Free PEG Suppresses Anaphylaxis to PEGylated Nanomedicine in Swine.

Covalent conjugation of poly(ethylene glycol) (PEG) is frequently employed to enhance the pharmacokinetics and biodistribution of various protein and nanoparticle therapeutics. Unfortunately, some PEGylated drugs can induce elevated levels of antibodies that can bind PEG, i.e., anti-PEG antibodies (APA), in some patients. APA in turn can reduce the efficacy and increase the risks of allergic reactions, including anaphylaxis. There is currently no intervention available in the clinic that specifically mitigates allergic reactions to PEGylated drugs without the use of broad immunosuppression. We previously showed that infusion of high molecular weight free PEG could safely and effectively suppress the induction of APA in mice and restore prolonged circulation of various PEGylated therapeutics. Here, we explored the effectiveness of free PEG as a prophylaxis against anaphylaxis induced by PEG-specific allergic reactions in swine. Injection of PEG-liposomes (PL) resulted in anaphylactoid shock (pseudoanaphylaxis) within 1-3 min in both naïve and PL-sensitized swine. In contrast, repeated injection of free PEG alone did not result in allergic reactions, and injection of free PEG effectively suppressed allergic reactions to PL, including in previously PL-sensitized swine. These results strongly support the further investigation of free PEG for reducing APA and allergic responses to PEGylated therapeutics.

Development of anti-PEG IgG/IgM/IgE ELISA assays for profiling anti-PEG immunoglobulin response in PEG-sensitized individuals and patients with alpha-gal allergy.

Polyethylene glycol (PEG) is frequently used in various protein and nanomedicine therapeutics. However, various studies have shown that select PEGylated therapeutics can induce production of anti-PEG antibodies (APA), potentially culminating in rapid clearance from the systemic circulation, loss of efficacy and possibly increased risks of allergic reactions. Although IgE is a frequent cause of immediate hypersensitivity reactions (IHR), the role of IgE APA in PEG-related IHR is not well understood, due in part to a lack of standardized assays for measuring IgE APA. Here, we developed a rigorous competitive ELISA method to measure the concentrations of various APA isotypes, including IgE, with picomolar sensitivities. In a small number of serum samples from patients with known PEG allergy, the assay allowed us to detect a strong correlation between IgG and IgE APA in individuals with history of allergic reactions to PEG or PEGylated drugs, but not between IgM and IgE APA. We detected appreciable levels of IgG and IgM APA in individuals with history of alpha-gal allergy, however, they were not elevated relative to those detected in other healthy controls, and we found no pre-existing IgE APA. While preliminary and should be further investigated, these results suggest that differences in the route and mechanism of PEG exposure may drive variability in APA response.

Pegloticase co-administered with high MW polyethylene glycol effectively reduces PEG-immunogenicity and restores prolonged circulation in mouse.

The interactions between polymers and the immune system remains poorly controlled. In some instances, the immune system can produce antibodies specific to polymer constituents. Indeed, roughly half of pegloticase patients without immunomodulation develop high titers of anti-PEG antibodies (APA) to the PEG polymers on pegloticase, which then quickly clear the drug from circulation and render the gout treatment ineffective. Here, using pegloticase as a model drug, we show that addition of high molecular weight (MW) free (unconjugated) PEG to pegloticase allows us to control the immunogenicity and mitigates APA induction in mice. Compared to pegloticase mixed with saline, mice repeatedly dosed with pegloticase containing different MW or amount of free PEG possessed 4- to 12- fold lower anti-PEG IgG, and 6- to 10- fold lower anti-PEG IgM, after 3 rounds of pegloticase dosed every 2 weeks. The markedly reduced APA levels, together with competitive inhibition by free PEG, restored the prolonged circulation of pegloticase to levels observed in APA-naïve animals. In contrast, mice with pegloticase-induced APA eliminated nearly all pegloticase from the circulation within just four hours post-injection. These results support the growing literature demonstrating free PEG may effectively suppress drug-induced APA, which in turn may offer sustained therapeutic benefits without requiring broad immunomodulation. We also showed free PEG effectively blocked the PEGylated protein from binding with cells expressing PEG-specific B cell receptors. It provides a template of how we may be able to tune the interactions and immunogenicity of other polymer-modified therapeutics. STATEMENT OF SIGNIFICANCE: A major challenge with engineering materials for drug delivery is their interactions with the immune system. For instance, our body can produce high levels of anti-PEG antibodies (APA). Unfortunately, the field currently lack tools to limit immunostimulation or overcome pre-existing anti-PEG antibodies, without using broad immunosuppression. Here, we showed that simply introducing free PEG into a clinical formulation of PEG-uricase can effectively limit induction of anti-PEG antibodies, and restore their prolonged circulation upon repeated dosing. Our work offers a readily translatable method to safely and effectively restore the use PEG-drugs in patients with PEG-immunity, and provides a template to use unconjugated polymers with low immunogenicity to regulate interactions with the immune system for other polymer-modified therapeutics.

Dermal Lymphatic Capillaries Do Not Obey Murray's Law.

Lymphatic vessels serve as a major conduit for the transport of interstitial fluid, immune cells, lipids and drugs. Therefore, increased knowledge about their development and function is relevant to clinical issues ranging from chronic inflammation and edema, to cancer metastasis to targeted drug delivery. Murray's Law is a widely-applied branching rule upheld in diverse circulatory systems including leaf venation, sponge canals, and various human organs for optimal fluid transport. Considering the unique and diverse functions of lymphatic fluid transport, we specifically address the branching of developing lymphatic capillaries, and the flow of lymph through these vessels. Using an empirically-generated dataset from wild type and genetic lymphatic insufficiency mouse models we confirmed that branching blood capillaries consistently follow Murray's Law. However surprisingly, we found that the optimization law for lymphatic vessels follows a different pattern, namely a Murray's Law exponent of ~1.45. In this case, the daughter vessels are smaller relative to the parent than would be predicted by the hypothesized radius-cubed law for impermeable vessels. By implementing a computational fluid dynamics model, we further examined the extent to which the assumptions of Murray's Law were violated. We found that the flow profiles were predominantly parabolic and reasonably followed the assumptions of Murray's Law. These data suggest an alternate hypothesis for optimization of the branching structure of the lymphatic system, which may have bearing on the unique physiological functions of lymphatics compared to the blood vascular system. Thus, it may be the case that the lymphatic branching structure is optimized to enhance lymph mixing, particle exchange, or immune cell transport, which are particularly germane to the use of lymphatics as drug delivery routes.

A PBPK model recapitulates early kinetics of anti-PEG antibody-mediated clearance of PEG-liposomes.

PEGylation is routinely used to extend the systemic circulation of various protein therapeutics and nanomedicines. Nonetheless, mounting evidence is emerging that individuals exposed to select PEGylated therapeutics can develop antibodies specific to PEG, i.e., anti-PEG antibodies (APA). In turn, APA increase both the risk of hypersensitivity to the drug as well as potential loss of efficacy due to accelerated blood clearance of the drug. Despite the broad implications of APA, the timescales and systemic specificity by which APA can alter the pharmacokinetics and biodistribution of PEGylated drugs remain not well understood. Here, we developed a physiologically based pharmacokinetic (PBPK) model designed to resolve APA's impact on both early- and late-phase pharmacokinetics and biodistribution of intravenously administered PEGylated drugs. Our model accurately recapitulates PK and biodistribution data obtained from PET/CT imaging of radiolabeled PEG-liposomes and PEG-uricase in mice with and without APA, as well as serum levels of PEG-uricase in humans. Our work provides another illustration of the power of high-resolution PBPK models for understanding the pharmacokinetic impacts of anti-drug antibodies and the dynamics with which antibodies can mediate clearance of foreign species.

Experimental Data and PBPK Modeling Quantify Antibody Interference in PEGylated Drug Carrier Delivery.

Physiologically-based pharmacokinetic (PBPK) modeling is a popular drug development tool that integrates physiology, drug physicochemical properties, preclinical data, and clinical information to predict drug systemic disposition. Since PBPK models seek to capture complex physiology, parameter uncertainty and variability is a prevailing challenge: there are often more compartments (e.g., organs, each with drug flux and retention mechanisms, and associated model parameters) than can be simultaneously measured. To improve the fidelity of PBPK modeling, one approach is to search and optimize within the high-dimensional model parameter space, based on experimental time-series measurements of drug distributions. Here, we employ Latin Hypercube Sampling (LHS) on a PBPK model of PEG-liposomes (PL) that tracks biodistribution in an 8-compartment mouse circulatory system, in the presence (APA+) or absence (naïve) of anti-PEG antibodies (APA). Near-continuous experimental measurements of PL concentration during the first hour post-injection from the liver, spleen, kidney, muscle, lung, and blood plasma, based on PET/CT imaging in live mice, are used as truth sets with LHS to infer optimal parameter ranges for the full PBPK model. The data and model quantify that PL retention in the liver is the primary differentiator of biodistribution patterns in naïve versus APA+ mice, and spleen the secondary differentiator. Retention of PEGylated nanomedicines is substantially amplified in APA+ mice, likely due to PL-bound APA engaging specific receptors in the liver and spleen that bind antibody Fc domains. Our work illustrates how applying LHS to PBPK models can further mechanistic understanding of the biodistribution and antibody-mediated clearance of specific drugs.

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

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 radio-labeled 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-h 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.

A semi-automated finite difference mesh creation method for use with immersed boundary software IB2d and IBAMR.

Numerous fluid-structure interaction problems in biology have been investigated using the immersed boundary method. The advantage of this method is that complex geometries, e.g., internal or external morphology, can easily be handled without the need to generate matching grids for both the fluid and the structure. Consequently, the difficulty of modeling the structure lies often in discretizing the boundary of the complex geometry (morphology). Both commercial and open source mesh generators for finite element methods have long been established; however, the traditional immersed boundary method is based on a finite difference discretization of the structure. Here we present a software library for obtaining finite difference discretizations of boundaries for direct use in the 2D immersed boundary method. This library provides tools for extracting such boundaries as discrete mesh points from digital images. We give several examples of how the method can be applied that include passing flow through the veins of insect wings, within lymphatic capillaries, and around starfish using open-source immersed boundary software.

Ordinary Differential Equation Models for Adoptive Immunotherapy.

Modified T cells that have been engineered to recognize the CD19 surface marker have recently been shown to be very successful at treating acute lymphocytic leukemias. Here, we explore four previous approaches that have used ordinary differential equations to model this type of therapy, compare their properties, and modify the models to address their deficiencies. Although the four models treat the workings of the immune system in slightly different ways, they all predict that adoptive immunotherapy can be successful to move a patient from the large tumor fixed point to an equilibrium with little or no tumor.

Estimating Tumor Growth Rates In Vivo.

In this paper, we develop methods for inferring tumor growth rates from the observation of tumor volumes at two time points. We fit power law, exponential, Gompertz, and Spratt's generalized logistic model to five data sets. Though the data sets are small and there are biases due to the way the samples were ascertained, there is a clear sign of exponential growth for the breast and liver cancers, and a 2/3's power law (surface growth) for the two neurological cancers.