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

Contribution of CARPA to polystyrene NP effects in pigs

Background: It has been proposed that many hypersensitivity reactions to nanopharmaceuticals represent complement (C)-activation-related pseudoallergy (CARPA), and that pigs provide a sensitive animal model to study the phenomenon. However, a recent study suggested that pulmonary hypertension, the pivotal symptom of porcine CARPA, is not mediated by C in cases of polystyrene nanoparticle (PS-NP)-induced reactions. Goals: To characterize PS-NPs and reexamine the contribution of CARPA to their pulmonary reactivity in pigs. Study design: C activation by 200, 500, and 750 nm (diameter) PS-NPs and their opsonization were measured in human and pig sera, respectively, and correlated with hemodynamic effects of the same NPs in pigs in vivo. Methods: Physicochemical characterization of PS-NPs included size, ζ-potential, cryo-transmission electron microscopy, and hydrophobicity analyses. C activation in human serum was measured by ELISA and opsonization of PS-NPs in pig serum by Western blot and flow cytometry. Pulmonary vasoactivity of PS-NPs was quantified in the porcine CARPA model. Results: PS-NPs are monodisperse, highly hydrophobic spheres with strong negative surface charge. In human serum, they caused size-dependent, significant rises in C3a, Bb, and sC5b-9, but not C4d. Exposure to pig serum led within minutes to deposition of C5b-9 and opsonic iC3b on the NPs, and opsonic iC3b fragments (C3dg, C3d) also appeared in serum. PS-NPs caused major hemodynamic changes in pigs, primarily pulmonary hypertension, on the same time scale (minutes) as iC3b fragmentation and opsonization proceeded. There was significant correlation between C activation by different PS-NPs in human serum and pulmonary hypertension in pigs. Conclusion: PS-NPs have extreme surface properties with no relevance to clinically used nanomedicines. They can activate C via the alternative pathway, entailing instantaneous opsonization of NPs in pig serum. Therefore, rather than being solely C-independent reactivity, the mechanism of PS-NP-induced hypersensitivity in pigs may involve C activation. These data are consistent with the “double-hit” concept of nanoparticle-induced hypersensitivity reactions involving both CARPA and C-independent pseudoallergy

Infusion Reactions Associated with the Medical Application of Monoclonal Antibodies: The Role of Complement Activation and Possibility of Inhibition by Factor H

Human application of monoclonal antibodies (mAbs), enzymes, as well as contrast media and many other particulate drugs and agents referred to as “nanomedicines”, can initiate pseudoallergic hypersensitivity reactions, also known as infusion reactions. These may in part be mediated by the activation of the complement system, a major humoral defense system of innate immunity. In this review, we provide a brief outline of complement activation-related pseudoallergy (CARPA) in general, and then focus on the reactions caused by mAb therapy. Because the alternative pathway of complement activation may amplify such adverse reactions, we highlight the potential use of complement factor H as an inhibitor of CARPA

Infusion Reactions Associated with the Medical Application of Monoclonal Antibodies: The Role of Complement Activation and Possibility of Inhibition by Factor H

Human application of monoclonal antibodies (mAbs), enzymes, as well as contrast media and many other particulate drugs and agents referred to as “nanomedicines”, can initiate pseudoallergic hypersensitivity reactions, also known as infusion reactions. These may in part be mediated by the activation of the complement system, a major humoral defense system of innate immunity. In this review, we provide a brief outline of complement activation-related pseudoallergy (CARPA) in general, and then focus on the reactions caused by mAb therapy. Because the alternative pathway of complement activation may amplify such adverse reactions, we highlight the potential use of complement factor H as an inhibitor of CARPA

Flow cytometric analysis of supravesicular structures in doxorubicin-containing pegylated liposomes

In an attempt to develop a quantitative assay for supravesicular structures (SVS) - such as aggregates, fused liposomes or solid lipid particles - in liposome preparations, forward vs. side scattering of liposomal doxorubicin (Doxil/Caelyx) was analyzed by flow cytometry. Based on calibration with fluorescent latex beads, the size resolution was between about 500 and 1000nm. Caelyx, just as structurally matched empty liposomes (Doxebo) produced dot plots clearly distinguishable from background, suggesting the presence of SVS in the above size region. A comparison of gated areas on the scattergrams obtained for different Caelyx preparations showed differences between current and expired samples, implying that SVS formation may be storage-time-dependent. Incubation of doxorubicin with Doxebo in a free drug and lipid concentration range that corresponds to that in Caelyx also led to varying SVS patterns, raising the possibility that free doxorubicin in Caelyx might contribute to SVS formation. Dynamic light scattering and transmission electron microscopic analysis of liposomes following gaiting and sorting of >500nm particles from Caelyx confirmed the presence of SVS, providing independent evidence for their stable existence. Based on a rough estimation, the amount of SVS in Caelyx is some 60 billionth part of all liposomes. These observations raise the possibility that the presence of an exceedingly small fraction of >500nm particles may be an intrinsic property of PEGylated small unilamellar liposomes, and that the described FACS analysis may be developed further as a quality assay for liposomal homogeneity

Increased synthesis of vascular endothelial growth factor in allergic airway inflammation in histidine decarboxylase knockout (HDC-/-) mice

Histamine and vascular endothelial growth factor (VEGF) have been implicated in the pathogenesis of allergic asthma; they enhance inflammation, vascular permeability, and mucus secretion. Histamine was suggested to alter the level of VEGF via the H2 receptors. Here the authors have applied histidine decarboxylase gene-targeted (HDC-/-) mice, lacking histamine, to investigate the effect of histamine deficiency on VEGF expression in an animal model of asthma. HDC-/- and wild-type (WT) mice were sensitized and challenged with ovalbumin (OVA). VEGF mRNA expression and protein level were determined in the lung. Number of VEGF-positive immune cells of bronchoalveolar lavage (BAL) and their intracellular VEGF content were measured by flow cytometry. VEGF protein level in the lung and in the BAL cells was increased in OVA treated (HDCova-/- as well as in WTova) animals compared to their controls. However, there was no difference in the VEGF levels between HDC-/- or WT animals, either in the lung or in the BAL cells. In conclusion, increased VEGF production of the lung or BAL immune cells can be induced by allergen provocation independently from the genetic background of the animals. These data suggest that VEGF-mediated allergic processes can persist in the absence of histamine

Pseudo-anaphylaxis to polyethylene glycol (PEG)-coated liposomes: Roles of anti-PEG IgM and complement activation in a porcine model of human infusion reactions

Polyethylene glycol (PEG)-coated nanopharmaceuticals can cause mild to severe hypersensitivity reactions (HSRs), which can occasionally be life threatening or even lethal. The phenomenon represents an unsolved immune barrier to the use of these drugs, yet its mechanism is poorly understood. This study showed that a single i.v. injection in pigs of a low dose of PEGylated liposomes (Doxebo) induced a massive rise of anti-PEG IgM in blood, peaking at days 7–9 and declining over 6 weeks. Bolus injections of PEG-liposomes during seroconversion resulted in anaphylactoid shock (pseudo-anaphylaxis) within 2–3 min, although similar treatments of naı̈ve animals led to only mild hemodynamic disturbance. Parallel measurement of pulmonary arterial pressure (PAP) and sC5b-9 in blood, taken as measures of HSR and complement activation, respectively, showed a concordant rise of the two variables within 3 min and a decline within 15 min, suggesting a causal relationship between complement activation and pulmonary hypertension. We also observed a rapid decline of anti-PEG IgM in the blood within minutes, increased binding of PEGylated liposomes to IgM+ B cells in the spleen of immunized animals compared to control, and increased C3 conversion by PEGylated liposomes in the serum of immunized pigs. These observations taken together suggest rapid binding of anti-PEG IgM to PEGylated liposomes, leading to complement activation via the classical pathway, entailing anaphylactoid shock and accelerated blood clearance of liposome–IgM complexes. These data suggest that complement activation plays a causal role in severe HSRs to PEGylated nanomedicines and that pigs can be used as a hazard identification model to assess the risk of HSRs in preclinical safety studies