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

Roadmap and strategy for overcoming infusion reactions to nanomedicines

Infusion reactions (IRs) are complex, immune-mediated side effects that mainly occur within minutes to hours of receiving a therapeutic dose of intravenously administered pharmaceutical products. These products are diverse and include both traditional pharmaceuticals (for example biological agents and small molecules) and new ones (for example nanotechnology-based products). Although IRs are not unique to nanomedicines, they represent a hurdle for the translation of nanotechnology-based drug products. This Perspective offers a big picture of the pharmaceutical field and examines current understanding of mechanisms responsible for IRs to nanomedicines. We outline outstanding questions, review currently available experimental evidence to provide some answers and highlight the gaps. We review advantages and limitations of the in vitro tests and animal models used for studying IRs to nanomedicines. Finally, we propose a roadmap to improve current understanding, and we recommend a strategy for overcoming the problem.National Cancer InstituteNational Institutes of Health | Ref. CA194058National Institutes of Health | Ref. EB022040Miskolc University. Applied Materials and Nanotechnology Center of ExcellenceXunta de Galicia | Ref. Xunta de Galici

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

Human serum albumin nanoparticles loaded with phthalocyanine dyes for potential use in photodynamic therapy of atherosclerotic plaques

Diseases caused by obstruction or rupture of vulnerable plaques in the arterial walls such as cardiovascular infarction or stroke are the leading cause of death in the world. In the present work, we developed human serum albuminnanoparticles loaded by physisorption with zinc phthalocyanine, TT1, mainly used for industrial application as near-infrared photosensitizer and compared these to HSA NPsloaded with the well-known silicone phthalocyanine (Pc4). The use of NIR light allows for better tissue penetration, while the use of nanoparticles permitshigh local concentrations. The particles were characterized and tested for toxicity and stability as well as for their potential use as a contrast agent and NIR photosensitizer for photodynamic therapy in cardiovascular disease. We focused on the distribution of the nanoparticles in RAW264.7macrophage cells and atherosclerotic mice. The nanoparticles had an average size of 120 nm according todynamic light scattering, good loading capacity for zinc phthalocyanine,and satisfying stability in 50% (v/v) fetal bovine serum for 8 hours and in an aqueous environment at 4°C for 4–6 weeks. Under light irradiation we found a high production of singlet oxygen and the products showed no dark toxicity in vitro with macrophages(the target cells in vulnerable plaques),but at a low μg/mL nanoparticleconcentration killed efficiently the macrophagesupon LED illumination. Injection of the contrast agentin atherosclerotic mice led to a visible fluorescence signal of zinc phthalocyaninein the atherosclerotic plaque at 30 minutes and in the lungs with afast clearance of the nanoparticles. Zinc phthalocyanine loaded human serum albumin nanoparticles present an interesting candidate for the visualization and potentially photodynamictreatment of macrophages in atherosclerotic plaquesThe research leading to these results has received funding from FP7-NMP CosmoPHOS-Nano under grant agreement No. 310337. Additional funding was received by the Spanish groups from MINECO (CTQ2017-85393-P) and ERA-NET/MINECO EuroNanoMed2017-191 / PCIN-2017-04

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

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