Site-specific PEGylation Research Articles

Computer-Aided Site-Specific PEGylation of PET Hydrolases for Enhanced PET Degradation.

The enormous accumulation of poly(ethylene terephthalate) (PET) waste has posed a serious threat to the environment and human health, and biodegradation with PET hydrolase (PETase) can be a possible solution. Herein, we propose site-specifically modifying PETase with amphiphilic polymers to improve the enzyme performance at ambient temperature. For this purpose, we devise a computer-aided strategy to prioritize the conjugation site, and polyethylene glycol (PEG) preparations of 0.55 to 10 kDa are site-specifically conjugated to PETase. The most active conjugate PETase-PEG 5k (PETase-5K) shows an increase of melting temperature (3.88 °C) and significantly improves PET degradation performance (3.5- and 3.1-fold increases at 30 and 40 °C, respectively). Experimental investigation and molecular dynamics simulations reveal that the site-specific PEGylation increases the hydrophobic solvent-accessible surface area and the binding capability to the PET surface, thickens the hydration layer, increases the intramolecular hydrogen bonding, reduces the interactions between water and the conjugated enzyme surface, and rigidifies the enzyme structure via hydrogen bonding and hydrophobic interactions between the polymer and the enzyme, thus leading to improved enzymatic performance of PETase-5K. We further validate the versatility of the site-specific PEGylation in one of the most evolved variants of PETase, FAST-PETase, by 1.8-fold improvement in PET degradation at 30 °C. The presented computer-aided site-specific conjugation strategy has opened a new avenue to enhancing PETase performance at ambient temperature, and the contribution of PEGylation to PETase unraveled in this work laid a foundation for the rational engineering of PET hydrolases.

A CD25-biased interleukin-2 for autoimmune therapy engineered via a semi-synthetic organism

BackgroundNatural cytokines are poorly suited as therapeutics for systemic administration due to suboptimal pharmacological and pharmacokinetic (PK) properties. Recombinant human interleukin-2 (rhIL-2) has shown promise for treatment of autoimmune (AI) disorders yet exhibits short systemic half-life and opposing immune responses that negate an appropriate therapeutic index.MethodsA semi-synthetic microbial technology platform was used to engineer a site-specifically pegylated form of rhIL-2 with enhanced PK, specificity for induction of immune-suppressive regulatory CD4 + T cells (Tregs), and reduced stimulation of off-target effector T and NK cells. A library of rhIL-2 molecules was constructed with single site-specific, biorthogonal chemistry-compatible non-canonical amino acids installed near the interface where IL-2 engages its cognate receptor βγ (IL-2Rβγ) signaling complex. Biorthogonal site-specific pegylation and functional screening identified variants that retained engagement of the IL-2Rα chain with attenuated potency at the IL-2Rβγ complex.ResultsPhenotypic screening in mouse identifies SAR444336 (SAR’336; formerly known as THOR-809), rhIL-2 pegylated at H16, as a potential development candidate that specifically expands peripheral CD4+ Tregs with upregulation of markers that correlate with their suppressive function including FoxP3, ICOS and Helios, yet minimally expands CD8 + T or NK cells. In non-human primate, administration of SAR’336 also induces dose-dependent expansion of Tregs and upregulated suppressive markers without significant expansion of CD8 + T or NK cells. SAR’336 administration reduces inflammation in a delayed-type hypersensitivity mouse model, potently suppressing CD4+ and CD8 + T cell proliferation.ConclusionSAR’336 is a specific Treg activator, supporting its further development for the treatment of AI diseases.

An imbalanced GLP-1R/GIPR co-agonist peptide with a site-specific N-terminal PEGylation to maximize metabolic benefits

Glycemic and body weight control gained from GLP-1R agonists remains an unmet need for diabetes and obesity treatment, leading to the development of GLP-1R/GIPR co-agonists. An imbalance in GLP-1R/GIPR agonism may extensively maximize the glucose- and weight-lowering effects. Hence, we prepared a potent and imbalanced GLP-1R/GIPR co-agonist, and refined its action time through a site-specific N-terminal PEGylation strategy. The pharmacological efficacy of these resulting long-acting co-agonists was interrogated both invitro and invivo. The results showed that peptide 1 possessed potent and imbalanced receptor-stimulating potency favoring GIP activity, but its hypoglycemic action was disrupted probably resulting from its short half-life. After PEGylation to improve the pharmacokinetics, the pharmacological effects were amplified compared to native peptide 1. Among the resulting derivatives, D-5K exhibited significant glycemic, HbA1c, body-weight, and food-intake control, outperforming GLP-1R mono-agonists. Based on its excellent pharmacological profiles, D-5K may hold the great therapeutic potential for diabetes and obesity treatment.

A biotechnological approach for the production of new protein bioplastics.

The future of biomaterial production will leverage biotechnology based on the domestication of cells as biological factories. Plants, algae, and bacteria can produce low-environmental impact biopolymers. Here, two strategies were developed to produce a biopolymer derived from a bioengineered vacuolar storage protein of the common bean (phaseolin; PHSL). The cys-added PHSL* forms linear-structured biopolymers when expressed in the thylakoids of transplastomic tobacco leaves by exploiting the formation of inter-chain disulfide bridges. The same protein without signal peptide (ΔPHSL*) accumulates in Escherichia coli inclusion bodies as high-molar-mass species polymers that can subsequently be oxidized to form disulfide crosslinking bridges in order to increase the stiffness of the biomaterial, a valid alternative to the use of chemical crosslinkers. The E. coli cells produced 300 times more engineered PHSL, measured as percentage of total soluble proteins, than transplastomic tobacco plants. Moreover, the thiol groups of cysteine allow the site-specific PEGylation of ΔPHSL*, which is a desirable functionality in the design of a protein-based drug carrier. In conclusion, ΔPHSL* expressed in E. coli has the potential to become an innovative biopolymer.

Multiple Mutations on α, β and γ Domains of Streptokinase Lead to the Generation of Highly Efficient Cysteine Analogues with Promising Features.

Streptokinase, one of the most widely used thrombolytic medicines, is a favorable protein for site-specific PEGylation as it lacks any cysteine residues in its amino acid sequence; however, any changes in the protein's structure should be carefully planned to avoid undesired changes in its function. This study aimed to design and produce novel di/tri-cysteine variants of streptokinase from previously developed cysteine analogues, Arg45, Glu263, and Arg319, as candidates for multiple site-specific PEGylation. Using bioinformatics tools and site-directed mutagenesis, we incorporated concurrent mutations at Arg45, Glu263, and Arg319 (carried out in our previous study) to create di/tri-cysteine variants of streptokinase proteins (SK45-319cys, SK263-319cys, and SK45-263-319cys) and evaluated their kinetic activity parameters by a colorimetric method, using H-D-Val-Leu-Lys-pNA.2HCl (S2251) as substrate. Based on the kinetic results, SK263-319cys with 44% enzyme efficiency increment compared to wild-type SK was the superior protein in terms of activity; as well, SK45-319cys and SK45-263-319cys showed 17 and 22% activity enhancement, respectively. Docking of the mutant streptokinase proteins with μ-plasmin demonstrated that changes in intermolecular interactions caused by amino acid substitution could be the reason for activity difference. The novel mutant proteins created in this study exhibit remarkable biological activity and may be uniquely suitable for simultaneous PEGylation on two/three domains. As well, PEGylated derivates of these variants might prove to be more proficient proteins, compared to the singlecysteine analogs of streptokinase; because of their more surface coverage and increased molecular weight.

Modified Asparaginase for Treatment of Cancer Historical Appraisal and Future Perspectives

Abstract: Asparaginase (ASNase) is widely used as an important component of first-line treatment for acute lymphoblastic leukemia (ALL). Although it is associated with a high rate of complete remission (~93%), challenges remain due to several side effects ranging from immune reactions to severe toxicity, largely associated with its higher immunogenicity and glutamine coactivity. Innovative products have therefore been devised to minimise these adverse reactions while increasing the enzymes’ pharmacokinetic properties, stability, and efficacy. This review focuses on commercially available formulations and others that remain in development, discussing the most recent strategies for preparing alternative formulations of the enzyme to be less immunogenic and have low glutaminase coactivity by using site-specific mutagenesis, PEGylation, and encapsulation techniques.

Site-specific N-terminal PEGylation-based controlled release of biotherapeutics: An application for GLP-1 delivery to improve pharmacokinetics and prolong hypoglycemic effects

PEGylation is a well-established technology for half-life extension in drug delivery. In this study, we aimed to develop a site-specific N-terminal PEGylation for biotherapeutics to achieve controlled release, using GLP-1 as a model. An additional threonine was introduced at N-terminal GLP-1. Followed by periodate oxidation, hydrazide-based PEGylation was achieved in a site-selective manner under reductive condition. Two homogenous monovalent mPEG5k-GLP-1 (peptide 4) and mPEG20k-GLP-1 (peptide 5) were successfully constructed. After PEGylation, the degradation by DPP-IV and rat plasma was obviously reduced. Their pharmacokinetic performances were enhanced at the expense of impaired GLP-1R stimulating potency, and their hypoglycemic effects were improved in different degrees. Compared with conventional strategies, this approach is devoid of the restriction and alteration of native peptide sequences, and can produce utterly homogenous conjugates with excellent selectivity and efficiency. It provides a practical controlled release approach for peptides by site-specific modification to achieve better pharmacological and therapeutic properties.

Polymer selection impacts the pharmaceutical profile of site-specifically conjugated Interferon-α2a.

Conjugation of poly(ethylene glycol) (PEG) to biologics is a successful strategy to favorably impact the pharmacokinetics and efficacy of the resulting bioconjugate. We compare bioconjugates synthesized by strain-promoted azide-alkyne cycloaddition (SPAAC) using PEG and linear polyglycerol (LPG) of about 20kDa or 40kDa, respectively, with an azido functionalized human Interferon-α2a (IFN-α2a) mutant. Site-specific PEGylation and LPGylation resulted in IFN-α2a bioconjugates with improved in vitro potency compared to commercial Pegasys. LPGylated bioconjugates had faster disposition kinetics despite comparable hydrodynamic radii to their PEGylated analogues. Overall exposure of the PEGylated IFN-α2a with a 40kDa polymer exceeded Pegasys, which, in return, was similar to the 40kDa LPGylated conjugates. The study points to an expanded polymer design space through which the selected polymer class may result in a different distribution of the studied bioconjugates.

Recombinant Expression, Purification and PEGylation of DNA Ligases

Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are commonly used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5'-phosphorylated end to a 3'-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source. The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases. We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins wereconjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected to various PEGylation conditions. In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-foldfor plasmid ligations and reduced the formation of protein aggregates. Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases.

Bioorthogonal PEGylation Prolongs the Elimination Half-Life of N-TIMP2 While Retaining MMP Inhibition.

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of the matrix metalloproteinase (MMP) family of proteins, whose members are key regulators of the proteolysis of extracellular matrix components and hence of multiple biological processes. In particular, imbalanced activity of matrix metalloproteinase-14 (MMP-14) may lead to the development of cancer and cardiovascular and other diseases. This study aimed to engineer TIMP2, one of the four homologous TIMPs, as a potential therapeutic by virtue of its ability to bind to the active-site Zn2+ of MMP-14. However, the susceptibility to degradation of TIMP2 and its small size, which results in a short circulation half-life, limit its use as a therapeutic. PEGylation was thus used to improve the pharmacokinetic profile of TIMP2. PEGylation of the MMP-targeting N-terminal domain of TIMP2 (N-TIMP2), via either cysteine or lysine residues, resulted in a significant decrease in N-TIMP2 affinity toward MMP-14 or multisite conjugation and conjugate heterogeneity, respectively. Our strategy designed to address this problem was based on incorporating a noncanonical amino acid (NCAA) into N-TIMP2 to enable site-specific mono-PEGylation. The first step was to incorporate the NCAA propargyl lysine (PrK) at position S31 in N-TIMP2, which does not interfere with the N-TIMP2-MMP-14 binding interface. Thereafter, site-specific PEGylation was achieved via a click chemistry reaction between N-TIMP2-S31PrK and PEG-azide-20K. Inhibition studies showed that PEGylated N-TIMP2-S31PrK did indeed retain its inhibitory activity toward MMP-14. The modified protein also showed improved serum stability vs non-PEGylated N-TIMP2. In vivo pharmacokinetic studies in mice revealed a significant 8-fold increase in the elimination half-life of PEGylated N-TIMP2 vs the non-PEGylated protein. This study shows that site-specific bioorthogonal mono-PEGylation extends the half-life of N-TIMP2 without impairing its biological activity, thereby highlighting the advantage of this strategy for generating potent PEGylated proteins.

Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability.

Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30–40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C.

Assessing site-specific PEGylation of TEM-1 β-lactamase with cell-free protein synthesis and coarse-grained simulation

PEGylation is a broadly used strategy to enhance the pharmacokinetic properties of therapeutic proteins. It is well established that the location and extent of PEGylation have a significant impact on protein properties. However, conventional PEGylation techniques have limited control over PEGylation sites. Emerging site-specific PEGylation technology provides control of PEG placement by conjugating PEG polymers via click chemistry reaction to genetically encoded non-canonical amino acids. Unfortunately, a method to rapidly determine the optimal PEGylation location has yet to be established. Here we seek to address this challenge. In this work, coarse-grained molecular dynamic simulations are paired with high-throughput experimental screening utilizing cell-free protein synthesis to investigate the effect of site-specific PEGylation on the two-state folder protein TEM-1 β-lactamase. Specifically, the conjugation efficiency, thermal stability, and enzymatic activity are studied for the enzyme PEGylated at several different locations. The results of this analysis confirm that the physical properties of the PEGylated protein vary considerably with PEGylation site and that traditional design recommendations are insufficient to predict favorable PEGylation sites. In this study, the best predictor of the most favorable conjugation site is coarse-grained simulation. Thus, we propose a dual combinatorial screening approach in which coarse-grained molecular simulation informs site selection for high-throughput experimental verification.

Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells.

The preferential activation of regulatory T (Treg) cells by interleukin-2 (IL-2), which selectively binds to the trimeric IL-2 receptor (IL-2R) on Treg cells, makes this cytokine a promising therapeutic for the treatment of autoimmune diseases. However, IL-2 has a narrow therapeutic window and a short half-life. Here, we show that the pharmacokinetics and half-life of IL-2 can be substantially improved by orthogonally conjugating the cytokine to poly(ethylene glycol) (PEG) moieties via a copper-free click reaction through the incorporation of azide-bearing amino acids at defined sites. Subcutaneous injection of a PEGylated IL-2 that optimally induced sustained Treg-cell activation and expansion over a wide range of doses through highly selective binding to trimeric IL-2R led to enhanced therapeutic efficacy in mouse models of lupus, collagen-induced arthritis and graft-versus-host disease without compromising the immune defences of the host against viral infection. Site-specific PEGylation could be used more generally to engineer cytokines with improved therapeutic performance for the treatment of autoimmune diseases.

Pyridine Borane as Alternative Reducing Agent to Sodium Cyanoborohydride for the PEGylation of L-asparaginase.

PEGylation is a reductive alkylation of a protein N-terminal/α-amine of protein with mPEG chain by reducing agent. To obtain quantitative and site-specific PEGylation, sodium cyanoborohydride is commonly used as a reducing agent. The reduction process of sodium cyanoborohydride produces highly poisonous hydrogen cyanide, which may render the final product toxic. Herein, we have studied various reducing agents such as dimethylamine borane, triethylamine borane, trimethylamine borane, pyridine borane, morpholine borane, 2-picoline borane, and 5-ethyl-2-methyl-pyridine borane were tested as alternatives to sodium cyanoborohydride for the PEGylation of L-asparaginase. The characterization of reacted pegaspargase was carried out by SDS-PAGE, Western blotting, SEC-HPLC, RP-HPLC, SEC-MALS, CD, enzyme activity, and cell proliferation assays using with lymphoblast cells and MTS/PMS as substrate. Pyridine borane was determined to be the best acceptable reducing agent for PEGylation in terms of purity and activity. As a result, instead of sodium cyanoborohydride, pyridine borane can be employed.

Enhanced Bioactivity of a Human GHR Antagonist Generated by Solid-Phase Site-Specific PEGylation.

Growth hormone (GH) has been implicated in cancer progression andis a potential target for anticancer therapy. Currently, pegvisomant is the only GH receptor (GHR) antagonist approved for clinical use. Pegvisomant is a mutated GH molecule (B2036) which is PEGylated on amine groups to extend serum half-life. However, PEGylation significantly reduces the bioactivity of the antagonist in mice. To improve bioactivity, we generated a series of B2036 conjugates with the site-specific attachment of 20, 30, or 40 kDa methoxyPEG maleimide (mPEG maleimide) by introduction of a cysteine residue at amino acid 144 (S144C). Recombinant B2036-S144C was expressed in Escherichia coli, purified, and then PEGylated using cysteine-specific conjugation chemistry. To avoid issues with dimerization due to the introduced cysteine, B2036-S144C was PEGylated while immobilized on an Ni-nitrilotriacetic (Ni-NTA) acid column, which effectively reduced disulfide-mediated dimer formation and allowed efficient conjugation to mPEG maleimide. Following PEGylation, the IC50 values for the 20, 30, and 40 kDa mPEG maleimide B2036-S144C conjugates were 66.2 ± 3.8, 106.1 ± 7.1, and 127.4 ± 3.6 nM, respectively. The circulating half-life of the 40 kDa mPEG conjugate was 58.3 h in mice. Subcutaneous administration of the 40 kDa mPEG conjugate (10 mg/kg/day) reduced serum insulin-like growth factor I (IGF-I) concentrations by 50.6%. This in vivo reduction in serum IGF-I was at a considerably lower dose compared to the higher doses required to observe comparable activity in studies with pegvisomant. In conclusion, we have generated a novel PEGylated GHR antagonist by the solid-phase site-specific attachment of mPEG maleimide at an introduced cysteine residue, which effectively reduces serum IGF-I in vivo.

Development of Site-Specific PEGylated Granulocyte Colony Stimulating Factor With Prolonged Biological Activity.

Currently, amino-terminal PEGylated human granulocyte colony stimulating factor (huG-CSF) is used to prevent and treat neutropenia. Although huG-CSF has been used as a drug for more than 20 years, it has three significant drawbacks: (i) it relies on PEG aldehyde for PEGylation of the alpha-amino group of the first amino acid, and this leads to non-specific PEGylation of the epsilon amino group of lysine residues within the G-CSF; (ii) longer-acting G-CSF variants are desirable to reduce the risk of chemotherapy-associated neutropenia; and (iii) G-CSF cannot be administered on the day of chemotherapy. In an attempt to overcome the above drawbacks, we engineered cysteine variants of G-CSF to facilitate the maleimide PEG-based site-specific PEGylation that leads to a highly homogenous PEGylated product. Importantly, we have demonstrated that 20 kDa thiol-reactive PEG conjugated by maleimide chemistry to the Cys2 G-CSF variant exhibits leukocyte proliferative activity similar to that of the commercially available G-CSF conjugated with aldehyde PEG in a neutropenia mice model. Moreover, we have demonstrated that PEGylation of the cysteine variant of huG-CSF with higher molecular weight PEGs, such as 30 kDa PEG and 40 kDa PEG, leads to significantly prolonged leukocyte proliferation activity compared to the variant conjugated with 20 kDa PEG. Importantly, even a half-dose of the engineered variant conjugated with 40 kDa PEG exhibited significantly longer biological activity than the commercially available 20 kDa PEGylated huG-CSF. Finally, we have demonstrated that administration of the engineered variant conjugated with 40 kDa PEG on the day of administration of cyclophosphamide for inducing neutropenia in mice can alleviate neutropenia through leukocyte proliferation. In summary, this study provides the design of site-specific PEGylated huG-CSF variants with improved therapeutic potential. It opens the possibility of long-acting and same-day prophylactic administration of G-CSF after chemotherapy drug regimens. These results may pave the way for the development of potential G-CSF derivatives possessing longer half-lives and favorable clinical attributes.

Genetic Code Expansion Enables Site-Specific PEGylation of a Human Growth Hormone Receptor Antagonist through Click Chemistry.

Regulation of human growth hormone (GH) signaling has important applications in the remediation of several diseases including acromegaly and cancer. Growth hormone receptor (GHR) antagonists currently provide the most effective means for suppression of GH signaling. However, these small 22 kDa recombinantly engineered GH analogues exhibit short plasma circulation times. To improve clinical viability, between four and six molecules of 5 kDa poly(ethylene glycol) (PEG) are nonspecifically conjugated to the nine amines of the GHR antagonist designated as B2036 in the FDA-approved therapeutic pegvisomant. PEGylation increases the molecular weight of B2036 and considerably extends its circulation time, but also dramatically reduces its bioactivity, contributing to high dosing requirements and increased cost. As an alternative to nonspecific PEGylation, we report the use of genetic code expansion technology to site-specifically incorporate the unnatural amino acid propargyl tyrosine (pglY) into B2036 with the goal of producing site-specific protein-polymer conjugates. Substitution of tyrosine 35 with pglY yielded a B2036 variant containing an alkyne functional group without compromising bioactivity, as verified by a cellular assay. Subsequent conjugation of 5, 10, and 20 kDa azide-containing PEGs via the copper-catalyzed click reaction yielded high purity, site-specific conjugates with >89% conjugation efficiencies. Site-specific attachment of PEG to B2036 is associated with substantially improved in vitro bioactivity values compared to pegvisomant, with an inverse relationship between polymer size and activity observed. Notably, the B2036-20 kDa PEG conjugate has a molecular weight comparable to pegvisomant, while exhibiting a 12.5 fold improvement in half-maximal inhibitory concentration in GHR-expressing Ba/F3 cells (103.3 nM vs 1289 nM). We expect that this straightforward route to achieve site-specific GHR antagonists will be useful for GH signal regulation.

Investigating the clearance of VWF A‐domains using site‐directed PEGylation and novel N‐linked glycosylation

BackgroundPrevious studies have demonstrated that the A1A2A3 domains of von Willebrand factor (VWF) play a key role in regulating macrophage‐mediated clearance in vivo. In particular, the A1‐domain has been shown to modulate interaction with macrophage low‐density lipoprotein receptor‐related protein‐1 (LRP1) clearance receptor. Furthermore, N‐linked glycans within the A2‐domain have been shown to protect VWF against premature LRP1‐mediated clearance. Importantly, however, the specific regions within A1A2A3 that enable macrophage binding have not been defined. Objective and MethodsTo address this, we utilized site‐directed PEGylation and introduced novel targeted N‐linked glycosylation within A1A2A3‐VWF and subsequently examined VWF clearance. ResultsConjugation with a 40‐kDa polyethylene glycol (PEG) moiety significantly extended the half‐life of A1A2A3‐VWF in VWF−/− mice in a site‐specific manner. For example, PEGylation at specific sites within the A1‐domain (S1286) and A3‐domain (V1803, S1807) attenuated VWF clearance in vivo, compared to wild‐type A1A2A3‐VWF. Furthermore, PEGylation at these specific sites ablated binding to differentiated THP‐1 macrophages and LRP1 cluster II and cluster IV in‐vitro. Conversely, PEGylation at other positions (Q1353‐A1‐domain and M1545‐A2‐domain) had limited effects on VWF clearance or binding to LRP1.Novel N‐linked glycan chains were introduced at N1803 and N1807 in the A3‐domain. In contrast to PEGylation at these sites, no significant extension in half‐life was observed with these N‐glycan variants. ConclusionsThese novel data demonstrate that site specific PEGylation but not site specific N‐glycosylation modifies LRP1‐dependent uptake of the A1A2A3‐VWF by macrophages. This suggests that PEGylation, within the A1‐ and A3‐domains in particular, may be used to attenuate LRP1‐mediated clearance of VWF.

SAT-283 Generation of a Long-Acting Human Growth Hormone Receptor Antagonist by Site-Specific Pegylation

Growth hormone (GH) is a peptide hormone that mediates actions through binding to a cell surface GH receptor (GHR), activating key signalling pathways including the JAK/STAT pathway. Excess GH secretion leads to acromegaly and tumoral expression has been implicated in cancer progression, suggesting that GH is also a potential target for anticancer therapy. Pegvisomant is the only GHR antagonist approved for clinical use. This antagonist is a PEGylated form of a mutated GH (B2036) that binds and blocks the receptor. Conjugation to polyethylene glycol (PEG) at multiple amine residues reduces in vitro bioactivity but extends the serum half-life resulting in improved in vivo bioactivity. We investigated whether we could generate a long-acting PEGylated GHR antagonist through site-specific conjugation of PEG. A codon optimised GHR antagonist, with an introduced free cysteine residue at amino acid site 144 (S144C), was generated by gene synthesis and recombinantly engineered by gene fusion with thioredoxin. Recombinant protein was expressed in E. coli and purified using a series of chromatographic methods. Antagonists were PEGylated using cysteine-specific conjugation chemistry. In vitro activity was determined using a Ba/F3-GHR viability assay, and in vivo pharmacokinetic and bioactivity was determined in mice. Fusion to thioredoxin was found to improve soluble protein expression at 30℃, resulting in dramatically increased yield. After a series of purification steps, including Ni-NTA, 3C protease cleavage and ion-exchange chromatography, a single band with a molecular mass of 22 kDa was observed by SDS-PAGE analysis. The recombinant antagonist was conjugated to 20 kDa or 30 kDa-PEG at amino acid site S144C. After purification, a single band with an effective molecular size of approximately 60 kDa (PEG-20kDa conjugate) or 70 kDa (PEG-30kDa conjugate) was observed by SDS-PAGE analysis. The unconjugated antagonist inhibited the proliferation of Ba/F3-GHR cells in a dose-dependent manner with a half maximal inhibitory concentration (IC50) of 10.1 ± 2.5 nM. Following PEGylation and purification, the PEG-20kDa and PEG-30kDa conjugates retained high in vitro bioactivity with an IC50 of 66.2 ± 3.8 nM and 106.1 ± 7.1 nM, respectively. Pharmacokinetic analysis demonstrated that PEGylation increased the serum half-life to approximately 15 hours in mice. Subcutaneous administration of the PEG-30kDa conjugate (10 mg/kg/day) reduced serum IGF-I levels in mice. In conclusion, we have generated a novel long-acting human GHR antagonist conjugate by introducing a free cysteine at a non-essential site of the antagonist and targeted attachment of PEG.

Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.