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. Author manuscript; available in PMC: 2022 Oct 10.
Published in final edited form as: J Control Release. 2021 Sep 2;338:804–812. doi: 10.1016/j.jconrel.2021.08.051

High MW polyethylene glycol prolongs circulation of pegloticase in mice with anti-PEG antibodies

Anne M Talkington 1, Morgan D McSweeney 2, Tao Zhang 3, Zibo Li 3,5, Andrew C Nyborg 4, Brian LaMoreaux 4, Eric W Livingston 5, Jonathan E Frank 5, Hong Yuan 3,5, Samuel K Lai 1,2,6,7
PMCID: PMC8794005  NIHMSID: NIHMS1741352  PMID: 34481925

Abstract

Pegloticase is an enzyme used to reduce serum uric acid levels in patients with chronic, treatment-refractory gout. Clinically, about 40% of patients develop high titers of anti-PEG antibodies (APA) after initial treatment, which in turn quickly eliminate subsequent doses of pegloticase from the systemic circulation and render the treatment ineffective. We previously found that pre-infusion with high MW free PEG (40 kDa) can serve as a decoy to saturate circulating APA, preventing binding to a subsequently administered dose of PEG-liposomes and restoring their prolonged circulation in mice, without any detectible toxicity. Here, we investigated the use of 40 kDa free PEG to restore the circulation of radiolabeled pegloticase in mice using longitudinal Positron Emission Tomography (PET) imaging over 4 days. Mice injected with pegloticase developed appreciable APA titers by Day 9, which further increased through Day 14. Compared to naïve mice, mice with pegloticase-induced APA rapidly cleared 89Zr-labeled pegloticase, with ~75% lower pegloticase concentrations in the circulation at four hours after treatment. The 96-hour AUC in APA+ mice was less than 30% of the AUC in naïve mice. In contrast, pre-infusion of free PEG into PEG-sensitized mice restored the AUC of pegloticase to ~80% of that seen in naïve mice, resulting in a similar biodistribution to pegloticase in naïve mice over time. These results suggest that pre-infusion of free PEG may be a promising strategy to enable the safe and efficacious use of pegloticase and other PEGylated drugs in patients that have previously failed therapy due to induced APA.

Keywords: Pegloticase, PEG, anti-PEG antibodies

Graphical Abstract

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Introduction

Pegloticase is currently the only FDA-approved treatment for severe, treatment-refractory chronic gout, a type of inflammatory arthritis affecting ~25,000–100,000 patients in the U.S. each year [1]. Pegloticase is a PEGylated mammalian enzyme that is a tetramer of uricase subunits and contains a total of forty covalently attached 10 kDa mPEG molecules per pegloticase molecule [2, 3]. Its mechanism of action is based on the circulating uricase enzyme converting circulating uric acid to allantoin, which is readily eliminated renally, resulting in a rapid, dramatic reduction in serum uric acid (sUA) and resolution of the tophi (urate deposits in the joints) responsible for the underlying inflammation from gout. Naturally, maintaining sustained serum levels of pegloticase adequate to suppress uric acid levels is essential for its efficacy. Unfortunately, ~89% of pegloticase treated patients develop measurable anti-PEG antibodies (APA), with ~40% developing high enough titers of APA to cause rapid elimination of subsequent doses of pegloticase from the circulation, rendering the treatment ineffective [4–8]. Unfortunately, there is no alternative intervention available for patients who develop strong APA responses.

Given the urgency of this unmet need, a number of companies and investigators have sought to advance alternatives to pegloticase. These interventions typically involve either modifying a uricase enzyme with different polymers [9–16], and/or combining a uricase molecule with some form of immunosuppression [17, 18]. When searching for a solution to overcome the challenges presented by APA, we were drawn to the safety and relative immunological inertness of free PEG (i.e., unmodified PEG molecules) administered intravenously [19–24]. This contrasts to when PEG is conjugated to select proteins or liposomes, which can be inherently immunogenic [15, 23, 25, 26]. We hypothesized that the infusion of free PEG could competitively inhibit pre-existing APA from binding PEGylated drugs, providing a safe, effective, and readily clinically translatable intervention that restores the safe and efficacious use of PEGylated drugs adversely impacted by APA. In support of our hypothesis, we found that, in mice with APA levels matching the highest APA titers naturally occurring in the general population (i.e., without induction by specific PEGylated therapeutics, >1 μg/mL APA), injection of 40 kDa free PEG (550 mg/kg, IV) increased the amount of circulating PEG-liposomes 48 hrs post-infusion by >100-fold compared to mice with APA that were treated with either PBS or 10 kDa free-PEG, restoring PEG-liposome concentrations to a level comparable to naïve mice with no APA [27]. Importantly, the toxicity profile following the chronic administration of free PEG to PEG-sensitized mice (including complete blood counts, renal function assays, and liver/kidney histology) were indistinguishable from control groups treated with PBS. More impressively, repeated dosing of free PEG did not stimulate runaway APA secretion, even in mice that had been previously sensitized to produce APA [28].

Building off these promising findings, we sought to investigate whether infusion of 40 kDa free PEG would restore the long-lasting circulation of pegloticase in the presence of pegloticase-induced APA. The mouse model originally used to assess the activity of pegloticase in vivo, a urate oxidase knockout mouse model, is exceptionally difficult. Even with recent advances, 40–65% of the homozygous mice are not viable past 5 weeks [29]. However, the pharmacokinetic (PK) profile of pegloticase is highly predictive of its efficacy; APA is not known to neutralize uricase activity [30, 31], and APA-mediated accelerated blood clearance is not associated with loss of enzymatic activity of uricase [31]. Thus, we decided to evaluate the effectiveness of pre-infusion of 40 kDa free PEG in restoring the long-lasting circulation of pegloticase as a proxy for restoration of efficacy, specifically by monitoring the PK profile of 89Zirconium-labeled pegloticase in real time using Positron Emission Tomography (PET) imaging in live animals. Similar to our prior studies, we found that pre-infusion of free PEG was an exceptionally simple yet effective means of restoring the prolonged systemic circulation of pegloticase in mice with high titers of APA.

Results

Mouse model of pegloticase-induced APA

Clinically, pegloticase is administered via IV infusion once every 2 weeks, and APA-mediated rapid elimination of pegloticase can be observed as quickly as clearance of the second dose, implying that high APA titers can be induced within 2 weeks of the first infusion [4]. Although pegloticase-induced APAs are readily apparent in clinical studies, the immunogenicity profile of pegloticase in mice has not been extensively characterized. To mimic clinical use in humans, we injected pegloticase via the tail vein into immunocompetent BALB/c mice on Day 0 and quantified anti-PEG IgG and IgM on both Day 9 and Day 14 (Figure 1A, B). In good agreement with clinical reports, mice developed detectable IgG and IgM APA even by Day 9, and then exhibited a rapid increase between Day 9 and Day 14 (Figure 1B, C). Indeed, anti-PEG IgG was increased from ~1.7 ± 0.9 μg/mL on Day 9 to ~10.4 ± 7.3 μg/mL by Day 14, whereas anti-PEG IgM was increased from ~0.4 ± 0.3 μg/mL on Day 9 to ~2.6 ± 2.3 μg/mL by Day 14. Our findings are consistent with the literature showing that anti-PEG immune responses to pegloticase increase after 1 week [32]. These induced levels of APA by pegloticase in mice parallel the highest levels of APA we had previously detected in blood samples from the general healthy population [33], but remains substantially lower than the APA levels induced by IV infusion of PEG-liposomes in mice [28, 34].

Figure 1. Study Design and Induction of APA.

Figure 1.

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(A) Study design and experimental timeline. (B) Average anti-PEG IgG and IgM in mice 9 and 14 days after initial pegloticase infusion on Day 0. Changes in (C) IgG and (D) IgM APA levels in mice from Day 9 to Day 14.

PET imaging for assessing the PK profile of 89Zr-labeled pegloticase

After confirming that pegloticase induced substantial APA titers within 2 weeks, we next performed a PET/CT study in the same animals assessing the impact of APA on the circulation of radiolabeled pegloticase (Figure 2). We assigned mice with APA (“APA+”) mice randomly into two groups (placebo control vs. free PEG pre-treatment; Figure S.1), and also included naïve BALB/c mice (i.e., not PEG-sensitized, or APA-) that served as a negative control to represent the normal PK profile of pegloticase. To assess the PK of pegloticase in the same mice over time, pegloticase was modified with chelator cheisothiocyanatobenzyl-desferrioxamine, which was then labeled with 89Zr, as previously described [35]. All mice received either saline or free PEG intravenously 45 min prior to injection with a bolus of 89Zr-pegloticase (4.1±1.3 MBq), followed immediately with a 60 min dynamic PET scan. CT was conducted afterwards. Repeated PET/CT scans were conducted at 4, 24, 48, 72, and 96 hours following the injection of 89Zr-pegloticase. Analysts and technicians were blinded to mice group information during image acquisition and analysis.

Figure 2. PET/CT Imaging of Mice.

Figure 2.

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PET/CT imaging was performed at different time points to assess pharmacokinetics and biodistribution of 89Zr-pegloticase in 3 cohorts: naïve mice infused with saline before pegloticase PEG-sensitized (APA+) mice infused with free PEG prior to pegloticase, and PEG-sensitized (APA+) mice infused with saline prior to pegloticase. Single PET coronal sections were overlayed on CT MIP images to represent the distribution of 89Zr-pegloticase over time.

Almost immediately, the APA-mediated clearance was apparent in the APA+, saline-treated cohort, as evidenced by the steep downward trend in plasma pegloticase levels in the first hour after injection, corresponding with an upward trend in hepatic signal as pegloticase was cleared to the liver (Figure 3A–B). In contrast, the plasma concentration of pegloticase in the naïve and free PEG-treated APA+ cohorts remained at a much higher level. This trend continued and became even more pronounced by 4 hours post injection. Early clearance was notable in the kidney and lung in the APA+, saline-treated mice, with corresponding accumulation in the liver during the first 4 hours post injection (Figure 3C–D). This is consistent with prior reports suggesting that APA may mediate accelerated blood clearance through the formation of small immune complexes, driving FcγRIIB-mediated clearance by liver sinusoidal endothelial cells or Kupffer cells [36, 37], or through alternative activation of complement, broadly driving phagocyte-mediated clearance [38]. After the protein drug is digested, the PEG component is believed to slowly cycle through rounds of pinocytosis, exocytosis, and lymphatic and plasma transport, as it is not easily biodegradable [39, 40]. It is ultimately released from the body via renal filtration or into feces via the liver [40, 41]. Signal in the muscle did not differ notably between the groups (Figure 3E).

Figure 3. Mean Pegloticase Levels Through 4h.

Figure 3.

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Mean pegloticase levels over the first 4h in (A) plasma, (B) liver, (C) kidney, (D) lung, and (E) muscle, as determined by PET/CT imaging. Solid black circle represents naïve mice preinfused with saline prior to pegloticase dosing; solid blue square represents PEG-sensitized mice pre-infused with 40 kDa free PEG prior to pegloticase dosing, and solid red triangle represents PEG-sensitized mice pre-infused with saline prior to pegloticase dosing. Error bars represent standard deviation.

The amount of pegloticase in the circulation in APA+, saline-treated mice continued to decline at a slower rate after the first four hours, and less than 5% of the injected dose remained in the circulation after 24 hrs (Figure 4A). Individually, the detectible levels of circulating pegloticase in the four APA+ mice were 66%, 20%, 7%, and 4% of the average concentration seen in naïve mice at 24 hrs (Figure 4A), with a greater degree of reduction corresponding to a greater APA titer observed at baseline (3, 5, 24, 18 μg/mL respectively). APA-mediated clearance of pegloticase resulted primarily in deposition in the liver, with nearly 60% of the injected dose found in the liver after 4 hrs, and ~80% after 24 hrs (Figure 4B). Due to the rapid hepatic accumulation in mice with APA at baseline who were not pre-treated with free PEG, less pegloticase was found in both the kidneys and lungs at all times during the study in these animals (Figure 4C, 4D). APA did not alter the amount of pegloticase found in the muscles, with a very low intensity of pegloticase-associated radioactivity in the observed section of muscle at all timepoints (Figure 4E).

Figure 4. Mean Pegloticase Levels Through 96h.

Figure 4.

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Mean pegloticase levels over the first 96h in (A) plasma, (B) liver, (C) kidney, (D) lung, and (E) muscle, as determined by PET/CT imaging. Solid black circle represents naïve mice preinfused with saline prior to pegloticase dosing; solid blue square represents PEG-sensitized mice pre-infused with 40 kDa free PEG prior to pegloticase dosing, and solid red triangle represents PEG-sensitized mice pre-infused with saline prior to pegloticase dosing. Error bars represent standard deviation. (F) Multiple comparisons highlighting statistical differences (adjusted p-values) in key organs and time points. Plasma pegloticase levels are not statistically different between the cohort treated with free PEG and the naïve mice through 96 hrs (Welch’s ANOVA, Dunnett’s T3 multiple comparisons). Liver uptake is not statistically different between the free PEG-treated cohort and naïve cohort through 96 hrs, while both of these groups have significantly less liver accumulation than the untreated cohort at the 96 hrs time point (Welch’s ANOVA and Dunnett’s T3, p < 0.05). Likewise, the free PEG-treated and naïve cohorts do not statistically differ through 96 hrs in the kidney and lung, whereas the untreated cohort with APA has greater clearance at the earlier time points (Welch’s ANOVA and Dunnett’s T3, p < 0.05). Muscle pegloticase levels do not significantly differ between cohorts (Welch’s ANOVA).

After PET-imaging at 96 hrs, we sacrificed the mice, collected the blood and organs, and assessed the amount of radioactivity in all tissues using a traditional gamma counter. The amount of pegloticase remaining in the blood as well as the relative amounts in different organs (measured via gamma counter) were in good agreement with the amounts estimated based on PET/CT imaging. These results validated our use of PET/CT imaging to assess the real-time pharmacokinetic profile of pegloticase in live animals.

Free PEG infusion restores prolonged circulation profile of pegloticase and delays distribution to the liver

Unlike APA+ mice treated with saline, the amount of circulating pegloticase in APA+ mice treated with free PEG was restored to nearly the same high concentrations seen in naïve mice over the first 48 hrs. Although differences began to emerge at the 72 hrs and 96 hrs time points, with a decline in the free PEG-treated animals relative to naïve controls, this decline was not statistically significantly different (applying Welch’s ANOVA followed by Dunnett’s T3 multiple comparisons test) (Figure 4A). The similar PK profiles were corroborated by a near-identical biodistribution profile seen between APA+, free PEG-treated mice vs. naïve mice over the first 48 hrs, with differences in amounts found in the liver, kidney and lung beginning to emerge only at the 72 hrs and 96 hrs time points (Figures 4B–D). The amount of pegloticase found in the muscles was not statistically different from naïve mice or APA+, saline-treated mice at any of the later time points (Welch’s ANOVA) (Figure 4E).

Based on the amounts of 89Zr-pegloticase detected in the blood and different organs over time, we calculated the AUC (trapezoidal) of pegloticase in different organs over the first 96 hrs for the different treatment groups, which we used to compare the effectiveness of free PEG in restoring the PK of pegloticase. Compared to naïve mice, the presence of APA reduced the blood AUC0–96h of pegloticase by over 70% (Figure 5A). In contrast, free PEG infusion mitigated much of that loss, with just a ~20% decline in AUC0–96h compared to naïve mice. Supportively, we observed very similar AUC values between naïve mice and APA+, free-PEG treated mice for all major organs, including the liver, kidney, lung, and muscle (Figure 5B–C). Given the good agreement between the estimations of circulating pegloticase when measured by gamma counter or PET, we believe PET/CT provided an accurate estimate of the effectiveness of free PEG.

Figure 5. AUC and % AUC Recovered.

Figure 5.

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AUC and % AUC recovered relative to naïve animals for (A) plasma, (B) liver, and (C) kidney, lung, and muscle. Error bars represent standard deviation. One-way ANOVA and multiple comparison testing (Kruskal-Wallis followed by Dunn’s; Brown-Forsythe and Welch followed by Dunnett’s T3) demonstrated a difference between the Naïve and APA+, saline cohorts (* p < 0.05), whereas the APA+, PEG cohort was not significantly different from Naïve. AUC in all organs in the free PEG-treated cohort is similar to AUC in the naïve cohort. Reported p-values are from Dunnett’s T3 and have been adjusted for multiple comparisons. There is some overlap between the behavior of the mouse with low APA in the saline-treated cohort and the mice with high APA in the free PEG-treated cohort.

Increased APA levels correlate to faster elimination of pegloticase from the circulation to the liver

We hypothesized that lower concentrations of APA at the time of administration of pegloticase would lead to slower clearance of pegloticase from the circulation, since lower APA levels should translate to either less APA bound to pegloticase, or a longer duration before a critical threshold of bound APA is achieved. We thus compared the concentration of APA measured at Day 14 (~2–3 days prior to the PET/CT study) to the resulting PK profiles and biodistribution in mice. In good agreement with our expectation, the mice with lower APA titers had greater levels of pegloticase detected in the circulation at all time points studied (Figure 6). Starting from 24 hrs post-administration, the amounts of 89 Zr-pegloticase detected in the circulation in mice with ≥ 5 μg/mL IgG APA were cleared with virtually identical speed and to comparable extent, suggesting a critical threshold for APA in the clearance of pegloticase. In contrast, in mice with ≤ 2.5 μg/mL IgG APA, high to modest quantities of pegloticase were detectable through the first 72 hrs. By the 96 hrs time point, the amount of pegloticase in the circulation was effectively at background level regardless of the initial IgG APA levels in the PEG-sensitized mice (Figure 6C, 6D). Not surprisingly, we saw a similar relationship between circulating APA levels and the amount of pegloticase accumulated in the liver at different time points; mice possessing the highest APA titers also had the greatest fraction of injected dose of pegloticase found in the liver, which became apparent as early as 24h.

Figure 6. %ID as a function of APA.

Figure 6.

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%ID of 89Zr-pegloticase signal in blood and liver as a function of IgG APA in serum, quantified by PET/CT imaging at (A) 24 hrs, (B) 48 hrs, (C) 72 hrs, and (D) 96 hrs post infusion. (E) Gamma counter measurements of radioactivity in excised organs at 96 hrs confirms the trends observed by PET/CT data.

Free PEG infusion is effective across a range of APA titers

At very high APA levels, it is mathematically more difficult for free PEG to saturate all circulating APA and competitively inhibit APA from binding and clearing pegloticase from the circulation. We thus correlated the measured APA levels to the observed PK. At modest APA titers that led to rapid clearance of pegloticase in placebo control mice, the infusion of free PEG afforded near-complete restoration of the pegloticase PK and biodistribution profiles (Figure 6). Even at the highest APA titer observed (17 μg/mL), free PEG infusion restored ~60% of the AUC for levels of pegloticase in the circulation. These results underscore the ability for free PEG infusion to overcome a range of APA titers to extend the circulation of PEG-uricase, allowing it to continue to reduce serum uric acid levels.

Discussion

As a result of the increasing number of PEGylated therapeutics authorized for human use, including the recent Pfizer/BioNTech and Moderna COVID mRNA vaccines, there has been increasing attention on the potential induction of APA, since APA may render select PEGylated therapies unsafe and/or not efficacious in some patients. For instance, Hershfield et al. found that over 60% of patients with APA experienced an adverse event after receiving pegloticase [4]. While pegloticase is the foremost example for APA-induced loss of efficacy of a PEGylated drug, there are other PEGylated drugs whose efficacy are also impacted by APA. This includes Oncospar, a PEG-asparaginase used as part of frontline therapy for acute lymphoblastic leukemia in young children. The high failure rate of response to Oncospar treatment, nearly 1 in 3, is strongly associated with the presence of APA [32, 42–44]. Evidence is emerging for some other PEGylated drugs, including pegnivacogin, where acute severe allergic response is attributed to APA-mediated complement activation [45]. However, not all PEGylated therapeutics have been shown to induce APA to an extent that impacts their safety or efficacy, and it has been suggested that the immunogenicity of the underlying protein or nanoparticle conjugated to PEG determines the relative strength of the APA response [15]. Regardless, there is considerable urgency in developing interventions that could mitigate the loss of efficacy and increased incidence of adverse events due to APA.

While an array of new molecules is under active clinical development, given the exorbitant costs and long timelines in advancing new therapeutic molecules through clinical studies, it is far more attractive to develop an intervention that is PEG-specific, and can restore the efficacy of a multitude of PEGylated therapeutics. Here, building off of our recent work utilizing free PEG as a decoy that binds circulating APA and competitively inhibits APA from binding to PEG-liposomes, we evaluated whether a similar strategy could also effectively restore the prolonged circulation of pegloticase. At low to moderate APA levels that are induced by pegloticase, we showed that a simple prior infusion of free PEG was able to restore nearly 80% of the AUC for amount of pegloticase in the circulation of naïve mice, with virtually no difference in the PK and biodistribution profiles over the first 48 hrs. Even at the highest APA titers, free PEG infusion appeared to recover ~60% of the AUC. Based on allometric scaling of mouse to humans, we believe this effect is likely meaningful clinically. The half-life of pegloticase in naïve mice here was ~10 hours in the alpha phase (0–4h) and ~48h in the beta phase (4–96h), in good agreement with other studies [2, 46], whereas the median half-life of pegloticase in patients is 214 hours, with the elimination likely driven by renal/urinary excretion [47]. In contrast, the average alpha half-life of pegloticase in APA+ mice without free PEG intervention was just ~2.5 hours, reflective of very rapid clearance into the liver. The beta half-life in this group of APA+ mice was ~38h, with slower clearance possibly attributed to less effective concentration of APA with the elimination of APA/pegloticase immunocomplexes. However, APA+ mice that received free PEG exhibited an early PK profile with very slow clearance in the alpha phase (~25h). The beta half-life in this group was ~27h, roughly similar to APA+ mice receiving saline, likely due to declining concentration of free PEG in the circulation over time. Scaling to human physiology, we believe these results suggest that free PEG intervention prior to administration of pegloticase may restore the prolonged circulation of pegloticase for many days in patients whose APA would otherwise quickly clear the drug and render the treatment ineffective.

While previous work from our lab has illustrated the ability of free PEG to extend the half-life of liposomal drugs in animals passively immunized with anti-PEG IgG [34], the current work represents the first time that free PEG dosing has been shown to be effective at restoring the PK and biodistribution of a clinically used PEGylated protein drug, in mice with a drug-induced APA response encompassing both anti-PEG IgG and IgM. Our approach with using free PEG to specifically saturate APA contrasts with current approaches focused on broad immunosuppression. Broad immunosuppression can carry significant side effects [48]. For instance, treatment with mTOR inhibitors has been associated with increased risk of infection or sepsis [49, 50], while low/moderate dose methotrexate appears to carry a modest risk of increased infection, as well as other adverse events [51, 52]. Our approach also contrasts markedly with the use of alternative polymers to replace PEG. Although many alternatives to PEG are under active investigation [10, 11, 13, 53], it remains unclear if any will possess the same degree of safety in humans as PEG, or even if they would be less immunogenic. Furthermore, given the enormous costs to bring a new treatment to market and the large number of PEGylated drugs in clinical development, we believe the most cost effective and readily translatable approach is to specifically inhibit APA from binding to PEGylated drugs, rather than develop new substitutes for each impacted PEGylated drug.

Although it may seem paradoxical to overcome anti-PEG immunity by administering additional doses of PEG, our approach is rooted in decades of prior studies that found PEG immunogenicity to primarily result from grafting PEG to proteins or lipids, and that immunogenicity of the compounds is related to the immunogenicity of the proteins themselves [15, 23, 26, 32]. In other words, PEG may act as a hapten that elicits an immune response only when attached to a large carrier such as a protein or lipid drug carriers. A large number of preclinical studies have investigated the safety of infusing simply high MW PEG: beyond the occasional observation of benign PEG-related vacuolation in select tissues, the infusion of free PEG has generally been exceptionally safe [54], and there is no evidence of deposition of immune complexes in the kidney that may lead to inflammation and glomerular disease [22, 55]. Thus, the infusion of free PEG effectively shifts the potential immunogenicity profile of a treatment, from the more immunogenic form of PEG associated with a protein or lipid, to the less immunogenic form of free PEG alone. It is certainly possible to saturate APA by simply dosing more PEGylated drugs. However, increasing the dose and/or adjusting the dosing frequency, particularly for drugs with dose-limited toxicity, is not only time-consuming and more expensive, but may yield unintended toxicities associated with the active molecule.

Based on the same principles of presenting PEG-decoys to saturate APA, one may obtain similar benefits of prolonging the systemic circulation of a PEGylated drug in APA+ animals by pretreating with empty PEG-liposomes. Indeed, Szebeni et al. elegantly showed that low-dose pre-infusion of Doxebo (empty PEG-liposomes) can block an anaphylactic response to subsequent doses of Doxil [56]. However, we believe free PEG offers unique advantages over PEG-liposomes. First, free PEG is far less immunostimulatory than PEG-liposomes, which can elicit much greater APA production [23, 25, 57, 58]. We can more readily achieve a substantial molar excess with free-PEG:APA than PEG-liposomes:APA; this in turn limits the formation of immune complexes with multiple anti-PEG B-cell receptors on APA-secreting B-cells, a necessary event to stimulate B-cell expansion and APA secretion. Finally, no PEGylated intervention can be more cost-effective than free-PEG, since USP-grade PEG is readily available at scale. All these factors contributed to our decision to pursue the use of free-PEG to overcome APA, rather than develop more complex interventions that may be scientifically novel but face significant developmental hurdles. Despite its simplicity, we found free PEG to limit short term APA induction to an extent not statistically different than PEG-liposomal doxorubicin (PL-dox) and rapamycin (PL-rapa) that directly kill or block proliferation of APA-specific B-cells, respectively [28]. Coupled with our current results, we believe free PEG infusion appears to be a promising intervention to overcome APA-mediated clearance of PEGylated therapeutics.

Methods

Mouse model and induction of APA.

Animal procedures used in this study were approved by the University of North Carolina at Chapel Hill’s Institutional Animal Care and Use Committee. BALB/c mice aged 4–5 weeks were obtained from Charles River Labs and allowed to acclimate so that they would be young adults by the time of study. To account for potential sex differences, half the mice in each cohort were male and half were female. On Day 0, 10 mice were injected IV with 150 μL pegloticase solution dosed at 0.9 mg/kg. This produced a controlled immune response study range through approximately 220 μg/mL anti-PEG IgG (Figure S.1).

Collection of plasma for quantitating APA titers.

At periodic time points (Day-1, Day 9, Day 14), mice were bled via mandibular bleed (200uL whole blood). The blood was kept in EDTA tubes on ice. Samples were then centrifuged at 2000 rcf for 15 minutes and the plasma was stored at −80 °C. Samples were thawed and kept at 4 °C for short-term processing. All plasma samples were subjected to exactly 1 freeze-thaw cycle.

Measurements of anti-PEG IgG and IgM.

Anti-PEG IgG and IgM levels were determined via competition ELISA based on previously established protocols [27]. 96-well plates were coated overnight at 4 °C with DSPE-PEG5000, and blocked with 5% milk in 1x PBS at room temperature for 1 hr. Plasma samples were diluted 50-fold in 1% milk with or without 8 kDa free PEG. Plates were incubated overnight at 4 °C, followed by washing. Then, goat anti-mouse IgG (Invitrogen, A28177, lot TG2596484) or IgM (ThermoFisher Life Technologies, 626820, lot QB215229) conjugated to HRP was added to the wells, incubated at room temperature for 1 hour, and washed. Finally, TMB was added to the wells, and after a waiting period, the conversion was stopped with 1N HCl. The plates were read at 450 and 570 nm, and 5-parameter logistic regression was performed on the standard curves. All individual specimens were measured in duplicate within an experiment, and 2 independent experiments were performed for all samples, with reported values an average of all measurements. All absorbance values, after subtracting the corresponding PEG-competition wells, were compared to APA isotype standards to determine the precise IgG and IgM APA in the specimens on a mass concentration basis. Standards were made from mouse anti-PEG IgG (Silver Lake, CH2076, lot K0868) and IgM (Academia Sinica, AGP4 (AGP4-PABM-A)).

Radiolabeling of pegloticase.

Pegloticase was provided by Horizon Therapeutics and stored according to instructions until use. To radiolabel pegloticase for PET imaging, we conjugated ~130 μg of pegloticase via Df chelation (p-SCN-Bn-Deferoxamine, lot B70510004–150407) targeting free amine groups on available lysine. The pegloticase-chelator conjugate was purified using a PD-10 column to remove unreacted chelator. The Df-pegloticase was then labeled with 89Zr in two batches [35]. The first batch produced 46% yield (3.5 mCi with 65 μg Df-pegloticase, received 1.6 mCi), and the second batch produced 80% yield (1 mCi with 65 μg Df-pegloticase, received 0.8 mCi). The samples were combined to form the solution dosed to the mice.

Mouse PET/CT Study.

On Day 16/17, the mice were prepared for PET/CT imaging using a small animal PET/CT imaging system (SuperArgus 4R, Sedecal Inc., Spain). The APA+ mice were randomly divided into two groups (saline placebo control vs. free PEG treatment). A group of naïve BALB/c mice (i.e., not PEG-sensitized) was included as a negative control. A 1.5%−2.5% isoflurane-oxygen gas mixture was used to anesthetize the mice. All mice received either saline (for naïve group and placebo group) or 40 kDa free PEG (550 mg/kg) by slow infusion through tail vein catheter over 2 min. Group information was blinded to the animal treatment handler. Mice were then placed in a multi-animal imaging cradle in groups of 4 for simultaneous imaging. After 45 min, the mice received a bolus injection of 89Zr-pegloticase (4.1±1.3 MBq, equivalent to ~4.7 μg of pegloticase in mass), followed immediately with dynamic PET scan for 60 mins. CT was conducted afterwards for anatomical reference. Repeated PET/CT scans were conducted at 4, 24, 48, 72, and 96 hours post injection of 89Zr-pegloticase. For the first 1-hour dynamic scan, images were binned into 19 frames with the following scheme: 6×10s, 4×30s, 2×60s, 3×300s, and 4×600s. The static scans at later time points were acquired for 20 minutes. 3D-OSEM algorithms with scatter, attenuation, and decay correction were used to reconstruct the PET images. For image analysis, PET and CT images were first registered and regions of interest (ROIs) were drawn in major organs for ROI-based PET image analysis. The standardized uptake value (SUV) and %ID/g were reported as the quantitative measure of the uptake level in each organ and tissue. %ID for each organ was further calculated by multiplying %ID/g with the estimated organ mass based on the empirical estimation [59–61]. All the analysis procedures were conducted using PMOD software (version 3.9). After the PET scan at 96 hrs, the mice were sacrificed. The liver, lung, spleen, kidney, and muscle tissue were harvested, as well as organs that did not account for substantial uptake including the heart and brain. Blood samples were centrifuged for separation into “plasma” and “blood cells and other sediments.” Heart and lung tissue were flushed with saline and dried with gauze before gamma counting. Gamma counter readings were performed on collected specimens to quantify 89Zr activity.

Supplementary Material

1

Figure S.1. Distribution of Induced APA in Mice. Distribution (mean and SD) of induced (A) IgG and (B) IgM levels in all mice. IgG and IgM levels were not statistically different between the two groups (two-tailed Welch’s t-test).

Highlights.

  • Pegloticase (PEGylated uricase) is a leading treatment for chronic gout

  • Anti-PEG antibodies induce accelerated blood clearance (ABC) of PEGylated uricase

  • Dosing free PEG alleviates APA-mediated ABC of pegloticase, recovering ~80% AUC

  • PEG dosing holds promise for restoring pegloticase use in non-responding patients

Acknowledgments

We like to thank the staff at the UNC Animal Studies Core.

Funding

This work was supported by The David and Lucile Packard Foundation (2013-39274, SKL), National Institutes of Health (T32-HL069768; AMT, R01 HL141934; SKL), P.E.O. International (AMT), and Eshelman Institute of Innovation (SKL). The BRIC Small Animal Imaging (SAI) core facility and UNC Lineberger Animal Core are supported in part by an NCI Grant P30-CA016086. The PET/CT equipment is supported by the NIH shared instrumentation grant (1S10OD023611, ZL).

Footnotes

Conflict of interest

S.K.L. and M.M. are inventors of patents on the use of free PEG to block APA. The terms of these arrangements are managed by UNC-CH in accordance with its conflict-of-interest policies. A.C.N. and B.L. are employees of, and have stock in, Horizon Therapeutics.

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References

  • [1].Guttmann A, Krasnokutsky S, Pillinger MH, Berhanu A, Pegloticase in gout treatment - safety issues, latest evidence and clinical considerations, Therapeutic advances in drug safety, 8 (2017) 379–388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Nyborg AC, Ward C, Zacco A, Chacko B, Grinberg L, Geoghegan JC, Bean R, Wendeler M, Bartnik F, O’Connor E, Gruia F, Iyer V, Feng H, Roy V, Berge M, Miner JN, Wilson DM, Zhou D, Nicholson S, Wilker C, Wu CY, Wilson S, Jermutus L, Wu H, Owen DA, Osbourn J, Coats S, Baca M, A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties, PLoS One, 11 (2016) e0167935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].HIGHLIGHTS OF PRESCRIBING INFORMATION: KRYSTEXXA® (pegloticase) in: Injection label, pp. 1–14. [Google Scholar]
  • [4].Hershfield MS, Ganson NJ, Kelly SJ, Scarlett EL, Jaggers DA, Sundy JS, Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients, Arthritis Research & Therapy, 16 (2014) R63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Lipsky PE, Calabrese LH, Kavanaugh A, Sundy JS, Wright D, Wolfson M, Becker MA, Pegloticase immunogenicity: the relationship between efficacy and antibody development in patients treated for refractory chronic gout, Arthritis Research & Therapy, 16 (2014) R60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Reinders MK, Jansen TLTA, New advances in the treatment of gout: review of pegloticase, Ther Clin Risk Manag, 6 (2010) 543–550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Sundy JS, Baraf HSB, Yood RA, Edwards NL, Gutierrez-Urena SR, Treadwell EL, Vázquez-Mellado J, White WB, Lipsky PE, Horowitz Z, Huang W, Maroli AN, Waltrip RW II, Hamburger SA, Becker MA, Efficacy and Tolerability of Pegloticase for the Treatment of Chronic Gout in Patients Refractory to Conventional Treatment: Two Randomized Controlled Trials, JAMA, 306 (2011) 711–720. [DOI] [PubMed] [Google Scholar]
  • [8].Gentry WM, Dotson MP, Williams BS, Hartley M, Stafford KR, Bottorff MB, Gandhi PK, Investigation of pegloticase-associated adverse events from a nationwide reporting system database, American Journal of Health-System Pharmacy, 71 (2014) 722–727. [DOI] [PubMed] [Google Scholar]
  • [9].Elsadek NE, Abu Lila AS, Ishida T, 5 - Immunological responses to PEGylated proteins: anti-PEG antibodies, in: Pasut G, Zalipsky S (Eds.) Polymer-Protein Conjugates, Elsevier, 2020, pp. 103–123. [Google Scholar]
  • [10].Zhang P, Sun F, Tsao C, Liu S, Jain P, Sinclair A, Hung H-C, Bai T, Wu K, Jiang S, Zwitterionic gel encapsulation promotes protein stability, enhances pharmacokinetics, and reduces immunogenicity, Proc Natl Acad Sci U S A, 112 (2015) 12046–12051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Knop K, Hoogenboom R, Fischer D, Schubert US, Poly(ethylene glycol) in Drug Delivery: Pros and Cons as Well as Potential Alternatives, Angewandte Chemie International Edition, 49 (2010) 6288–6308. [DOI] [PubMed] [Google Scholar]
  • [12].Abbina S, Parambath A, 14 - PEGylation and its alternatives: A summary, in: Parambath A (Ed.) Engineering of Biomaterials for Drug Delivery Systems, Woodhead Publishing, 2018, pp. 363–376. [Google Scholar]
  • [13].Qi Y, Chilkoti A, Protein-Polymer Conjugation—Moving Beyond PEGylation, Current opinion in chemical biology, 28 (2015) 181–193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Barz M, Luxenhofer R, Zentel R, Vicent MJ, Overcoming the PEG-addiction: well-defined alternatives to PEG, from structure–property relationships to better defined therapeutics, Polymer Chemistry, 2 (2011) 1900–1918. [Google Scholar]
  • [15].Li B, Yuan Z, Hung H-C, Ma J, Jain P, Tsao C, Xie J, Zhang P, Lin X, Wu K, Jiang S, Revealing the Immunogenic Risk of Polymers, Angewandte Chemie International Edition, 57 (2018) 13873–13876. [DOI] [PubMed] [Google Scholar]
  • [16].van Witteloostuijn SB, Pedersen SL, Jensen KJ, Half-Life Extension of Biopharmaceuticals using Chemical Methods: Alternatives to PEGylation, ChemMedChem, 11 (2016) 2474–2495. [DOI] [PubMed] [Google Scholar]
  • [17].Zhang P, Jain P, Tsao C, Wu K, Jiang S, Proactively Reducing Anti-Drug Antibodies via Immunomodulatory Bioconjugation, Angewandte Chemie International Edition, 58 (2019) 2433–2436. [DOI] [PubMed] [Google Scholar]
  • [18].Kishimoto TK, Ferrari JD, LaMothe RA, Kolte PN, Griset AP, O’Neil C, Chan V, Browning E, Chalishazar A, Kuhlman W, Fu F.-n., Viseux N, Altreuter DH, Johnston L, Maldonado RA, Improving the efficacy and safety of biologic drugs with tolerogenic nanoparticles, Nature Nanotechnology, 11 (2016) 890–899. [DOI] [PubMed] [Google Scholar]
  • [19].Carpenter CP, Woodside MD, Kinkead ER, King JM, Sullivan LJ, Response of dogs to repeated intravenous injection of polyethylene glycol 4000 with notes on excretion and sensitization, Toxicology and applied pharmacology, 18 (1971) 35–40. [DOI] [PubMed] [Google Scholar]
  • [20].Smyth HF Jr., Carpenter CP, Shaffer CB, The toxicity of high molecular weight polyethylene glycols; chronic oral and parenteral administration, Journal of the American Pharmaceutical Association. American Pharmaceutical Association, 36 (1947) 157–160. [DOI] [PubMed] [Google Scholar]
  • [21].Stidl R, Fuchs S, Bossard M, Siekmann J, Turecek PL, Putz M, Safety of PEGylated recombinant human full-length coagulation factor VIII (BAX 855) in the overall context of PEG and PEG conjugates, Haemophilia, 22 (2016) 54–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Webster R, Elliott V, Park BK, Walker D, Hankin M, Taupin P, PEG and PEG conjugates toxicity: towards an understanding of the toxicity of PEG and its relevance to PEGylated biologicals, in: Veronese FM (Ed.) PEGylated Protein Drugs: Basic Science and Clinical Applications, Birkhäuser Basel, Basel, 2009, pp. 127–146. [Google Scholar]
  • [23].Mima Y, Hashimoto Y, Shimizu T, Kiwada H, Ishida T, Anti-PEG IgM Is a Major Contributor to the Accelerated Blood Clearance of Polyethylene Glycol-Conjugated Protein, Molecular pharmaceutics, 12 (2015) 2429–2435. [DOI] [PubMed] [Google Scholar]
  • [24].Richter AW, Åkerblom E, Antibodies against Polyethylene Glycol Produced in Animals by Immunization with Monomethoxy Polyethylene Glycol Modified Proteins, International Archives of Allergy and Immunology, 70 (1983) 124–131. [DOI] [PubMed] [Google Scholar]
  • [25].Ishida T, Wang X, Shimizu T, Nawata K, Kiwada H, PEGylated liposomes elicit an anti-PEG IgM response in a T cell-independent manner, Journal of Controlled Release, 122 (2007) 349–355. [DOI] [PubMed] [Google Scholar]
  • [26].Wan X, Zhang J, Yu W, Shen L, Ji S, Hu T, Effect of protein immunogenicity and PEG size and branching on the anti-PEG immune response to PEGylated proteins, Process Biochemistry, 52 (2017) 183–191. [Google Scholar]
  • [27].McSweeney MD, Price LSL, Wessler T, Ciociola EC, Herity LB, Piscitelli JA, DeWalle AC, Harris TN, Chan AKP, Saw RS, Hu P, Jennette JC, Forest MG, Cao Y, Montgomery SA, Zamboni WC, Lai SK, Overcoming anti-PEG antibody mediated accelerated blood clearance of PEGylated liposomes by pre-infusion with high molecular weight free PEG, Journal of Controlled Release, 311–312 (2019) 138146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].McSweeney MD, Shen L, DeWalle AC, Joiner JB, Ciociola EC, Raghuwanshi D, Macauley MS, Lai SK, Pre-treatment with high molecular weight free PEG effectively suppresses anti-PEG antibody induction by PEG-liposomes in mice, Journal of Controlled Release, (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Lu J, Hou X, Yuan X, Cui L, Liu Z, Li X, Ma L, Cheng X, Xin Y, Wang C, Zhang K, Wang X, Ren W, Sun R, Jia Z, Tian Z, Mi Q-S, Li C, Knockout of the urate oxidase gene provides a stable mouse model of hyperuricemia associated with metabolic disorders, Kidney International, 93 (2018) 69–80. [DOI] [PubMed] [Google Scholar]
  • [30].Burns CM, Wortmann RL, Gout therapeutics: new drugs for an old disease, The Lancet, 377 (2011) 165–177. [DOI] [PubMed] [Google Scholar]
  • [31].Zhang C, Fan K, Ma X, Wei D, Impact of large aggregated uricases and PEG diol on accelerated blood clearance of PEGylated canine uricase, PLoS One, 7 (2012) e39659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Zhang P, Sun F, Liu S, Jiang S, Anti-PEG antibodies in the clinic: Current issues and beyond PEGylation, Journal of Controlled Release, 244 (2016) 184–193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Yang Q, Jacobs TM, McCallen JD, Moore DT, Huckaby JT, Edelstein JN, Lai SK, Analysis of Pre-existing IgG and IgM Antibodies against Polyethylene Glycol (PEG) in the General Population, Analytical Chemistry, 88 (2016) 11804–11812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].McSweeney MD, Wessler T, Price LSL, Ciociola EC, Herity LB, Piscitelli JA, Zamboni WC, Forest MG, Cao Y, Lai SK, A minimal physiologically based pharmacokinetic model that predicts anti-PEG IgG-mediated clearance of PEGylated drugs in human and mouse, Journal of Controlled Release, 284 (2018) 171–178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Vosjan MJWD, Perk LR, Visser GWM, Budde M, Jurek P, Kiefer GE, van Dongen GAMS, Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine, Nature Protocols, 5 (2010) 739–743. [DOI] [PubMed] [Google Scholar]
  • [36].Pyzik M, Sand KMK, Hubbard JJ, Andersen JT, Sandlie I, Blumberg RS, The Neonatal Fc Receptor (FcRn): A Misnomer?, Frontiers in Immunology, 10 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [37].Ganesan LP, Kim J, Wu Y, Mohanty S, Phillips GS, Birmingham DJ, Robinson JM, Anderson CL, FcγRIIb on liver sinusoidal endothelium clears small immune complexes, J Immunol, 189 (2012) 4981–4988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Merkel OM, Urbanics R, Bedőcs P, Rozsnyay Z, Rosivall L, Toth M, Kissel T, Szebeni J, In vitro and in vivo complement activation and related anaphylactic effects associated with polyethylenimine and polyethylenimine-graft-poly(ethylene glycol) block copolymers, Biomaterials, 32 (2011) 4936–4942. [DOI] [PubMed] [Google Scholar]
  • [39].Yamaoka T, Tabata Y, Ikada Y, Distribution and Tissue Uptake of Poly(ethylene glycol) with Different Molecular Weights after Intravenous Administration to Mice, Journal of Pharmaceutical Sciences, 83 (1994) 601–606. [DOI] [PubMed] [Google Scholar]
  • [40].Baumann A, Tuerck D, Prabhu S, Dickmann L, Sims J, Pharmacokinetics, metabolism and distribution of PEGs and PEGylated proteins: quo vadis?, Drug discovery today, 19 (2014) 1623–1631. [DOI] [PubMed] [Google Scholar]
  • [41].Bjørnsdottir I, Sternebring O, Kappers WA, Selvig H, Kornø HT, Kristensen JB, Bagger MA, Pharmacokinetics, tissue distribution and excretion of 40kDa PEG and PEGylated rFVIII (N8-GP) in rats, European Journal of Pharmaceutical Sciences, 87 (2016) 58–68. [DOI] [PubMed] [Google Scholar]
  • [42].Armstrong JK, Hempel G, Koling S, Chan LS, Fisher T, Meiselman HJ, Garratty G, Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients, Cancer, 110 (2007) 103–111. [DOI] [PubMed] [Google Scholar]
  • [43].Kloos R, van der Sluis IM, Mastrobattista E, Hennink W, Pieters R, Verhoef J-J, Acute lymphoblastic leukaemia patients treated with PEGasparaginase develop antibodies to PEG and the succinate linker, British Journal of Haematology, 189 (2020) 442–451. [DOI] [PubMed] [Google Scholar]
  • [44].Liu Y, Smith CA, Panetta JC, Yang W, Thompson LE, Counts JP, Molinelli AR, Pei D, Kornegay NM, Crews KR, Swanson H, Cheng C, Karol SE, Evans WE, Inaba H, Pui CH, Jeha S, Relling MV, Antibodies Predict Pegaspargase Allergic Reactions and Failure of Rechallenge, Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 37 (2019) 2051–2061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Povsic TJ, Lawrence MG, Lincoff AM, Mehran R, Rusconi CP, Zelenkofske SL, Huang Z, Sailstad J, Armstrong PW, Steg PG, Bode C, Becker RC, Alexander JH, Adkinson NF, Levinson AI, Pre-existing anti-PEG antibodies are associated with severe immediate allergic reactions to pegnivacogin, a PEGylated aptamer, Journal of Allergy and Clinical Immunology, 138 (2016) 1712–1715. [DOI] [PubMed] [Google Scholar]
  • [46].Bomalaski JS, Holtsberg FW, Ensor CM, Clark MA, Uricase formulated with polyethylene glycol (uricase-PEG 20): biochemical rationale and preclinical studies, The Journal of Rheumatology, 29 (2002) 1942. [PubMed] [Google Scholar]
  • [47].Product Information Annex: KRYSTEXXA 8 mg concentrate for solution for infusion, in: Product Information, European Medicines Agency, European Public Assessment Report, pp. 1–27. [Google Scholar]
  • [48].Youssef J, Novosad SA, Winthrop KL, Infection Risk and Safety of Corticosteroid Use, Rheumatic diseases clinics of North America, 42 (2016) 157–x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [49].Shi G, Ozog S, Torbett BE, Compton AA, mTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3, Proc Natl Acad Sci U S A, 115 (2018) E10069–e10078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [50].Fisher A, Seguel JM, de la Torre AN, Wilson D, Merchant A, Arora RK, Koneru B, Effect of sirolimus on infection incidence in liver transplant recipients, Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society, 10 (2004) 193–198. [DOI] [PubMed] [Google Scholar]
  • [51].Vanni KMM, Berliner N, Paynter NP, Glynn RJ, MacFadyen J, Colls J, Lu F, Xu C, Ridker PM, Solomon DH, Adverse Effects of Low-Dose Methotrexate in a Randomized Double-Blind PlaceboControlled Trial: Adjudicated Hematologic and Skin Cancer Outcomes in the Cardiovascular Inflammation Reduction Trial, ACR Open Rheumatology, 2 (2020) 697–704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [52].Boerbooms AM, Kerstens PJ, van Loenhout JW, Mulder J, van de Putte LB, Infections during low-dose methotrexate treatment in rheumatoid arthritis, Seminars in arthritis and rheumatism, 24 (1995) 411–421. [DOI] [PubMed] [Google Scholar]
  • [53].Barz M, Luxenhofer R, Zentel R, Vicent MJ, Overcoming the PEG-addiction: well-defined alternatives to PEG, from structure-property relationships to better defined therapeutics, Polymer Chemistry, 2 (2011) 1900–1918. [Google Scholar]
  • [54].Ivens IA, Achanzar W, Baumann A, Brändli-Baiocco A, Cavagnaro J, Dempster M, Depelchin BO, Irizarry Rovira AR, Dill-Morton L, Lane JH, Reipert BM, Salcedo T, Schweighardt B, Tsuruda LS, Turecek PL, Sims J, PEGylated Biopharmaceuticals: Current Experience and Considerations for Nonclinical Development, Toxicologic pathology, 43 (2015) 959–983. [DOI] [PubMed] [Google Scholar]
  • [55].Bendele A, Seely J, Richey C, Sennello G, Shopp G, Short Communication: Renal Tubular Vacuolation in Animals Treated with Polyethylene-Glycol-Conjugated Proteins, Toxicological Sciences, 42 (1998) 152–157. [DOI] [PubMed] [Google Scholar]
  • [56].Bavli Y, Winkler I, Chen BM, Roffler S, Cohen R, Szebeni J, Barenholz Y, Doxebo (doxorubicinfree Doxil-like liposomes) is safe to use as a pre-treatment to prevent infusion reactions to PEGylated nanodrugs, Journal of controlled release : official journal of the Controlled Release Society, 306 (2019) 138–148. [DOI] [PubMed] [Google Scholar]
  • [57].Ishida T, Kiwada H, [Accelerated blood clearance (ABC) phenomenon induced by administration of PEGylated liposome], Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan, 128 (2008) 233–243. [DOI] [PubMed] [Google Scholar]
  • [58].Wang X, Ishida T, Kiwada H, Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes, Journal of controlled release : official journal of the Controlled Release Society, 119 (2007) 236–244. [DOI] [PubMed] [Google Scholar]
  • [59].Davies B, Morris T, Physiological Parameters in Laboratory Animals and Humans, Pharmaceutical Research, 10 (1993) 1093–1095. [DOI] [PubMed] [Google Scholar]
  • [60].Brown RP, Delp MD, Lindstedt SL, Rhomberg LR, Beliles RP, Physiological parameter values for physiologically based pharmacokinetic models, Toxicology and industrial health, 13 (1997) 407–484. [DOI] [PubMed] [Google Scholar]
  • [61].Kaliss N, Pressman D, Plasma and blood volumes of mouse organs, as determined with radioactive iodoproteins, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.), 75 (1950) 16–20. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

1

Figure S.1. Distribution of Induced APA in Mice. Distribution (mean and SD) of induced (A) IgG and (B) IgM levels in all mice. IgG and IgM levels were not statistically different between the two groups (two-tailed Welch’s t-test).

NIHMS1741352-supplement-1.png (70.7KB, png)

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