Polyethylene glycol (PEG) is a flexible, hydrophilic simple polymer that is physically attached to peptides, proteins, nucleic acids, liposomes, and nanoparticles to reduce renal clearance, block ...antibody and protein binding sites, and enhance the half-life and efficacy of therapeutic molecules. Some naïve individuals have pre-existing antibodies that can bind to PEG, and some PEG-modified compounds induce additional antibodies against PEG, which can adversely impact drug efficacy and safety. Here we provide a framework to better understand PEG immunogenicity and how antibodies against PEG affect pegylated drug and nanoparticles. Analysis of published studies reveals rules for predicting accelerated blood clearance of pegylated medicine and therapeutic liposomes. Experimental studies of anti-PEG antibody binding to different forms, sizes, and immobilization states of PEG are also provided. The widespread use of SARS-CoV-2 RNA vaccines that incorporate PEG in lipid nanoparticles make understanding possible effects of anti-PEG antibodies on pegylated medicines even more critical.
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IJS, KILJ, NUK, PNG, UL, UM
Polyethylene glycol (PEG) is a biocompatible polymer that is often attached to therapeutic molecules to improve bioavailability and therapeutic efficacy. Although antibodies with specificity for PEG ...may compromise the safety and effectiveness of PEGylated medicines, the prevalence of pre-existing anti-PEG antibodies in healthy individuals is unclear. Chimeric human anti-PEG antibody standards were created to accurately measure anti-PEG IgM and IgG antibodies by direct ELISA with confirmation by a competition assay in the plasma of 1504 healthy Han Chinese donors residing in Taiwan. Anti-PEG antibodies were detected in 44.3% of healthy donors with a high prevalence of both anti-PEG IgM (27.1%) and anti-PEG IgG (25.7%). Anti-PEG IgM and IgG antibodies were significantly more common in females as compared to males (32.0% vs 22.2% for IgM, p < 0.0001 and 28.3% vs 23.0% for IgG, p = 0.018). The prevalence of anti-PEG IgG antibodies was higher in younger (up to 60% for 20 year olds) as opposed to older (20% for >50 years) male and female donors. Anti-PEG IgG concentrations were negatively associated with donor age in both females (p = 0.0073) and males (p = 0.026). Both anti-PEG IgM and IgG strongly bound PEGylated medicines. The described assay can assist in the elucidation of the impact of anti-PEG antibodies on the safety and therapeutic efficacy of PEGylated medicines.
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The early and massive vaccination campaign in Israel with the mRNA-LNP Comirnaty® (Pfizer-BioNTech) vaccine against the SARS-CoV-2 virus made available large amounts of data regarding the efficacy ...and safety of this vaccine. Adverse reactions to mRNA-based SARS-CoV-2 vaccines are rare events, but due to large mediatic coverage they became feared and acted as a potential source of delay for the vaccination of the Israeli population. The experience with the reactogenicity of the polyethylene glycol (PEG) moiety of PEGylated liposomes, PEGylated proteins and other PEGylated drugs raised the fear that similar adverse effects can be associated with the PEG lipid which is an essential component of currently used mRNA-LNP vaccines against COVID-19. In this study we quantified the levels of anti-PEG IgG, IgM and IgE present in the blood of 79 volunteers immediately before and 3 weeks after receiving a first dose of Comirnaty® vaccine. Our in vitro results show that different humanized anti-PEG antibodies bind the PEGylated nano-liposomes in a concentration-dependent manner, but they bind with a lower affinity to the Comirnaty vaccine, despite it having a high mole% of neutral PEG2000-lipid on its surface. We found an increase in IgG concentration in the blood 3 weeks after the first vaccine administration, but no increase in IgM or IgE. In addition, no severe signs of adverse reactions to the Comirnaty vaccine were observed in the population studied despite the significant pre-existing high titers of IgG before the first dose of vaccine in 2 donors.
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•Anti-PEG IgG, IgM and IgE were quantified using an established quantitative assay.•Anti-PEG IgG antibodies significantly increased 3 weeks after Comirnaty injection.•Anti-PEG IgM did not rise 3 weeks after Comirnaty and no anti-PEG IgE were detected.•The affinity of humanized anti-PEG IgG and IgM for Comirnaty is lower than for Doxil.•Very low IgM titers were found in the population before Comirnaty administration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Conjugation of polyethylene glycol (PEG) to therapeutic molecules can improve bioavailability and therapeutic efficacy. However, some healthy individuals have pre-existing anti-PEG antibodies and ...certain patients develop anti-PEG antibody during treatment with PEGylated medicines, suggesting that genetics might play a role in PEG immunogenicity. Here we perform genome-wide association studies for anti-PEG IgM and IgG responses in Han Chinese with 177 and 140 individuals, defined as positive for anti-PEG IgM and IgG responses, respectively, and with 492 subjects without either anti-PEG IgM or IgG as controls. We validate the association results in the replication cohort, consisting of 211 and 192 subjects with anti-PEG IgM and anti-PEG IgG, respectively, and 596 controls. We identify the immunoglobulin heavy chain (IGH) locus to be associated with anti-PEG IgM response at genome-wide significance (P = 2.23 × 10
). Our findings may provide novel genetic markers for predicting the immunogenicity of PEG and efficacy of PEGylated therapeutics.Some individuals develop antibodies against the polyethylene glycol that is commonly used in therapeutic preparations. Here the authors conduct a GWAS in Han Chinese and find the IGH locus is associated with anti-PEG IgM.
Anti-polyethylene glycol (PEG) antibodies are present in many healthy individuals as well as in patients receiving polyethylene glycol-functionalized drugs. Antibodies against PEG-coated nanocarriers ...can accelerate their clearance, but their impact on nanodrug properties including nanocarrier integrity is unclear. Here, we show that anti-PEG IgG and IgM antibodies bind to PEG molecules on the surface of PEG-coated liposomal doxorubicin (Doxil, Doxisome, LC-101, and Lipo-Dox), resulting in complement activation, formation of the membrane attack complex (C5b-9) in the liposomal membrane, and rapid release of encapsulated doxorubicin from the liposomes. Drug release depended on both classical and alternative pathways of complement activation. Doxorubicin release of up to 40% was also observed in rats treated with anti-PEG IgG and PEG-coated liposomal doxorubicin. Our results demonstrate that anti-PEG antibodies can disrupt the membrane integrity of PEG-coated liposomal doxorubicin through activation of complement, which may alter therapeutic efficacy and safety in patients with high levels of pre-existing antibodies against PEG.
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The increasing use in the last decade of PEGylated nanodrugs such as Doxil® has seen a rise in the number of associated occurrences of hypersensitivity reactions (HSRs). These reactions (also called ...infusion reactions or IR), can range from harmless symptoms to life-threatening reactions. Current means to prevent IR include the prophylactic use of antihistamines and steroids, but they cannot ensure total prevention. We previously showed that an intravenous injection of doxorubicin-free Doxil-like PEGylated nano-liposomes (Doxebo) prior to Doxil treatment suppresses Doxil-induced complement activation-related pseudoallergy (CARPA) in pigs, a model of human hypersensitivity reactions to Doxil. However, in order to use Doxebo to prevent Doxil-induced IR, we have to prove its safety and that it does not affect Doxil's performance. Here we show that Doxebo itself does not have toxic effects on the host or tumor, and it does not interfere with Doxil's antitumor activity in mice. Blood, microscopic and macroscopic organ evaluation of rats after repeated administration confirm the lack of intrinsic adverse effect of Doxebo. Likewise, the repeated injection of Doxebo before Doxil did not impact Doxil's pharmacokinetics in plasma and therefore does not cause accelerated blood clearance (ABC). Taken together with our previous publications, these data suggest that the injection of Doxebo prior to Doxil administration can help protect against Doxil-induced IR without adversely affecting treatment efficacy and safety.
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•Doxebo (empty Doxil-like liposomes) bind anti-PEG antibodies similarly to Lipodox.•Doxebo does not affect tumor growth and does not interfere with Lipodox treatment.•Repeated administration in mice and rats showed the innocuousness of Doxebo.•Doxebo as pre-treatment slightly increased the plasmatic doxorubicin AUC in mice.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Nanomedicines and macromolecular drugs can induce hypersensitivity reactions (HSRs) with symptoms ranging from flushing and breathing difficulties to hypothermia, hypotension, and death in the most ...severe cases. Because many normal individuals have pre-existing antibodies that bind to poly(ethylene glycol) (PEG), which is often present on the surface of nanomedicines and macromolecular drugs, we examined if and how anti-PEG antibodies induce HSRs to PEGylated liposomal doxorubicin (PLD). Anti-PEG IgG but not anti-PEG IgM induced symptoms of HSRs including hypothermia, altered lung function, and hypotension after PLD administration in C57BL/6 and nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Hypothermia was significantly reduced by blocking FcγRII/III, by depleting basophils, monocytes, neutrophils, or mast cells, and by inhibiting secretion of histamine and platelet-activating factor. Anti-PEG IgG also induced hypothermia in mice after administration of other PEGylated liposomes, nanoparticles, or proteins. Humanized anti-PEG IgG promoted binding of PEGylated nanoparticles to human immune cells and induced secretion of histamine from human basophils in the presence of PLD. Anti-PEG IgE could also induce hypersensitivity reactions in mice after administration of PLD. Our results demonstrate an important role for IgG antibodies in induction of HSRs to PEGylated nanomedicines through interaction with Fcγ receptors on innate immune cells and provide a deeper understanding of HSRs to PEGylated nanoparticles and macromolecular drugs that may facilitate development of safer nanomedicines.
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Triple-negative breast cancer (TNBC) lacks effective treatment options due to the absence of traditional therapeutic targets. The epidermal growth factor receptor (EGFR) has emerged as a promising ...target for TNBC therapy because it is overexpressed in about 50% of TNBC patients. Here we describe a PEG engager that simultaneously binds polyethylene glycol and EGFR to deliver PEGylated nanomedicines to EGFR
TNBC. The PEG engager displays conditional internalization by remaining on the surface of TNBC cells until contact with PEGylated nanocarriers triggers rapid engulfment of nanocargos. PEG engager enhances the anti-proliferative activity of PEG-liposomal doxorubicin to EGFR
TNBC cells by up to 100-fold with potency dependent on EGFR expression levels. The PEG engager significantly increases retention of fluorescent PEG probes and enhances the antitumour activity of PEGylated liposomal doxorubicin in human TNBC xenografts. PEG engagers with specificity for EGFR are promising for improved treatment of EGFR
TNBC patients.
Increasing frequency of human exposure to PEG-related products means that healthy people are likely to have pre-existing anti-PEG antibodies (pre-αPEG Ab). However, the influence of pre-αPEG Abs on ...the pharmacokinetics (PK) and therapeutic efficacy of LipoDox is unknown.
We generated two pre-αPEG Ab mouse models. First, naïve mice were immunized with PEGylated protein to generate an endogenous αPEG Ab titer (endo αPEG). Second, monoclonal αPEG Abs were passively transferred (αPEG-PT) into naïve mice to establish a αPEG titer. The naïve, endo αPEG and αPEG-PT mice were intravenously injected with
in-labeled LipoDox to evaluate its PK. Tumor-bearing naïve, endo αPEG and αPEG-PT mice were intravenously injected with
in-labeled LipoDox to evaluate its biodistribution. The therapeutic efficacy of LipoDox was estimated in the tumor-bearing mice.
The areas under the curve (AUC)
of LipoDox in endo αPEG and αPEG-PT mice were 11.5- and 15.6- fold less, respectively, than that of the naïve group. The biodistribution results suggested that pre-αPEG Ab can significantly reduce tumor accumulation and accelerate blood clearance of
In-labeled LipoDox from the spleen. The tumor volumes of the tumor-bearing endo αPEG and αPEG-PT mice after treatment with LipoDox were significantly increased as compared with that of the tumor-bearing naïve mice.
Pre-αPEG Abs were found to dramatically alter the PK and reduce the tumor accumulation and therapeutic efficacy of LipoDox. Pre-αPEG may have potential as a marker to aid development of personalized therapy using LipoDox and achieve optimal therapeutic efficacy.
Antibodies that bind polyethylene glycol (PEG) can be induced by pegylated biomolecules and also exist in a significant fraction of healthy individuals who have never received pegylated medicines. ...The binding affinity of antibodies against PEG (anti-PEG antibodies) likely varies depending on if they are induced or naturally occurring. Anti-PEG antibodies can accelerate the clearance of pegylated medicines from the circulation, resulting in loss of drug efficacy, but it is unknown how accelerated blood clearance is affected by anti-PEG antibody affinity. We identified a panel of anti-PEG IgG and IgM antibodies with binding avidities ranging over several orders of magnitude to methoxy polyethylene glycol-epoetin beta (PEG-EPO), which is used to treat patients suffering from anemia. Formation of in vitro immune complexes between PEG-EPO and anti-PEG IgG or IgM antibodies was more obvious as antibody affinity increased. Likewise, high affinity anti-PEG antibodies produced greater accelerated blood clearance of PEG-EPO as compared to low affinity antibodies. The molar ratio of anti-PEG antibody to PEG-EPO that accelerates drug clearance in mice correlates with antibody binding avidity. Our study indicates that the bioactivity of PEG-EPO may be reduced due to rapid clearance in patients with either high concentrations of low affinity or low concentrations of high affinity anti-PEG IgG and IgM antibodies.
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•The affinity of anti-PEG IgG and IgM determines their biological impact.•Affinity of anti-PEG antibodies alters in vitro immune complex formation with PEG-EPO.•Accelerated clearance of PEG-EPO correlates with anti-PEG antibody avidity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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