anti-PEG Immune Responses Research Articles

Impact of PEG sensitization on the efficacy of PEG hydrogel-mediated tissue engineering

While poly(ethylene glycol) (PEG) hydrogels are generally regarded as biologically inert blank slates, concerns over PEG immunogenicity are growing, and the implications for tissue engineering are unknown. Here, we investigate these implications by immunizing mice against PEG to stimulate anti-PEG antibody production and evaluating bone defect regeneration after treatment with bone morphogenetic protein-2-loaded PEG hydrogels. Quantitative analysis reveals that PEG sensitization increases bone formation compared to naive controls, whereas histological analysis shows that PEG sensitization induces an abnormally porous bone morphology at the defect site, particularly in males. Furthermore, immune cell recruitment is higher in PEG-sensitized mice administered the PEG-based treatment than their naive counterparts. Interestingly, naive controls that were administered a PEG-based treatment also develop anti-PEG antibodies. Sex differences in bone formation and immune cell recruitment are also apparent. Overall, these findings indicate that anti-PEG immune responses can impact tissue engineering efficacy and highlight the need for further investigation.

An injectable PEG-like conjugate forms a subcutaneous depot and enables sustained delivery of a peptide drug

Many biologics have a short plasma half-life, and their conjugation to polyethylene glycol (PEG) is commonly used to solve this problem. However, the improvement in the plasma half-life of PEGylated drugs' is at an asymptote because the development of branched PEG has only had a modest impact on pharmacokinetics and pharmacodynamics. Here, we developed an injectable PEG-like conjugate that forms a subcutaneous depot for the sustained delivery of biologics. The PEG-like conjugate consists of poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) conjugated to exendin, a peptide drug used in the clinic to treat type 2 diabetes. The depot-forming exendin-POEGMA conjugate showed greater efficacy than a PEG conjugate of exendin as well as Bydureon, a clinically approved sustained-release formulation of exendin. The injectable depot-forming exendin-POEGMA conjugate did not elicit an immune response against the polymer, so that it remained effective and safe for long-term management of type 2 diabetes upon chronic administration. In contrast, the PEG conjugate induced an anti-PEG immune response, leading to early clearance and loss of efficacy upon repeat dosing. The exendin-POEGMA depot also showed superior long-term efficacy compared to Bydureon. Collectively, these results suggest that an injectable POEGMA conjugate of biologic drugs that forms a drug depot under the skin, providing favorable pharmacokinetic properties and sustained efficacy while remaining non-immunogenic, offers significant advantages over other commonly used drug delivery technologies.

The mechanisms of anti-PEG immune response are different in the spleen and the lymph nodes.

Polyethylene glycol (PEG) is a common ingredient in nanomedicines and pharmaceuticals. Recent studies show that approximately 20-70% of humans have anti-PEG antibodies that can recognize the polymer. Because these anti-PEG antibodies can reduce the effectiveness of certain PEGylated therapeutics, understanding how these immunoglobulins are produced is important. In this work, we investigate the mechanisms of the anti-PEG immune response, following the injection of polymeric nanoparticles by different routes of administration. We observed that the extent of systemic absorption and splenic deposition cannot predict the production of anti-PEG IgM - possibly because redundant biological pathways can be involved. Data obtained by surgically removing the spleen or depleting the complement activity suggest that the mechanisms behind the anti-PEG immune response differ between intravenous and subcutaneous injections. While B cells from the spleen appear to necessitate complement proteins to interact with nanoparticles, internalization by follicular B cells from the lymph nodes is unaffected by depletion of the cascade. This study confirms that the biological mechanisms involved in the immune recognition of nanomedicines varies based on the administration route. This knowledge can be utilized to use nanomedicines to engage the immune system in differentiated ways.

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.

Effect of gangliosides on structure and integrity of polyethylene glycol (PEG)-stabilized liposomes

The in vivo efficacy and tolerance of polyethylene glycol (PEG)-decorated drug nanocarriers, such as liposomes, is compromised bytheir tendency to induce the generation of PEG-specific immunoglobulin M (IgM) antibodies. Recently, a number of independent studies have reported on an attenuated anti-PEG immune response upon incorporation of gangliosides in the membrane of PEGylated liposomes.In the present study we investigate the effect of gangliosides on the self-assembled structures found in lipid dispersions based on hydrogenated egg phosphatidylcholine (HEPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG(2000)). Results from cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS) investigations show that gangliosides promote structural transitions from liposomes to bilayer disks. In case of samples comprising 5 mol% PEG-conjugated lipids (PEG-lipid), inclusion of 2.5 mol% ganglioside (porcine ganglioside extract) results in the presence of a small but significant amount of disks. With increasing ganglioside content the population of disks grows at the expense of the liposomes. Comparative investigations using isolated ganglioside components reveal that disialoganglioside GD1a is more potent than monosialoganglioside GM1 in promoting disk formation. Experiments involving liposome encapsulated carboxyfluorescein confirm that the ganglioside-induced structural transformations have a detrimental effect on the total entrapped aqueous volume of the samples. The reported coexistence of liposomes and bilayer disks may if overlooked have important implications for the therapeutic efficacy and immunogenicity of ganglioside-supplemented liposomal formulations.

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.

Induction of Anti-PEG Immune Responses by PEGylation of Proteins

Modification of proteins with polyethylene glycol (PEG) (PEGylation) is a gold standard technique that improves the solubility, pharmacokinetics, and immunogenicity of modified proteins. To date more than 10 PEGylated protein formulations have been approved, and more than 20 PEGylated drugs are entering clinical trials. PEG has been considered non-immunogenic and non-toxic, but several studies have shown that PEG acquires immunogenicity following attachment to nanoparticles. The administration of PEGylated liposomes, micelles and proteins induces the production of antibodies against PEG (anti-PEG antibodies) in animals and human subjects. Indeed, approximately 20% of healthy human subjects possess pre-existing anti-PEG antibodies prior to treatment with PEGylated therapeutics. The induced and pre-existing anti-PEG antibodies cause not only the elimination of PEGylated proteins from blood circulation, but also allergic responses via the release of anaphylatoxins. Consequently, therapeutic outcomes for PEGylated proteins are impaired. The utility of PEGylated proteins could be improved by attenuating the PEG-related immune response. On the other hand, anti-PEG immune responses might be exploited for vaccine applications. Our recent studies demonstrated that anti-PEG antibodies mediate the delivery of antigens encapsulated in PEGylated liposomes, and enhance antigen-specific immune responses. In this review, we summarize anti-PEG antibody induction by PEGylated proteins and alterations in anti-PEG IgM-mediated pharmacokinetics and pharmacodynamics. These findings extend our knowledge of PEG-related immune responses.

Therapeutic Protein PEPylation: The Helix of Nonfouling Synthetic Polypeptides Minimizes Antidrug Antibody Generation.

Polymer conjugation is a clinically proven approach to generate long acting protein drugs with decreased immune responses. Although poly(ethylene glycol) (PEG) is one of the most commonly used conjugation partners due to its unstructured conformation, its therapeutic application is limited by its poor biodegradability, propensity to induce an anti-PEG immune response, and the resultant accelerated blood clearance (ABC) effect. Moreover, the prevailing preference of unstructured polymers for protein conjugation still lacks strong animal data support with appropriate control reagents. By using two biodegradable synthetic polypeptides with similar structural compositions (l-P(EG3Glu) and dl-P(EG3Glu)) for site-specific protein modification, in the current study, we systematically investigate the effect of the polymer conformation on the in vivo pharmacological performances of the resulting conjugates. Our results reveal that the conjugate l20K-IFN, interferon (IFN) modified with the helical polypeptide l-P(EG3Glu) shows improved binding affinity, in vitro antiproliferative activity, and in vivo efficacy compared to those modified with the unstructured polypeptide analogue dl-P(EG3Glu) or PEG. Moreover, l20K-IFN triggered significantly less antidrug and antipolymer antibodies than the other two. Importantly, the unusual findings observed in the IFN series are reproduced in a human growth hormone (GH) conjugate series. Subcutaneously infused l20K-GH, GH modified with l-P(EG3Glu), evokes considerably less anti-GH and antipolymer antibodies compared to those modified with dl-P(EG3Glu) or PEG (dl20K-GH or PEG20K–GH). As a result, repeated injections of dl20K-GH or PEG20K-GH, but not l20K-GH, result in a clear ABC effect and significantly diminished drug availability in the blood. Meanwhile, immature mouse bone marrow cells incubated with the helical l20K-GH exhibit decreased drug uptake and secretion of proinflammatory cytokines compared to those treated with one of the other two GH conjugates bearing unstructured polymers. Taken together, the current study highlights an urgent necessity to systematically reassess the pros and cons of choosing unstructured polymers for protein conjugation. Furthermore, our results also lay the foundation for the development of next-generation biohybrid drugs based on helical synthetic polypeptides.

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

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. 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. 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. 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.

Heparosan-coated liposomes for drug delivery.

Liposomal encapsulation is a useful drug delivery strategy for small molecules, especially chemotherapeutic agents such as doxorubicin. Doxil® is a doxorubicin-containing liposome ("dox-liposome") that passively targets drug to tumors while reducing side effects caused by free drug permeating and poisoning healthy tissues. Polyethylene glycol (PEG) is the hydrophilic coating of Doxil® that protects the formulation from triggering the mononuclear phagocyte system (MPS). Evading the MPS prolongs dox-liposome circulation time thus increasing drug deposition at the tumor site. However, multiple doses of Doxil® sometimes activate an anti-PEG immune response that enhances liposome clearance from circulation and causes hypersensitivity, further limiting its effectiveness against disease. These side effects constrain the utility of PEG-coated liposomes in certain populations, justifying the need for investigation into alternative coatings that could improve drug delivery for better patient quality of life and outcome. We hypothesized that heparosan (HEP; [-4-GlcA-β1-4-GlcNAc-α1-]n) may serve as a PEG alternative for coating liposomes. HEP is a natural precursor to heparin biosynthesis in mammals. Also, bacteria expressing an HEP extracellular capsule during infection escape detection and are recognized as "self," not a foreign threat. By analogy, coating drug-carrying liposomes with HEP should camouflage the delivery vehicle from the MPS, extending circulation time and potentially avoiding immune-mediated clearance. In this study, we characterize the postmodification insertion of HEP-lipids into liposomes by dynamic light scattering and coarse-grain computer modeling, test HEP-lipid immunogenicity in rats, and compare the efficacy of drug delivered by HEP-coated liposomes to PEG-coated liposomes in a human breast cancer xenograft mouse model.

Effect of protein immunogenicity and PEG size and branching on the anti-PEG immune response to PEGylated proteins

Abstract PEGylation has successfully improved the pharmacological properties of therapeutic proteins. However, polyethylene glycol (PEG) has been burdened by immunogenicity that renders a negative clinical effect on therapeutic proteins. The anti-PEG immune response to PEGylated proteins possibly depends on the nature of proteins and the conjugated methoxy PEG (mPEG). Thus, it is necessary to investigate the effects of protein immunogenicity, the extent of PEGylation, the molecular weight (Mw), and the branching of mPEG on the anti-PEG immune response. Ovalbumin, tetanus toxoid (TT), TT–TT conjugate, and TT–bovine serum albumin conjugate were used as target proteins. PEGylated proteins with different extents of PEGylation were obtained by fractionation of the PEGylated TT with size exclusion chromatography. The PEGylated proteins with different Mw and branching of mPEG were obtained by modification of TT with linear mPEG (5 kDa and 20 kDa) and branched mPEG (20 kDa). The PEGylated proteins elicited high levels of anti-PEG antibodies (predominantly IgM and IgG1). The anti-PEG immune response depended on the immunogenicity of proteins, the extent of PEGylation, and the Mw of mPEG. In contrast, branching of mPEG had an insignificant effect on the anti-PEG immune response to the PEGylated proteins.

Anti-PEG antibodies alter the mobility and biodistribution of densely PEGylated nanoparticles in mucus.

Antibodies that specifically bind polyethylene glycol (PEG) can lead to rapid elimination of PEGylated therapeutics from the systemic circulation. We have recently shown that virus-binding IgG can immobilize viruses in mucus via multiple low-affinity crosslinks between IgG and mucins. However, it remains unclear whether anti-PEG antibodies in mucus may also alter the penetration and consequently biodistribution of PEGylated nanoparticles delivered to mucosal surfaces. We found that both anti-PEG IgG and IgM can readily bind nanoparticles that were densely coated with PEG polymer to minimize adhesive interactions with mucus constituents. Addition of anti-PEG IgG and IgM into mouse cervicovaginal mucus resulted in extensive trapping of mucus-penetrating PEGylated nanoparticles, with the fraction of mobile particles reduced from over 95% to only 34% and 7% with anti-PEG IgG and IgM, respectively. Surprisingly, we did not observe significant agglutination induced by either antibody, suggesting that particle immobilization is caused by adhesive crosslinks between mucin fibers and IgG or IgM bound to individual nanoparticles. Importantly, addition of corresponding control antibodies did not slow the PEGylated nanoparticles, confirming anti-PEG antibodies specifically bound to and trapped the PEGylated nanoparticles. Finally, we showed that trapped PEGylated nanoparticles remained largely in the luminal mucus layer of the mouse vagina even when delivered in hypotonic formulations that caused untrapped particles to be drawn by the flow of water (advection) through mucus all the way to the epithelial surface. These results underscore the potential importance of elucidating mucosal anti-PEG immune responses for PEGylated therapeutics and biomaterials applied to mucosal surfaces. PEG, generally considered a 'stealth' polymer, is broadly used to improve the circulation times and therapeutic efficacy of nanomedicines. Nevertheless, there is increasing scientific evidence that demonstrates both animals and humans can generate PEG-specific antibodies. Here, we show that anti-PEG IgG and IgM can specifically immobilize otherwise freely diffusing PEG-coated nanoparticles in fresh vaginal mucus gel ex vivo by crosslinking nanoparticles to the mucin mesh, and consequently prevent PEG-coated nanoparticles from accessing the vaginal epithelium in vivo. Given the increasing use of PEG coatings to enhance nanoparticle penetration of mucosal barriers, our findings demonstrate that anti-PEG immunity may be a potential concern not only for systemic drug delivery but also for mucosal drug delivery.

Anti-PEG immunity: emergence, characteristics, and unaddressed questions.

The modification of protein and nanoparticle therapeutics with polyethylene glycol (PEG), a flexible, uncharged, and highly hydrophilic polymer, is a widely adopted approach to reduce RES clearance, extend circulation time, and improve drug efficacy. Nevertheless, an emerging body of literature, generated by numerous research groups, demonstrates that the immune system can produce antibodies that specifically bind PEG, which can lead to the 'accelerated blood clearance' of PEGylated therapeutics. In animals, anti-PEG immunity is typically robust but short-lived and consists of a predominantly anti-PEG IgM response. Rodent studies suggest that the induction of anti-PEG antibodies (α-PEG Abs) primarily occurs through a type 2 T-cell independent mechanism. Although anti-PEG immunity is less well-studied in humans, the presence of α-PEG Abs has been correlated with reduced efficacy of PEGylated therapeutics in clinical trials. The prevalence of anti-PEG IgG and reports of memory immune responses, as well as the existence of α-PEG Abs in healthy untreated individuals, suggests that the mechanism(s) and features of human anti-PEG immune responses may differ from those of animal models. Many questions, including the incidence rate of pre-existing α-PEG Abs and immunological mechanism(s) of α-PEG Ab formation in humans, must be answered in order to fully address the potential complications of anti-PEG immunity.