Protein Polyethylene Glycol Research Articles

Freezing-mediated formation of supraproteins using depletion forces

HypothesisLong-acting formulations such as microparticles, injectable depots and implantable devices can realize spatiotemporally controlled delivery of protein drugs to extend their therapeutic in vivo half-lives. To efficiently encapsulate the protein drugs into such drug delivery systems, (sub)micron-sized protein particles are needed. The formation of micronized supraproteins can be induced through the synergistic combination of attractive depletion forces and freezing. The size of the supraproteins can be fine-tuned from submicron to several microns by adjusting the ice crystallization rate through the freeze-quench depth, which is set by the target temperature.MethodsSupraprotein micron structures were prepared from protein solutions under various conditions in the presence and absence of nonadsorbing polyethylene glycol. Scanning electron microscopy and dynamic light scattering were employed to determine the sizes of the supraproteins and real-time total internal reflection fluorescent microscopy was used to follow the supraprotein formation during freezing. The protein secondary structure was measured before and after micronization by circular dichroism. A phase diagram of a protein–polyethylene glycol mixture was theoretically predicted to investigate whether the depletion interaction can elucidate the phase behavior.FindingsMicronized protein supraparticles could be prepared in a controlled manner by rapid freeze-drying of aqueous mixtures of bovine serum albumin, horseradish peroxidase and lysozyme mixed with polyethylene glycol. Upon freezing, the temperature quench initiates a phase separation process which is reminiscent of spinodal decomposition. This demixing is subsequently arrested during droplet phase separation to form protein-rich microstructures. The final size of the generated protein microparticles is determined by a competition between phase separation and cooling rate, which can be controlled by target temperature. The experimental phase diagram of the aqueous protein–polyethylene glycol dispersion aligns with predictions from depletion theory for charged colloids and nonadsorbing polymers.

Self-Immolative Hydroxybenzylamine Linkers for Traceless Protein Modification

Traceless self-immolative linkers are widely used for the reversible modification of proteins and peptides. This article describes a new class of traceless linkers based on ortho- or para-hydroxybenzylamines. The introduction of electron-donating substituents on the aromatic core stabilizes the quinone methide intermediate, thus providing a platform for payload release that can be modulated. To determine the extent to which the electronics affect the rate of release, we prepared a small library of hydroxybenzylamine linkers with varied electronics in the aromatic core, resulting in half-lives ranging from 20 to 144 h. Optimization of the linker design was carried out with mechanistic insights from density functional theory (DFT) and the in silico design of an intramolecular trapping agent through the use of DFT and intramolecular distortion energy calculations. This resulted in the development of a faster self-immolative linker with a half-life of 4.6 h. To demonstrate their effectiveness as traceless linkers for bioconjugation, reversible protein-polyethylene glycol conjugates with a model protein lysozyme were prepared, which had reduced protein activity but recovered ≥94% activity upon traceless release of the polymer. This new class of linkers with tunable release rates expands the traceless linkers toolbox for a variety of bioconjugation applications.

Tetrafluorination of Aromatic Azide Yields a Highly Efficient Staudinger Reaction: Kinetics and Biolabeling.

The development of highly efficient bioorthogonal reactions is of paramount importance for the research fields of biomaterials and chemical biology. We found that the o,o'-difluorinated aromatic azide was able to react with triphenylphosphine to produce water-stable phosphanimine. To further improve the efficiency of this kind of nonhydrolysis Staudinger reaction, a tetrafluorinated aromatic azide was employed to develop a faster nonhydrolysis Staudinger reaction with a rate of up to 51 m-1 s-1 , as revealed by high-performance liquid chromatography (HPLC) analysis and fluorescence kinetics. As a proof-of-concept study, the highly efficient Staudinger reaction was successfully used for chemoselective fluorescence labeling of proteins and nucleic acids (DNA and RNA) as well as for protein polyethyleneglycol (PEG)ylation. We believe that this bioorthogonal reaction can provide a broadly useful tool for various bioconjugations.

Modelling of electrokinetic phenomena for capture of PEGylated ribonuclease A in a microdevice with insulating structures.

Synthesis of PEGylated proteins results in a mixture of protein-polyethylene glycol (PEG) conjugates and the unreacted native protein. From a ribonuclease A (RNase A) PEGylation reaction, mono-PEGylated RNase A (mono-PEG RNase A) has proven therapeutic effects against cancer, reason for which there is an interest in isolating it from the rest of the reaction products. Experimental trapping of PEGylated RNase A inside an electrokinetically driven microfluidic device has been previously demonstrated. Now, from a theoretical point of view, we have studied the electrokinetic phenomena involved in the dielectrophoretic streaming of the native RNase A protein and the trapping of the mono-PEG RNase A inside a microfluidic channel. To accomplish this, we used two 3D computational models, a sphere and an ellipse, adapted to each protein. The effect of temperature on parameters related to trapping was also studied. A temperature increase showed to rise the electric and thermal conductivities of the suspending solution, hindering dielectrophoretic trapping. In contrast, the dynamic viscosity of the suspending solution decreased as the temperature rose, favoring the dielectrophoretic manipulation of the proteins. Also, our models were able to predict the magnitude and direction of the velocity of both proteins indicating trapping for the PEGylated conjugate or no trapping for the native protein. In addition, a parametric sweep study revealed the effect of the protein zeta potential on the electrokinetic response of the protein. We believe this work will serve as a tool to improve the design of electrokinetically driven microfluidic channels for the separation and recovery of PEGylated proteins in one single step.

517. Improving CAR T Cell Efficacy for Solid Tumors By Nanogel-Based Delivery of Immunomodulatory Proteins

Adoptive T cell therapies utilizing T lymphocytes transduced to stably express an anti-CD19 chimeric antigen receptor (CAR) have shown remarkable success against refractory B cell malignancies in multiple clinical trials. Studies are underway to adapt this technology towards the treatment of solid tumors, however, the immunosuppressive tumor microenvironment of many solid tumors may represent a significant barrier to CAR T cell efficacy. In this study, we investigate a means to enhance CAR T cell function by loading them with nanogel “backpacks” containing the T and NK cell stimulatory molecule IL-15N72D:IL-15RaSu/Fc superagonist complex (IL-15sa, also referred to as ALT-803 and currently in clinical trials). The IL15sa nanogel is a crosslinked, protein-polyethylene glycol (PEG) complex that breaks down slowly under physiological conditions allowing for pseudoautocrine stimulation of the CAR T cells with the IL15sa cargo. The effects of IL15sa nanogel loading on CAR T cell function were tested in a glioblastoma multiforme tumor model. Human T lymphocytes were lentivirally-transduced with a CAR targeting the epidermal growth factor variant III (EGFRvIII) antigen, as is currently being tested in early human clinical trials. EGFRvIII is expressed in up to 30% of glioblastoma multiforme cells but not on normal cells. EGFRvIII CAR T cells were loaded with a fluorophore-labeled IL15sa nanogel and examined in vitro for nanogel breakdown and effects on CAR T cell functions. The nanogel was detected on labeled cells for up to five days. Nanogels loaded with either control or IL-15sa did not alter antigen-specific cytolytic activity of EGFRvIII CAR T cells towards glioblastoma target cells. EGFRvIII CAR T cells loaded with control nanogel proliferated with the same kinetics as non-loaded CAR T cells upon co-culture with EGFRvIII+ glioblastoma targets. In contrast, IL15sa nanogel loaded CAR T cells had increased proliferation upon co-culture with antigenbearing targets. Additionally, IL15sa nanogel loaded EGFRvIII CAR T cells proliferated more with repeated stimulation of EGFRvIII-expressing glioblastoma cells. Studies are currently underway to test the efficacy of IL15sa nanogel-loaded EGFRvIII CAR T cells in vivo using a xenogeneic murine model of glioblastoma. These results indicate that nanogel loading of T cells can be a viable way of delivering immunostimulatory agents with CAR T cells. Nanogel loading of immune-modulating or tumoricidal drugs coupled to CAR T cells represents a novel platform for enhancing T cell function, and potentially for delivering systemically toxic drugs, into a specific and hostile tumor microenvironment.

Abstract 2792: Engineering T lymphocytes with protein nanogels for cancer immunotherapy

Abstract Adoptive cell transfer (ACT) with autologous tumor-reactive T-lymphocytes is a promising strategy in cancer immunotherapy, but treatment of solid tumors is limited by the rapid decline in function of the transplanted T cells. In order to maintain high numbers of viable tumor-specific cytotoxic T cells in tumors, co-administration of supporting immunostimulant agents together with transferred cells is typically necessary in clinical practice. When given systemically at high doses, these agents could enhance the in vivo viability of donor cells, improve their therapeutic function and lead to overall improved efficacy against cancer. However, high doses of such agents can also result in serious side effects. For example, the use of Interleukin-2 (IL-2) as an adjuvant drug significantly enhances the efficacy of ACT in melanoma, where IL-2 provides key adjuvant signals to donor T cells but also elicits severe dose-limiting inflammatory toxicity and expands regulatory T cells (Tregs). One approach to focus adjuvant drug action on the transferred cells and lower systemic toxicity is to genetically engineer donor cells to secrete their own supporting factors. However, the technical difficulty of engineering safe autocrine production of potent adjuvant molecules such as cytokines is a challenge. Thus, an inexpensive technology platform that would allow simple, safe and efficient delivery of adjuvant drugs specifically to therapeutic cells over a defined window of time could be broadly useful in ACT and other cell therapies for cancer. Here, we developed a novel strategy to deliver therapeutic proteins in T cell therapies, via the chemical conjugation of protein-polyethylene glycol (PEG) nanogels (NGs) directly onto the plasma membrane of donor cells, enabling continuous pseudoautocrine release of cargo proteins for stimulation of transferred T-cells. These PEG NGs loaded with IL-2 contained very high level of drug (∼80 wt% of NG was the protein cargo) and released native IL-2 in physiological conditions in a sustained manner through breakdown of a degradable linker between the IL-2 cargo and PEG chains of the NG. T-cells with optimized number of NGs conjugated per cell maintained their cellular functions and cancer targeting and killing capability. We demonstrated that this IL2-PEG NG-T cell conjugation strategy could augment T-cell expansion in vivo and result in markedly improved efficacy against metastatic B16F10 melanoma in mice. This approach provides a readily generalizable strategy to provide autocrine protein drug support to donor cells to enhance the safety and efficacy of ACT. Citation Format: Li Tang, Yiran Zheng, Darrell J. Irvine. Engineering T lymphocytes with protein nanogels for cancer immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2792. doi:10.1158/1538-7445.AM2014-2792

Avidity-controlled hydrogels for injectable co-delivery of induced pluripotent stem cell-derived endothelial cells and growth factors

To translate recent advances in induced pluripotent stem cell biology to clinical regenerative medicine therapies, new strategies to control the co-delivery of cells and growth factors are needed. Building on our previous work designing Mixing-Induced Two-Component Hydrogels (MITCHs) from engineered proteins, here we develop protein–polyethylene glycol (PEG) hybrid hydrogels, MITCH-PEG, which form physical gels upon mixing for cell and growth factor co-delivery. MITCH-PEG is a mixture of C7, which is a linear, engineered protein containing seven repeats of the CC43 WW peptide domain (C), and 8-arm star-shaped PEG conjugated with either one or two repeats of a proline-rich peptide to each arm (P1 or P2, respectively). Both 20kDa and 40kDa star-shaped PEG variants were investigated, and all four PEG-peptide variants were able to undergo a sol–gel phase transition when mixed with the linear C7 protein at constant physiological conditions due to noncovalent hetero-dimerization between the C and P domains. Due to the dynamic nature of the C–P physical crosslinks, all four gels were observed to be reversibly shear-thinning and self-healing. The P2 variants exhibited higher storage moduli than the P1 variants, demonstrating the ability to tune the hydrogel bulk properties through a biomimetic peptide-avidity strategy. The 20kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40kDa variants. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) adopted a well-spread morphology within three-dimensional MITCH-PEG cultures, and MITCH-PEG provided significant protection from cell damage during ejection through a fine-gauge syringe needle. In a mouse hindlimb ischemia model of peripheral arterial disease, MITCH-PEG co-delivery of hiPSC-ECs and VEGF was found to reduce inflammation and promote muscle tissue regeneration compared to a saline control.

Transactivating-transduction protein-polyethylene glycol modified liposomes traverse the blood-spinal cord and blood-brain barriers.

Naive liposomes can cross the blood-brain barrier and blood-spinal cord barrier in small amounts. Liposomes modified by a transactivating-transduction protein can deliver antibiotics for the treatment of acute bacterial infection-induced brain inflammation. Liposomes conjugated with polyethylene glycol have the capability of long-term circulation. In this study we prepared transactivating-transduction protein-polyethylene glycol-modified liposomes labeled with fluorescein isothiocyanate. Thus, liposomes were characterized by transmembrane, long-term circulation and fluorescence tracing. Uptake, cytotoxicity, and the ability of traversing blood-spinal cord and blood-brain barriers were observed following coculture with human breast adenocarcinoma cells (MCF-7). Results demonstrated that the liposomes had good biocompatibility, and low cytotoxicity when cocultured with human breast adenocarcinoma cells. Liposomes could traverse cell membranes and entered the central nervous system and neurocytes through the blood-spinal cord and blood-brain barriers of rats via the systemic circulation. These results verified that fluorescein isothiocyanate-modified transactivating-transduction protein-polyethylene glycol liposomes have the ability to traverse the blood-spinal cord and blood-brain barriers.

Rapid quantification of protein–polyethylene glycol conjugates by multivariate evaluation of chromatographic data

Size exclusion chromatography (SEC) is often applied for characterization of protein–polyethylene glycol (PEG) conjugates regarding the number of attached PEG chains (PEGamers). SEC analysis is advantageous as it is precise, robust, and straightforward to establish. However, most SEC based assays have a maximal throughput of a few samples per hour. We present a strategy to increase analytical throughput based on combining a short column with a fast flow rate, and finally multivariate calibration in order to compensate for the resolution lost in the trade off for speed. Different multivariate approaches were compared and multilinear regression was shown to result in the most precise calibrations. Further, a dynamic calibration approach was developed in order to account for changes in column performance over time. In this way, it was possible to establish a highly precise assay for protein PEGamer quantification with a throughput of 30 samples per hour.

The evolution of drug discovery: from traditional medicines to modern drugs

Foreword Preface and Acknowledgment EVOLUTION OF DRUG DISCOVERY Antiquity Great Periods of Drug Discovery Vaccines THE FIRST SYNTHETIC DRUGS AND THE BIRTH OF CHEMOTHERAPY Aspirin, Phenacetin, and Paracetamol Discovery of Pyrazolones Cocaine and Local Anesthetics Barbiturates and Related Compounds Fathers of Chemotherapy FROM CASUAL OBSERVATIONS TO SYSTEMATIC SEARCH From Antihistamines to Antipsychotics and Antidepressants: The Origins of Psychopharmacology Benzodiazepines Structural Variations on Sulfanilamide Azoles as a Source of Antibacterials, Antifungals, and Antihelmintics The Long Fight Against Cancer Sildenafil - The First Inhibitor of Rectile Dysfunction NATURE AS A SOURCE OF DRUGS: DRUGS FROM NATURAL PRODUCTS Plant Sources: Derivatives and Related Drugs (Selected Examples) Animal Sources: Derivatives and Related Drugs Drugs from Microbiological Sources Drugs from Marine Origin: The Contribution of the Sea to the Therapeutic Arsenal RATIONALIZING THE DRUG DISCOVERY PROCESS Early Rational Drug Discovery Approaches: L-Dopa and L-Alpha-Methyldopa Receptor-Based Drug Discovery Antimetabolites in the Fight Against Bacteria, Viruses, and Cancer Cyclooxygenase(COX) Inhibitors STRUCTURE-BASED DRUG DISCOVERY Searching for the Ideal Antihypertensive Agent: Renin-Angiotensin-Aldosterone System (RAAS) HIV Protease Inhibitors Drugs Effective Against the Influenza Virus (the Flu) Fight Against Cancer New Avenues for the Development of Antidiabetic Agents. Dipeptidyl-Peptidase-IV Inhibitors: Sitagliptin and Vildaglptin BIOPHARMACEUTICALS Monoclonal Antibodies (MAbs) Recombinant DNA Technology (Genetic Engineering) Some Recombinant Protein Drugs Protein-Polyethyleneglycol (PEG) Conjugates (Protein PEGylation) DRUG DISCOVERY IN THE POSTGENOMIC ERA: CONCLUDING REMARKS Chemical/Genomic Strategy Summary

Formulation, lyophilization and solid-state properties of a pegylated protein

In this paper the importance of formulation and process parameters on the solid-state properties of a lyophilized, pegylated growth hormone antagonist (pegvisomant) was studied. The degree of solid-state disorder (amorphicity), protein/polyethylene glycol (PEG)/sucrose interactions, and dissolution characteristics of the resultant cakes were examined. Using isothermal microcalorimetry (IMC) and differential scanning calorimetry (DSC), it was shown that in co-lyophilized pegylated protein/sucrose systems there was an interaction between sucrose and pegylated protein molecules. This interaction was evidenced by a decrease in the melting temperature ( T m) and melting enthalpy of PEG as a function of sucrose concentration. It was also shown that the sum of the heat of interaction with water for the individual constituents, lyophilized pegylated protein and lyophilized sucrose, was higher than the heat of interaction for the co-lyophilized system. As the concentration of sucrose was increased, the degree of solid-state disorder increased and the solid dissolved faster. A correlation was found among heat of interaction with water, degree of solid-state disorder, and dissolution time. Pegylation caused a shorter dissolution time, lower moisture content, increased amorphicity, and a more rapid moisture-induced crystallization of sucrose.

Direct Electrochemistry and Catalytic Activity of Hemoglobin and Myoglobin Entrapped in PEG Film

Direct electrochemistry and electrocatalysis of two heme proteins, hemoglobin (Hb) and myoglobin (Mb), incorporated in polyethylene glycol (PEG) films, were studied by cyclic voltammetry. The two proteins exhibited a pair of well‐defined, quasi‐reversible cyclic voltammetric peaks with the apparent formal potential at about −0.21 V (Hb) and −0.22 V (Mb), respectively, vs. saturated calomel electrode (SCE) in pH 5.0 acetate buffer solution, characteristic of the h eme Fe(III)/Fe(II) redox couples, indicating enhanced electron transfer between the proteins and the substrate electrode in the PEG film environment. The protein–PEG films could also exhibit excellent stability. Meanwhile, positions of Soret absorption band of the proteins in the PEG films suggested that the heme proteins kept their secondary structure similar to their native state in the medium pH range. Oxygen, trichloroacetic acid, nitric oxide, and hydrogen peroxide could all be catalytically reduced by Hb or Mb in PEG films.

Protein carboxyl amidation increases the potential extent of protein polyethylene glycol conjugation

Chemical coupling of polyethylene glycol (PEG) to therapeutic proteins reduces their immunogenicity and prolongs their circulating half-life. The limitation of this approach is the number and distribution of sites on proteins available for PEGylation (the N terminus and the ε-amino group of lysines). To increase the extent of PEGylation, we have developed a method to increase the number of PEGylation sites in a model protein, recombinant methionine α,γ-lyase (recombinant methioninase; rMETase), an enzyme cancer therapeutic cloned from Pseudomonas putida. rMETase was first PEGylated with methoxypolyethylene glycol succinimidyl glutarate-5000 with a molar ratio of PEG:rMETase of 15:1. The carboxyl groups of the initially PEGylated protein were then conjugated with diaminobutane, resulting in carboxyl amidation. This reaction was catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, a water-soluble carbodiimide. The steric hindrance provided by the PEG chains already coupled to the protein prevented cross-linking between rMETase molecules during the carboxyl amidation reaction. The carboxyl-amidated PEGylated rMETase was hyper-PEGylated at a molar ratio of PEG to PEG-rMETase of 60:1. Biochemical analysis indicated that 13 PEG chains were coupled to each subunit of rMETase after hyper-PEGylation compared with 6–8 PEG chains attached to the non-carboxyl-amidated PEG-rMETase. Approximately 15–20% of the non-PEGylated rMETase activity was retained in the hyper-PEGylated molecule. Immunogenicity of the hyper-PEG-rMETase was significantly reduced relative to PEG-rMETase and rMETase. Initial results suggest that hyper-PEGylation may become a new strategy for PEGylation of protein biologics.

Protein carboxyl amidation increases the potential extent of protein polyethylene glycol conjugation

Chemical coupling of polyethylene glycol (PEG) to therapeutic proteins reduces their immunogenicity and prolongs their circulating half-life. The limitation of this approach is the number and distribution of sites on proteins available for PEGylation (the N terminus and the ε-amino group of lysines). To increase the extent of PEGylation, we have developed a method to increase the number of PEGylation sites in a model protein, recombinant methionine α,γ-lyase (recombinant methioninase; rMETase), an enzyme cancer therapeutic cloned from Pseudomonas putida . rMETase was first PEGylated with methoxypolyethylene glycol succinimidyl glutarate-5000 with a molar ratio of PEG:rMETase of 15:1. The carboxyl groups of the initially PEGylated protein were then conjugated with diaminobutane, resulting in carboxyl amidation. This reaction was catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, a water-soluble carbodiimide. The steric hindrance provided by the PEG chains already coupled to the protein prevented cross-linking between rMETase molecules during the carboxyl amidation reaction. The carboxyl-amidated PEGylated rMETase was hyper-PEGylated at a molar ratio of PEG to PEG-rMETase of 60:1. Biochemical analysis indicated that 13 PEG chains were coupled to each subunit of rMETase after hyper-PEGylation compared with 6–8 PEG chains attached to the non-carboxyl-amidated PEG-rMETase. Approximately 15–20% of the non-PEGylated rMETase activity was retained in the hyper-PEGylated molecule. Immunogenicity of the hyper-PEG-rMETase was significantly reduced relative to PEG-rMETase and rMETase. Initial results suggest that hyper-PEGylation may become a new strategy for PEGylation of protein biologics.

Evaluation of nonideality from gel chromatographic partition coefficients

Frontal gel chromatography has been used to measure partition coefficients which enable a quantitative evaluation of the thermodynamic nonideality of small solutes generated by the presence of high concentrations of macromolecular solutes. Equivalence of results obtained by the present method and by equilibrium dialysis is demonstrated in a comparison of results for dextran sulfate-NaCl and dextran-sorbitol systems. Interaction coefficients obtained for dextran-sorbitol and protein-polyethylene glycol 4000 systems yields results which are in reasonable agreement with those predicted on the statistical-mechanical basis of excluded volume. Because of its greater versatility in regard to the range of systems that may be studied, the frontal gel chromatographic procedure is likely to be of particular value for the quantitative characterization of thermodynamic nonideality arising from excluded volume effects in concentrated mixtures of macromolecular solutes.