PEGylated) liposomes with regards to liposome formulation, TLR agonist, administration route, administration schedule, biodistribution, blood clearance, and anti-PEG antibodies. We found that administration of TLR agonists formulated in PEGylated liposomes led to high anti-PEG antibody titers, which upon multiple intravenous administrations, resulted in accelerated blood clearance and acute hypersensitivity reactions. The latter was found to be associated with anti-PEG IgG antibody and not anti-PEG IgM antibody opsonization. This study highlights the need to carefully design and evaluate nanoparticle delivery systems for immunotherapy as anti-nanoparticle immune responses may challenge the therapeutic application.

Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement

PEGylation of lipid-based nanoparticles and other nanocarriers is widely used to increase their stability and plasma half-life. However, either pre-existing or de novo formed anti-PEG antibodies can induce hypersensitivity reactions and accelerated blood clearance through binding to the nanoparticle surfaces, leading to activation of the complement system.In this study, we investigated the consequences and mechanisms of complement activation by anti-PEG antibodies interacting with different types of PEGylated lipid-based nanoparticles. By using both liposomes loaded with different (model) drugs and LNPs loaded with mRNA, we demonstrate that complement activation triggered by anti-PEG antibodies can compromise the bilayer/surface integrity, leading to premature drug release or exposure of their mRNA contents to serum proteins. Anti-PEG antibodies also can induce deposition of complement fragments onto the surface of PEGylated lipid-based nanoparticles and induce the release of fluid phase complement activation products.The role of the different complement pathways activated by lipid-based nanoparticles was studied using deficient sera and/or inhibitory antibodies. We identified a major role for the classical complement pathway in the early activation events leading to the activation of C3. Our data also confirm the essential role of amplification of C3 activation by alternative pathway components in the lysis of liposomes.Finally, the levels of pre-existing anti-PEG IgM antibodies in plasma of healthy donors correlated with the degree of complement activation (fixation and lysis) induced upon exposure to PEGylated liposomes and mRNA-LNPs.Taken together, anti-PEG antibodies trigger complement activation by PEGylated lipid-based nanoparticles, which can potentially compromise their integrity, leading to premature drug release or cargo exposure to serum proteins.

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.

Time Interval of Two Injections and First-Dose Dependent of Accelerated Blood Clearance Phenomenon Induced by PEGylated Liposomal Gambogenic Acid: The Contribution of PEG-Specific IgM

Repeated injection of PEGylated liposomes can cause the disappearance of long circulating property because of the induction of anti-PEG IgM antibody referred to as “accelerated blood clearance (ABC) phenomenon.” Although ABC phenomenon typically occurs when entrapped drugs are chemotherapeutic agent with low cytotoxic, there is little evidence of accelerated blood clearance of PEGylated herbal-derived compound on repeated injection. Herein, we investigated the blood concentration of PEGylated liposomal gambogenic acid (PEG-GEA-L), a model PEGylated liposomal herbal extract, on its repeated injection to rats. We found time interval between injections had considerable impact on the magnitude of ABC phenomenon induced by PEG-GEA-L. When time interval was prolonged from 3 days to 7 days, ABC phenomenon could be attenuated. Furthermore, its magnitude was enhanced accompanied by a marked rise in the accumulation of PEG-GEA-L in the liver and spleen in a first-dose–dependent manner. Consistently, the level of anti-PEG IgM significantly increased with the first dose of PEG-GEA-L and decreased with the extended time interval between injections, which implies anti-PEG IgM is a major contributor to the ABC phenomenon. Notably, the increased expression of liver anti-PEG IgM was accompanied by an increased expression of efflux transporters in the induction process of the ABC phenomenon.

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.

Mixed PEGylated surfactant modifying system decrease the accelerated blood clearance phenomenon of nanoemulsions in rats

The accelerated blood clearance (ABC) phenomenon which is induced by repeated injection of poly (ethylene glycol) (PEG)-coated colloidal carriers gives clinical challenge to the promising drug delivery system. It is necessary to decrease this unexpected immunological response. A novel 4-arm poly (ethylene glycol-5000)4-cholesteryl methyl amide (4-arm PEG5000-CHMA) has been synthesized. The structure of 4-arm PEG5000-CHMA was confirmed by IR and 1H-NMR spectrum. The pharmacokinetics of the tocopheryl nicotinate (TN)-loaded nanoemulsions modified with 4-arm PEG5000-CHMA or/and 1, 2-distearoyl-Sn-glycero-3-phosphoethanolamine-n-[methoxy(poly-ethyleneglycol)-2000] (mPEG2000-DSPE) have been studied. Furthermore, the ABC phenomenon has been detailed investigated in rats by TN-loaded nanoemulsions modified with 4-arm PEG5000-CHMA and mPEG2000-DSPE (CPNE). The plasma levels of TN and anti-PEG IgM antibody were determined by HPLC and ELISA, respectively. The circulation time of the CPNEs were comparable to the mPEG2000-DSPE coated nanoemulsions. Moreover, the ABC phenomenon can be decreased by CPNEs. This study designs a method to decrease the ABC phenomenon and develops a clinical promising nanoemulsion for therapeutic or imaging purpose.

Impact of anti-PEG IgM antibodies on the pharmacokinetics of pegylated asparaginase preparations in mice

The potential impact of pre-existing anti-PEG antibodies on the asparaginase activity kinetics of two pegylated l-asparaginase preparations - pegylated recombinant l-asparaginase (PEG-rASNase MC0609) and pegaspargase (pegylated Escherichia colil-asparaginase) - was investigated in immune competent, naïve B6D2F1-hybrid mice. To generate anti-PEG antibodies, mice were pre-sensitised by repeated injections of 40kDa PEG-Diol without being conjugated to a carrier. Successful PEG-Diol pre-sensitisation was verified by analysis of anti-PEG antibody titers in serum. 88–100% of animals developed PEG-specific anti-PEG IgM antibodies after PEG-Diol pre-sensitisation. All animals positive for anti-PEG IgM antibodies and control animals (without prior PEG-Diol pre-sensitisation) were treated once with PEG-rASNase MC0609 or pegaspargase, and asparaginase enzyme activity levels and immunogenicity of both preparations were analysed. Known serum asparaginase activity profiles were measured after treatment with PEG-rASNase MC0609 or pegaspargase in all treatment groups. No rapid decrease of asparaginase activity was observed - irrespective of successful PEG-Diol pre-sensitisation and presence of acquired anti-drug-IgG and/or anti-PEG IgM antibodies.In conclusion, the pharmacokinetics of pegylated l-asparaginase was unaffected by the presence of pre-existing anti-PEG IgM antibodies in immune competent B6D2F1-hybrid mice Probably the titre or affinity of these anti-PEG IgM antibodies were too low to influence the pharmacokinetics of PEG-rASNase MC0609 or pegaspargase or anti-PEG IgM antibodies bound to PEG-ASNase without neutralising capabilities. Thus, early loss of asparaginase activity as observed in serum of ALL patients is a complex process and cannot be explained solely by the existence of pre-existing anti-PEG antibodies.

Accelerated Blood Clearance of PEGylated PLGA Nanoparticles Following Repeated Injections: Effects of Polymer Dose, PEG Coating, and Encapsulated Anticancer Drug

To investigate accelerated blood clearance (ABC) induction upon repeated injections of PLGA-PEG nanoparticles as a commonly used polymeric drug carrier. Etoposide-loaded PLGA-PEG NPs were developed and administered as the test dose to rats pre-injected with various NP treatments at certain time intervals. Pharmacokinetic parameters of etoposide and production of anti-PEG IgM antibody were evaluated. A notable ABC effect was induced by a wide range of polymer doses (0.1 to 20mg) of empty NPs, accompanied by IgM secretion. However, a further increase in polymer dose resulted not only in the abrogation of the observed ABC induction but also in distinctly a higher value for AUC of the NPs relative to the control. The data from the PEG-negative group verified the fundamental role of PEG for ABC induction. The first injection of etoposide-containing PEGylated nanoparticles (a cell cycle phase-specific drug) produced a strong ABC phenomenon. Three sequential administrations of etoposide-loaded NPs abolished ABC, although a high level of IgM was still detected, which suggests saturation with insignificant poisoning of immune cells. The presented results demonstrate the importance of clinical evaluations for PLGA-PEG nanocarriers that consider the administration schedule in multiple drug delivery, particularly in cancer chemotherapy.

Impact of Large Aggregated Uricases and PEG Diol on Accelerated Blood Clearance of PEGylated Canine Uricase

BackgroundUricase has proven therapeutic value in treating hyperuricemia but sufficient reduction of its immunogenicity may be the largest obstacle to its chronic use. In this study, canine uricase was modified with 5 kDa mPEG-SPA and the impact of large aggregated uricases and cross-linked conjugates induced by difunctional PEG diol on immunogenicity was investigated.Methods and FindingsRecombinant canine uricase was first expressed and purified to homogeneity. Source 15Q anion-exchange chromatography was used to separate tetrameric and aggregated uricase prior to pegylation, while DEAE anion-exchange chromatography was used to remove Di-acid PEG (precursor of PEG diol) from unfractionated 5 kDa mPEG-propionic acid. Tetrameric and aggregated uricases were separately modified with the purified mPEG-SPA. In addition, tetrameric uricases was modified with unfractionated mPEG-SPA, resulting in three types of 5 kDa mPEG-SPA modified uricase. The conjugate size was evaluated by dynamic light scattering and transmission electron microscope. The influence of differently PEGylated uricases on pharmacokinetics and immunogenicity were evaluated in vivo. The accelerated blood clearance (ABC) phenomenon previously identified for PEGylated liposomes occurred in rats injected with PEGylated uricase aggregates. Anti-PEG IgM antibodies, rather than neutralizing antibodies, were found to mediate the ABC.ConclusionsThe size of conjugates is important for triggering such phenomena and we speculate that 40–60 nm is the lower size limit that can trigger ABC. Removal of the uricase aggregates and the PEG diol contaminant and modifying with small PEG reagents enabled ABC to be successfully avoided and sufficient reduction in the immunogenicity of 5 kDa mPEG-modified tetrameric canine uricase.

Size-dependent induction of accelerated blood clearance phenomenon by repeated injections of polymeric micelles

An accelerated blood clearance (ABC) phenomenon is induced by repeated injections of poly(ethylene glycol)-modified (PEGylated) liposomes. We previously indicated that the phenomenon was induced by polymeric micelles possessing PEG chains like as liposomes, although, the induction mechanism of the ABC phenomenon is not fully elucidated. In the present study, we investigate whether repeat-injection of the polymeric micelles having PEG chains trigger the phenomenon or not. Two polymeric micelles, PM-30 (polymeric micelles with 33.6nm in diameter) and PM-75 (76.2nm), were prepared with PEG-poly[Asp(pentyl)] and PEG-poly[Asp(nonyl)], respectively. We firstly examined the ABC-triggering effect of these micelles, and observed that both polymeric micelles, especially PM-75, induced the production of anti-PEG IgM antibody in treated mice. Then, PM-30 or PM-75 was preadministered into mice as a preconditioning. Seven days later, AlexaFluor594-labeled PM-30 or PM-75 was administered to determine the susceptibility of the phenomenon. As a result, rapid clearance of AlexaFluor594-labeled PM-75 from the bloodstream and accumulation in the liver were observed in PM-75 pretreated mice. Although, the ABC phenomenon of AlexaFluor594-labeled PM-30 was less obvious in PM-30 pretreated mice. Our present results indicated that the repeated injections of polymeric micelles caused the ABC phenomenon in a size-dependent manner.

T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes

Repeated injection of polyethyleneglycol-modified (PEGylated) liposomes causes a rapid clearance of them from the bloodstream, this phenomenon is called accelerated blood clearance (ABC). In the present study, we focused on the immune system responsible for the ABC phenomenon. PEGylated liposomes were preadministered to BALB/c mice and [ 3H]-labeled ones were then administered to them 3 days after the preadministration. Consistent with our previous results, the preadministration with PEGylated liposomes triggered the rapid clearance of [ 3H]-labeled PEGylated liposomes from the bloodstream, but that with PEGylated liposomes encapsulating doxorubicin (Dox) did not. In addition, we found that the ABC phenomenon was observed when a mixture of free Dox and PEGylated liposomes was preadministered. These data indicate that immune cells responsible for the ABC phenomenon might be selectively damaged by the Dox encapsulated in PEGylated liposomes. The ABC phenomenon was also observed in BALB/c nu/ nu mice, but not in BALB/c SCID mice. The amount of anti-PEG IgM antibody induced by the stimulation with the PEGylated liposomes was significantly increased in the BALB/c nu/ nu mice, but not in the BALB/c SCID ones. These data indicate that a T cell-independent B cell response would play a significant role in the ABC phenomenon. Furthermore, the present study suggests that PEGylated liposomes might be recognized by B cells as a thymus-independent type 2 (TI-2) antigen. The present study provides important information for the future development of liposomal medicines.

Accelerated Blood Clearance Phenomenon Upon Repeated Injection of PEG-modified PLA-nanoparticles

We recently developed prostaglandin E(1) (PGE(1))-encapsulated nanoparticles, prepared with a poly(lactide) homopolymer (PLA, Mw = 17,500) and monomethoxy poly(ethyleneglycol)-PLA block copolymer (PEG-PLA) (NP-L20). In this study, we tested whether the accelerated blood clearance (ABC) phenomenon is observed with NP-L20 and other PEG-modified PLA-nanoparticles in rats. The plasma levels of PGE(1) and anti-PEG IgM antibody were determined by EIA and ELISA, respectively. Second injections of NP-L20 were cleared much more rapidly from the circulation than first injections, showing that the ABC phenomenon was induced. This ABC phenomenon, and the accompanying induction of anti-PEG IgM antibody production, was optimal at a time interval of 7 days between the first and second injections. Compared to NP-L20, NP-L33s that were prepared with PLA (Mw = 28,100) and have a smaller particle size induced production of anti-PEG IgM antibody to a lesser extent. NP-L20 but not NP-L33s gave rise to the ABC phenomenon with a time interval of 14 days. NP-L33s showed a better sustained-release profile of PGE(1) than NP-L20. This study revealed that the ABC phenomenon is induced by PEG-modified PLA-nanoparticles. We consider that NP-L33s may be useful clinically for the sustained-release and targeted delivery of PGE(1).

Glucan-enhanced immunity to protozoan infections

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.