Anti-PEG antibodies : Properties, formation, testing and role in adverse immune reactions to PEGylated nano-biopharmaceuticals

Conjugation of polyethylene glycols (PEGs) to proteins or drug delivery nanosystems is a widely accepted method to increase the therapeutic index of complex nano-biopharmaceuticals. Nevertheless, these drugs and agents are often immunogenic, triggering the rise of anti-drug antibodies (ADAs). Among these ADAs, anti-PEG IgG and IgM were shown to account for efficacy loss due to accelerated blood clearance of the drug (ABC phenomenon) and hypersensitivity reactions (HSRs) entailing severe allergic symptoms with occasionally fatal anaphylaxis. In addition to recapitulating the basic information on PEG and its applications, this review expands on the physicochemical factors influencing its immunogenicity, the prevalence, features, mechanism of formation and detection of anti-PEG IgG and IgM and the mechanisms by which these antibodies (Abs) induce ABC and HSRs. In particular, we highlight the in vitro, animal and human data attesting to anti-PEG Ab-induced complement (C) activation as common underlying cause of both adverse effects. A main message is that correct measurement of anti-PEG Abs and individual proneness for C activation might predict the rise of adverse immune reactions to PEGylated drugs and thereby increase their efficacy and safety

Causative factors behind poloxamer 188 (Pluronic F68, Flocor™)-induced complement activation in human sera A protective role against poloxamer-mediated complement activation by elevated serum lipoprotein levels

AbstractPoloxamer 188 is a complex polydisperse mixture of non-ionic macromolecules. Adverse non-IgE-mediated hypersensitivity reactions occur in some individuals following intravenous injection of poloxamer 188-based pharmaceuticals, presumably via complement activation. Here we have delineated potential causal chemical and biological interactive factors behind poloxamer 188-induced complement activation in human serum specimens. We identified the molecular constituents inherent in poloxamer 188 preparations and studied their effect on generation of the two complement split products, SC5b-9 and Bb. Poloxamer 188 activated complement at sub-micellar concentrations and the results indicated the potential involvement of all three known complement activation pathways. The poloxamer-induced rise of SC5b-9 in human sera was abolished in the presence of a recombinant truncated soluble form of complement receptor type 1, thus confirming the role of C3/C5 convertases in the activation process. Poloxamer 188-mediated complement activation is an intrinsic property of these macromolecules and was independent of the degree of sample polydispersity, as opposed to other non-polymeric constituents. Poloxamer 188 preparations also contained unsaturated chains of diblock copolymers capable of generating SC5b-9 in human sera; this effect was terminated following the removal of double bonds by catalytic hydrogenation. By quasi-elastic light scattering, we established interaction between poloxamer and lipoproteins; interestingly, poloxamer-induced rise in SC5b-9 was significantly suppressed when serum HDL and LDL cholesterol levels were increased above normal to mimic two relevant clinical situations. This observation was consistent with previously reported data from patients with abnormal or elevated lipid profiles where no or poor complement activation by poloxamer 188 occurred. Our findings could provide the basis of novel approaches to the prevention of poloxamer-mediated complement activation

PEGylated liposomes : immunological responses

A commonly held view is that nanocarriers conjugated to polyethylene glycol (PEG) are non-immunogenic. However, many studies have reported that unexpected immune responses have occurred against PEG-conjugated nanocarriers. One unanticipated response is the rapid clearance of PEGylated nanocarriers upon repeat administration, called the accelerated blood clearance (ABC) phenomenon. ABC involves the production of antibodies toward nanocarrier components, including PEG, which reduces the safety and effectiveness of encapsulated therapeutic agents. Another immune response is the hypersensitivity or infusion reaction referred to as complement (C) activation-related pseudoallergy (CARPA). Such immunogenicity and adverse reactivities of PEGylated nanocarriers may be of potential concern for the clinical use of PEGylated therapeutics. Accordingly, screening of the immunogenicity and CARPA reactogenicity of nanocarrier-based therapeutics should be a prerequisite before they can proceed into clinical studies. This review presents PEGylated liposomes, immunogenicity of PEG, the ABC phenomenon, C activation and lipid-induced CARPA from a toxicological point of view, and also addresses the factors that influence these adverse interactions with the immune system

Physiological and Pathological Factors Affecting Drug Delivery to the Brain by Nanoparticles.

The prevalence of neurological/neurodegenerative diseases, such as Alzheimer's disease is known to be increasing due to an aging population and is anticipated to further grow in the decades ahead. The treatment of brain diseases is challenging partly due to the inaccessibility of therapeutic agents to the brain. An increasingly important observation is that the physiology of the brain alters during many brain diseases, and aging adds even more to the complexity of the disease. There is a notion that the permeability of the blood-brain barrier (BBB) increases with aging or disease, however, the body has a defense mechanism that still retains the separation of the brain from harmful chemicals in the blood. This makes drug delivery to the diseased brain, even more challenging and complex task. Here, the physiological changes to the diseased brain and aged brain are covered in the context of drug delivery to the brain using nanoparticles. Also, recent and novel approaches are discussed for the delivery of therapeutic agents to the diseased brain using nanoparticle based or magnetic resonance imaging guided systems. Furthermore, the complement activation, toxicity, and immunogenicity of brain targeting nanoparticles as well as novel in vitro BBB models are discussed

Complement activation as a bioequivalence issue relevant to the development of generic liposomes and other nanoparticulate drugs

Liposomes are known to activate the complement (C) system, which can lead in vivo to a hypersensitivity syndrome called C activation-related pseudoallergy (CARPA). CARPA has been getting increasing attention as a safety risk of i.v. therapy with liposomes, whose testing is now recommended in bioequivalence evaluations of generic liposomal drug candidates. This review highlights the adverse consequences of C activation, the unique symptoms of CARPA triggered by essentially all i.v. administered liposomal drugs, and the various features of vesicles influencing this adverse immune effect. For the case of Doxil, we also address the mechanism of C activation and the opsonization vs. long circulation (stealth) paradox. In reviewing the methods of assessing C activation and CARPA, we delineate the most sensitive porcine model and an algorithm for stepwise evaluation of the CARPA risk of i.v. liposomes, which are proposed for standardization for preclinical toxicology evaluation of liposomal and other nanoparticulate drug candidates

Understanding the Role of Anti-PEG Antibodies in the Complement Activation by Doxil in Vitro

Infusion reactions (IRs) are common immune-mediated side effects in patients treated with a variety of drug products, including, but not limited to, nanotechnology formulations. The mechanism of IRs is not fully understood. One of the best studied mechanisms of IRs to nanomedicines is the complement activation. However, it is largely unknown why some patients develop reactions to nanomedicines while others do not, and why some nanoparticles are more reactogenic than others. One of the theories is that the pre-existing anti-polyethylene glycol (PEG) antibodies initiate the complement activation and IRs in patients. In this study, we investigated this hypothesis in the case of PEGylated liposomal doxorubicin (Doxil), which, when used in a clinical setting, is known to induce IRs; referred to as complement activation-related pseudoallergy (CARPA) in sensitive individuals. We conducted the study in vitro using plasma derived from C57BL/6 mice and twenty human donor volunteers. We used mouse plasma to test a library of well-characterized mouse monoclonal antibodies with different specificity and affinity to PEG as it relates to the complement activation by Doxil. We determined the levels of pre-existing polyclonal antibodies that bind to PEG, methoxy-PEG, and PEGylated liposomes in human plasma, and we also assessed complement activation by Doxil and concentrations of complement inhibitory factors H and I in these human plasma specimens. The affinity, specificity, and other characteristics of the human polyclonal antibodies are not known at this time. Our data demonstrate that under in vitro conditions, some anti-PEG antibodies contribute to the complement activation by Doxil. Such contribution, however, needs to be considered in the context of other factors, including, but not limited to, antibody class, type, clonality, epitope specificity, affinity, and titer. In addition, our data contribute to the knowledge base used to understand and improve nanomedicine safety