Anti-polyethylene Glycol Research Articles

Humoral immune response against SARS-CoV-2 and polyethylene glycol elicited by anti-SARS-CoV-2 mRNA vaccine, and effect of pre-existing anti-polyethylene glycol antibody in patients with hematological and autoimmune diseases

BackgroundThe effects of vaccination are modified by hematological and autoimmune diseases and/or treatment. Anti-SARS-CoV-2 mRNA vaccine contains polyethylene glycol (PEG), it is largely unknown whether PEG influences the effects of vaccination or induces a humoral response. This study examined whether anti-PEG antibodies before vaccination (pre-existing) influenced the acquisition of SARS-CoV-2 antibodies and evaluated the relationship between the development of anti-SARS-CoV-2 antibodies and anti-PEG antibodies after SARS-CoV-2 vaccination in hematological and autoimmune diseases. MethodsAnti-SARS-CoV-2 antibody IgG, anti-PEG IgG, and IgM titers were evaluated in patients with hematological and autoimmune diseases after the second dose of BNT162B2. Anti-PEG IgG and IgM titers were also measured before vaccination to examine changes after vaccination and the relationship with vaccine efficacy. ResultsIn patients with hematological (n = 182) and autoimmune diseases (n = 96), anti-SARS-CoV-2 and anti-PEG antibody titers were evaluated after a median of 33 days from 2nd vaccination. The median anti-SARS-CoV-2 antibody titers were 1901 AU/mL and 3832 AU/mL in patients with hematological and autoimmune disease, respectively. Multiple regression analysis showed that age and days from 2nd vaccination were negatively associated with anti-SARS-CoV-2 antibody titers. Anti-CD20 antibody treatment was negatively correlated with anti-SARS-CoV-2 antibody titers in hematological disease, and C-reactive protein (CRP) was positively correlated with anti-SARS-CoV-2 antibody titers in autoimmune disease. Baseline anti-PEG antibody titers were significantly higher in patients with autoimmune disease but were not correlated with anti-SARS-CoV-2 antibody titers. Patients with increased anti-PEG IgG acquired higher anti-SARS-CoV-2 antibody titers in patients with autoimmune disease. ConclusionsAnti-SARS-CoV-2 antibody acquisition was suboptimal in patients with hematological disease, but both anti-SARS-CoV-2 antibody and anti-PEG IgG were acquired in patients with autoimmune disease, reflecting robust humoral immune response. Pre-existing anti-PEG antibody titers did not affect anti-SARS-CoV-2 antibody acquisition.

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.

Safety and efficacy of damoctocog alfa pegol prophylaxis in patients with severe haemophilia A: Results of an interventional, post-marketing study.

Damoctocog alfa pegol (BAY 94-9027, Jivi® ) is an approved extended half-life factor VIII (FVIII) for treatment of previously treated patients with haemophilia A aged ≥12 years. We report the final results of an interventional, post-marketing study of damoctocog alfa pegol prophylaxis in patients with severe haemophilia A. In this open-label, interventional, post-marketing, phase 4 trial (NCT04085458), previously FVIII-treated patients with severe haemophilia A aged ≥18 years received damoctocog alfa pegol for ≥100 exposure days (EDs). Patients initially received 45IU/kg every 5 days (recommended) or 40IU/kg twice-weekly. At Visit3, patients' doses could be increased, or treatment frequency adapted. The primary endpoint was FVIII inhibitor development (titre ≥.6Bethesda units). Secondary endpoints included anti-polyethylene glycol (PEG) antibody development, treatment-emergent adverse events (AEs) and annualized bleeding rate (ABR). Overall, 36 patients were enrolled; 32 patients received treatment, of whom, 27 completed the study. No patients developed FVIII inhibitors; three tested transiently positive for low-titre anti-PEG antibodies without clinical relevance. Three patients reported study-drug-related AEs of mild or moderate intensity. Two patients discontinued the study due to AEs. No deaths occurred. Most patients (70%) were treated with E5D/E7D regimens. The median (Q1;Q3) total ABR (N=30) was 3.0 (.0;9.0) pre-study and 1.8(.7;5.9) during the study. Damoctocog alfa pegol individualized prophylaxis regimens were well-tolerated with no immunogenicity concerns. ABRs improved following the switch from pre-study prophylaxis to damoctocog alfa pegol prophylaxis. These results support the favourable safety and efficacy profile of damoctocog alfa pegol prophylaxis.

A singlicate immunogenicity method to detect anti-polyethylene glycol antibodies: pre- and post-dose of PEGylated therapies.

Aim: Preexisting anti-polyethylene glycol (PEG) antibodies (APAs) may affect the efficacy and safety of PEGylated compounds. Omontys® and Krystexxa® withdrawal and SARS-CoV-2 RNA vaccine anaphylaxis have all been linked to APAs. This project aimed to develop and validate a method to detect total antibodies against PEG, pre- and postdose. Materials & methods: The repetitive, linear PEG structure prevented the use of a bridging homogenous format, hence the requirement to use a solid phase extraction and acid dissociation assay coupled with the Meso Scale Discovery® platform. Results & conclusion: Using singlicate analysis, the method was validated to successfully detect APA pre- and postdose, with a crucial aspect of the method being the preparation of an appropriate negative control.

Role of anti-polyethylene glycol (PEG) antibodies in the allergic reactions to PEG-containing Covid-19 vaccines: Evidence for immunogenicity of PEG

A small fraction of recipients who receive polyethylene-glycol (PEG)-containing COVID-19 mRNA-LNP vaccines (Comirnaty and Spikevax) develop hypersensitivity reactions (HSRs) or anaphylaxis. A causal role of anti-PEG antibodies (Abs) has been proposed, but not yet been proven in humans.We used ELISA for serial measurements of SARS-CoV-2 neutralizing Ab (anti-S) and anti-PEG IgG/IgM Ab levels before and after the first and subsequent booster vaccinations with mRNA-LNP vaccines in a total of 291 blood donors. The HSRs in 15 subjects were graded and correlated with anti-PEG IgG/IgM, just as the anti-S and anti-PEG Ab levels with each other. The impacts of gender, allergy, mastocytosis and use of cosmetics were also analyzed. Serial testing of two or more plasma samples showed substantial individual variation of anti-S Ab levels after repeated vaccinations, just as the levels of anti-PEG IgG and IgM, which were over baseline in 98–99 % of unvaccinated individuals. About 3-4 % of subjects in the strongly left-skewed distribution had 15–45-fold higher values than the median, referred to as anti-PEG Ab supercarriers. Both vaccines caused significant rises of anti-PEG IgG/IgM with >10-fold rises in about ∼10 % of Comirnaty, and all Spikevax recipients. The anti-PEG IgG and/or IgM levels in the 15 vaccine reactors (3 anaphylaxis) were significantly higher compared to nonreactors. Serial testing of plasma showed significant correlation between the booster injection-induced rises of anti-S and anti-PEG IgGs, suggesting coupled anti-S and anti-PEG immunogenicity.Conclusions: The small percentage of people who have extremelevels of anti-PEG Ab in their blood may be at increased risk for HSRs/anaphylaxis to PEGylated vaccines and other PEGylated injectables. This risk might be further increased by the anti-PEG immunogenicity of these vaccines. Screening for anti-PEG Ab “supercarriers” may help predicting reactors and thus preventing these adverse phenomena.

The Level of Anti-Polyethylene Glycol (PEG) Immunoglobulin G Antibodies in Human Serum Does Not Correlate with Efficiency of PEGylated Nanoparticle Uptake by Monocytes.

The presence of anti-polyethylene glycol (PEG) antibodies can limit the clinical efficacy of PEGylated drugs and cause anaphylactic reactions in patients. Monocytes/macrophages are effector cells involved in IgG-mediated passive systemic anaphylaxis. We studied the influence of human blood serum on the efficiency of uptake of PEGylated nanoparticles by human blood monocytes. It has been shown that magnetic nanoparticles modified with PEG-3000 and solid lipid nanoparticles containing PEG-2000 are avidly internalized by human blood monocytes in vitro, the uptake efficiency depends on the features (composition) of donor blood serum, but does not correlate with the level of the IgG antibody against PEG.

Is PEGylation of Drugs Associated with Hypersensitivity Reactions? An Analysis of the Italian National Spontaneous Adverse Drug Reaction Reporting System.

Evidence highlights the allergenic potential of PEGylated drugs because of the production of anti-polyethylene glycol immunoglobulins. We investigated the risk of hypersensitivity reactions of PEGylated drugs using the Italian spontaneous adverse drug reaction reporting system database. We selected adverse drug reaction reports attributed to medicinal products containing PEGylated active substances and/or PEGylated liposomes from the Italian Spontaneous Reporting System in the period between its inception and March 2021. As comparators, we extracted adverse drug reaction reports of medicinal products containing the same non-PEGylated active substances and/or non-PEGylated liposomes (or compounds belonging to the same mechanistic class). A descriptive analysis of reports of hypersensitivity reactions was performed. Reporting rates and time to onset of hypersensitivity reactions were also calculated in the period between January 2009 and March 2021. As a measure of disproportionality, we calculated the reporting odds ratio. Overall, 3865 adverse drug reaction reports were related to PEGylated medicinal products and 11,961 to their non-PEGylated comparators. Around two-thirds of patients were female and reports mostly concerned patients aged between 46 and 64 years. The frequency of hypersensitivity reactions reporting was higher among PEGylated versus non-PEGylated medicinal products (11.7% vs 9.4%, p < 0.0001). The hypersensitivity reaction reporting rates were higher for PEGylated medicinal products versus non-PEGylated medicinal products, with reporting rate ratios that ranged from 1.4 (95% confidence interval 0.8-2.5) for pegfilgrastim versus filgrastim to 20.0 (95% confidence interval 2.8-143.5) for peginterferon alpha-2a versus interferon alpha-2a. The median time to onset of hypersensitivity reactions was 10 days (interquartile range: 0-61) for PEGylated medicinal products, and 36 days (interquartile range: 3-216) for non-PEGylated comparators. Statistically significant reporting odds ratios were observed when comparing the reporting of hypersensitivity reactions for PEGylated versus non-PEGylated medicinal products (reporting odds ratio: 1.3; 95% confidence interval 1.1-1.4). However, when using all other drugs as comparators, the disproportionality analysis showed no association with hypersensitivity reactions for PEGylated nor non-PEGylated medicinal products, thus suggesting that many other triggers of drug-induced hypersensitivity reactions play a major role. The findings of this analysis of the Italian spontaneous adverse drug reaction database suggest a potential involvement for PEGylation in triggering drug-related hypersensitivity reactions, especially clinically relevant reactions. However, when comparing both PEGylated and non-PEGylated drugs under study to all other drugs no disproportionate reporting of hypersensitivity reactions was observed, probably due to a masking effect owing to the presence in the same database of other medicinal products increasing the threshold required to highlight a safety signal when the entire database is used as a reference.

Atomic Force Microscopic Imaging of mRNA-lipid Nanoparticles in Aqueous Medium

The efficacy of mRNA-lipid nanoparticles (mRNA-LNPs) depends on several factors, including their size and morphology. This study presents a new technique to characterize mRNA-LNPs in an aqueous medium using atomic force microscopy (AFM). This method utilizes an anti-polyethylene glycol antibody to immobilize mRNA-LNPs onto a glass substrate without corruption, which cannot be avoided with conventional procedures using solid substrates such as mica and glass. The obtained AFM images showed spherical and bleb-like structures of mRNA-LNPs, consistent with previous observations made using cryo-transmission electron microscopy. The AFM method also revealed the predominant existence of nanoparticles with a diameter < 60 nm, which were not detectable by dynamic light scattering and nanoparticle tracking analysis. As mRNA-LNPs are usually not monodisperse, but rather polydisperse, the AFM method can provide useful complementary information about mRNA-LNPs in their development and quality assessment.

A Maleimide-Terminally Modified PEGylated Liposome Induced the Accelerated Blood Clearance Independent of the Production of Anti-PEG IgM Antibodies.

PEGylated liposomes (PL) lose their long-circulating characteristic when administered repeatedly, called the accelerated blood clearance (ABC) phenomenon. The ABC phenomenon is generally thought to occur when the anti-polyethylene glycol (PEG) antibody (anti-PEG immunoglobulin M (IgM)) expressed in the spleen B cells triggered by the first dose of PL binds to the second and subsequent doses of PL, leading to activation of the complement system. MAL-PEG-DSPE, a PEG lipid with a maleimide (MAL) group at the PEG terminal, is used in various studies as a linker for ligand-bound liposomes such as antibody-modified liposomes. However, most ABC phenomenon research used PL with a terminal methoxy group (PL-OCH3). In this study, we prepared MAL-PEG-DSPE liposomes (PL-MAL) to evaluate the effect of PL-MAL on the ABC phenomenon induction compared to PL-OCH3. Pharmacokinetic, anti-PEG IgM secretion and complement activation analyses of these liposomes were conducted in mice. Interestingly, despite C3 bound to the surface of the initially administered PL-MAL, the administered PL-MAL showed high blood retention, demonstrating the same results as PL-OCH3. On the other hand, although the secretion of anti-PEG IgM induced by PL-MAL was lower than PL-OCH3, the second dose of PL-MAL rapidly disappeared from the blood. These results suggest that the antibody produced from the first dose of PL-MAL binds to the second dose of PL-MAL, thereby activating C3 to act as an opsonin which promotes phagocytic uptake. In conclusion, PL-MAL induced the ABC phenomenon independent of the production of IgM antibodies against PEG. This study provides valuable findings for further studies using ligand-bound liposomes.

A case of coronavirus disease 2019 messenger RNA vaccine tolerance and immune response despite presence of anti-polyethylene glycol antibodies

The role of anti-polyethylene glycol (PEG) immunoglobulin (Ig)M, IgG, or IgE antibodies in coronavirus disease 2019 (COVID-19) messenger RNA (mRNA) vaccine anaphylaxis is unknown. We highlight a case with preexisting anti-PEG antibodies that tolerated vaccination.

Discovery in polyethylene glycol immunogenicity: The characteristic of intergenerational inheritance of anti-polyethylene glycol IgG

Several studies have reported the prevalence of anti-polyethylene glycol (PEG) antibodies (APAs) in healthy people, highlighting the widespread existence of APAs. The prevalence of anti-PEG immunoglobulin (Ig)G is significantly negatively correlated with age. Here, we used Wistar rats as model organism to examine whether APAs in parental rats can affect the production of antibodies in their offspring. After being pre-stimulated with blank PEGylated nanoemulsions (PE) to induce APAs production, parental rats were paired in cages. The presence of antibodies in the parents and offspring was detected using enzyme-linked immunosorbent assay. The presence of antibodies in the parental rats led to significant anti-PEG IgG positivity in their offspring, indicating that anti-PEG IgG exhibits intergenerational inheritance. Moreover, anti-PEG IgG in the offspring rats could bind to PE and accelerate its blood clearance. To the best of our knowledge, this is the first report on the intergenerational properties of APAs.

A mouse model for studying the effect of blood anti-PEG IgMs levels on the in vivo fate of PEGylated liposomes

The presence of anti-polyethylene glycol (PEG) antibodies in the systemic circulation might have potential implications for the therapeutic activity of PEGylated products in vivo in the clinic. In order to study the effect of pre-existing anti-PEG antibodies on the in vivo fate and the therapeutic efficiency of PEGylated therapeutics, we developed a BALB/c mouse model by virtue of the intraperitoneal (i.p.) inoculation of hybridoma cells (HIK-M09 and HIK-M11), secreting monoclonal anti-PEG IgM, mimicking the presence of pre-existing anti-PEG antibodies in the blood. In the model, the titers of anti-PEG IgM in the blood increased as a function of hybridoma cells numbers and time after i.p. inoculation. The in vivo levels of anti-PEG IgM decreased in a dose-dependent manner, following i.v. administration of empty PEGylated liposomes. C26 tumor-bearing mice with measurable levels of anti-PEG IgM, receiving i.v. injection of DiR-labeled empty PEGylated liposomes, showed lower levels of liposomal tumor accumulation and higher levels of liver and spleen accumulation, compared to C26 tumor-bearing mice without measurable anti-PEG IgM. This specifies that the presence of anti-PEG IgM in the murine circulation induced accelerated blood clearance of PEGylated liposomes and reduced their tumor accumulation. The biodistribution and antitumor efficacy of commercially available doxorubicin (DXR)-containing PEGylated liposomes, Doxil®, were scrutinized in the anti-PEG IgM mouse model. In C26 tumor-bearing mice having circulating anti-PEG IgM, at 24 h after injection almost no DXR was observed in blood and tumor, and increased DXR accumulation was observed in spleen and liver, compared to tumor-bearing mice with no circulating anti-PEG IgM. The antitumor efficacy of Doxil® was significantly compromised in the C26 tumor-bearing mice in the presence of anti-PEG IgM. These results demonstrate that the anti-PEG IgM mouse model could be a useful prognostic indicator for the therapeutic effectiveness of different formulations of PEGylated therapeutics in pre-clinical studies.

Using Bio-Layer Interferometry to Evaluate Anti-PEG Antibody-Mediated Complement Activation.

The purpose of this study was to develop a Bio-layer interferometry (BLI) system that could be an alternative approach for the direct evaluation of anti-polyethylene glycol (PEG) immunoglobulin M (IgM)-mediated complement activation of the accelerated blood clearance (ABC) phenomenon. Complement activation is well known to play an important role in the clearance of PEGylated and non-PEGylated nanomedicines following intravenous injection. This complement system is also thought to be responsible for the ABC phenomenon wherein repeated injections of PEGylated products are bound by anti-PEG antibodies. This study used three different sources of anti-PEG antibodies: HIK-M09 monoclonal antibodies (mAbs); HIK-M11 mAbs; and antiserum containing polyclonal anti-PEG IgMs. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (mPEG2000-DSPE) was immobilized as an antigen on aminopropyl silane biosensor chips of BLI. All anti-PEG IgMs in the sources increased the signals (thickness of the layer around the sensor tip) regarding binding of anti-PEG antibodies to PEG on the chips. In all anti-PEG IgM sources, further increases in the signals were observed when incubated in naïve mouse serum, which is a complement source, but not in heat inactivated (56 °C, 30 min) mouse serum, which abolishes complement activity. These findings show that the complement activation mediated via anti-PEG IgMs, which occurred on the sensor chips, was detected via BLI analysis. The complement activation induced by all anti-PEG IgM sources was confirmed via conventional enzyme-linked immunosorbent assay (ELISA), which is the conventional mode for detection of complement activation. Our study results show that BLI is a simple alternative method for the detection of complement activation.

Polyethylene Glycol Immunogenicity: Theoretical, Clinical, and Practical Aspects of Anti-Polyethylene Glycol Antibodies.

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.

Pseudo-Anaphylactic Reactions to Pfizer BNT162b2 Vaccine: Report of 3 Cases of Anaphylaxis Post Pfizer BNT162b2 Vaccination

Anaphylactic reactions were observed after Singapore’s national coronavirus disease 2019 (COVID-19) vaccination programme started in December 2020. We report the clinical and laboratory features of three patients in our institution who developed anaphylactic reactions after receiving the Pifzer BNT162b2 vaccine. IgM and IgG antibodies, but not IgE antibodies to the Pfizer BNT162b2 vaccine, were detected in all subjects. Similarly, mild to high elevated levels of anti-polyethylene glycol (PEG) IgG (1035–19709 U/mL, vs. vaccine-naive < 265 U/mL, vaccine-tolerant < 785 U/mL) and IgM (1682–5310 U/mL, vs. vaccine-naive < 1011 U/mL, vaccine-tolerant < 1007 U/mL) were detected in two out of three patients via commercial ELISA. High levels of serum anaphylatoxin C3a (79.0 ± 6.3 μg/mL, mean ± SD, vs. normal < 10 μg/mL) were observed in all three patients during the acute phase of the reaction, while tryptase levels, a marker of mast cell activation, were not elevated. Finally, one patient with the highest levels of anti-PEG IgG, IgM, and anti-Pfizer BNT162b2 IgG and IgM exhibited an enhanced Th2 cytokine serum profile during an acute reaction, with high levels of IL-4 (45.7 pg/mL, vs. vaccine-naive/tolerant < 2.30 pg/mL), IL-33 (86.4 pg/mL, vs. vaccine-naive/tolerant < 5.51 pg/mL) and IL-10 (22.9 pg/mL, vs. vaccine-naive/tolerant < 12.49 pg/mL) diminishing over time following corticosteroid treatment. Taken together, we propose these cases of anaphylaxis described are driven by a complement activation-related pseudoallergy (CAPRA), rather than classical IgE-mediated mechanisms.

Potential mechanisms of anaphylaxis to COVID-19 mRNA vaccines

Anaphylaxis to vaccines is historically a rare event. The coronavirus disease 2019 pandemic drove the need for rapid vaccine production applying a novel antigen delivery system: messenger RNA vaccines packaged in lipid nanoparticles. Unexpectedly, public vaccine administration led to a small number of severe allergic reactions, with resultant substantial public concern, especially within atopic individuals. We reviewed the constituents of the messenger RNA lipid nanoparticle vaccine and considered several contributors to these reactions: (1) contact system activation by nucleic acid, (2) complement recognition of the vaccine-activating allergic effector cells, (3) preexisting antibody recognition of polyethylene glycol, a lipid nanoparticle surface hydrophilic polymer, and (4) direct mast cell activation, coupled with potential genetic or environmental predispositions to hypersensitivity. Unfortunately, measurement of anti–polyethylene glycol antibodies in vitro is not clinically available, and the predictive value of skin testing to polyethylene glycol components as a coronavirus disease 2019 messenger RNA vaccine-specific anaphylaxis marker is unknown. Even less is known regarding the applicability of vaccine use for testing (in vitro/vivo) to ascertain pathogenesis or predict reactivity risk. Expedient and thorough research-based evaluation of patients who have suffered anaphylactic vaccine reactions and prospective clinical trials in putative at-risk individuals are needed to address these concerns during a public health crisis.

Flow cytometry analysis of anti-polyethylene glycol antibodies in human plasma.

Polyethylene glycol (PEG) is a biocompatible polymer used in biotherapeutics to increase bioavailability, reduce the frequency of administration, and optimize pharmacokinetics. Anti-PEG antibodies have been detected in healthy individuals and may decrease efficacy and alter the pharmacokinetics of PEGylated therapeutics; however, the prevalence of anti-PEG antibodies is unclear. In this study, a flow cytometry assay was optimized to detect anti-PEG IgG and IgM in human blood plasma. Three hundred (300) plasma samples from healthy blood donors were screened; anti-PEG IgG or IgM was detected in 65.3% of the total population, with 21.3% having anti-PEG IgG, 19.0% having anti-PEG IgM, and 25.0% having both anti-PEG IgG and IgM. The presence of anti-PEG IgG and IgM was confirmed using a 0.5% Tween-20 interference assay, a 20 kDa PEGylated polystyrene bead binding assay, and Western blotting of purified plasma from human IgG and IgM purification columns. The concentrations of anti-PEG IgG and IgM in positive samples ranged from 39 ng/mL to 18.7 μg/mL and 26 ng/mL to 11.6 μg/mL, respectively. The highest prevalence of both anti-IgG and anti-IgM was in individuals 18-24 years of age. The prevalence of anti-PEG IgG and IgM tended to be higher in women but did not differ among races. Age, sex, and race were not associated with the concentrations of anti-PEG IgG or IgM. No correlation was found between anti-PEG IgG and IgM concentrations. Our study indicates that flow cytometry can be used to detect anti-PEG IgG and IgM antibodies in human plasma.

The origins of flowering plants and pollinators.

New research raises questions about when flowering plants and their pollinators evolved

Impact of Pre-Existing or Induced Anti-PEG IgM on the Pharmacokinetics of Peginterferon Alfa-2a (Pegasys) in Mice.

PEGylation had been used successfully to improve the circulation half-lives and some physicochemical properties of protein therapeutics. However, anti-polyethylene glycol (anti-PEG) antibodies, either pre-existing or treatment-induced, can negatively affect the pharmacokinetics and pharmacological efficacy of PEGylated proteins. We have examined anti-PEG immune responses in mice for peginterferon alfa-2a (Pegasys), a clinically approved PEGylated protein therapeutic, at both the recommended dose (equivalent to 3 μg/kg in mice) and at higher doses (150 μg/kg) for single or repeated subcutaneous (s.c.) administrations. The effect of treatment-induced anti-PEG IgM on serum concentrations of Pegasys, following repeated administrations, was evaluated. In addition, the effect of pre-existing anti-PEG IgM elicited by a different PEGylated protein, PEG-OVA, on the systemic clearance of Pegasys, was investigated. At a s.c. dose of 3 μg/kg, single injections of Pegasys barely elicited anti-PEG immune responses. Four repeated doses of 150 μg/kg Pegasys elicited anti-PEG IgM production, depending on dose frequency, and triggered the rapid clearance of subsequent doses. In addition, anti-PEG-IgM produced in response to prior administration of PEG-OVA caused a rapid blood clearance of Pegasys. Our results, therefore, underscore the importance of screening for both pre-existing and treatment-induced anti-PEG antibodies in patients prior to and during treatment with PEGylated protein drugs.

Immunogenicity and safety of a proposed pegfilgrastim biosimilar MSB11455 versus the reference pegfilgrastim Neulasta® in healthy subjects: A randomized, double-blind trial.

MSB11455 is a proposed biosimilar to the currently licensed reference pegfilgrastim (Neulasta®). This study was designed primarily to compare the immunogenicity of MSB11455 and Neulasta®. As secondary objectives, the safety and tolerability of MSB11455 and Neulasta® were also compared. Healthy adult subjects were randomized to either MSB11455 or Neulasta®, stratified by antipolyethylene glycol (PEG) antibody status at screening and study site. Subjects received a single subcutaneous dose of MSB11455 or Neulasta® (both 6 mg/0.6 mL) on day 1 of each of two study periods (same product in both periods), separated by a washout of 28‐35 days. Immunogenicity samples were taken predose and up to day 84 post–first dose. Noninferiority was confirmed if the upper limit of the exact one‐sided adjusted 95% confidence interval (CI) for the difference in antidrug antibody (ADA)‐positive rates was < 10%. Safety was assessed throughout the study. Overall, 336 subjects were randomized and treated (N = 168 in each group). Noninferiority of MSB11455 over Neulasta® was demonstrated for immunogenicity; the difference in confirmed treatment‐induced ADA‐positive rate between MSB11455 and Neulasta® was −0.6% (upper limit of the exact one‐sided adjusted 95% CI: 6.25%). ADAs were mostly directed against the PEG moiety of pegfilgrastim. No filgrastim‐specific neutralizing antibodies were detected in either treatment group. Safety and tolerability were as expected for pegfilgrastim, and comparable between treatments. This study supports and strengthens the available evidence for the biosimilarity of MSB11455 to Neulasta®.