Conjugation to 10 kDa Linear PEG Extends Serum Half-Life and Preserves the Receptor-Binding Ability of mmTRAIL with Minimal Stimulation of PEG-Specific Antibodies

Nie, Qianxue; Jia, Dianlong; Yang, Hao; Feng, Yanru; Fan, Qi-Wen; Shi, Qiuxiao; Wan, Lin; Lu, Xiaofeng

doi:10.1021/acs.molpharmaceut.6b00964

Cited by 11 publications

(10 citation statements)

References 29 publications

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“…One disadvantage of nanobodies for prevention and treatment of viral disease is their short serum half-life and rapid renal clearance. Though we did not evaluate the serum half-life of 4-arm PEG10K conjugated nanobodies here, PEG conjugation can significantly increase serum half-life of molecules, including antibodies [44,45]. This would provide an additional, significant benefit over and above improved neutralization potency.…”

Section: Discussionmentioning

confidence: 97%

Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs

et al. 2020

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Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC50 in the low picomolar range.

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Section: Discussionmentioning

confidence: 97%

Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs

et al. 2020

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“…Therefore, PEGylation creates a steric shield surrounding the particle, reducing opsonization and subsequent clearance by the reticuloendothelial system (Butcher, et al 2016). This approach has been used to endow therapeutic proteins (Hirotsu, et al 2017, Liu, et al 2011, Nie, et al 2017), drug-carrying nanoparticles (Haynes and Huang 2016, Liu, et al 2017, Salmaso and Caliceti 2013), and imaging contrast agents (Borden, et al 2006, Garg, et al 2013, Malinge, et al 2017, Zhou, et al 2016) with improved pharmacokinetics, and several of these agents have been translated to clinical use (Appis, et al 2015, Marchal, et al 2015).…”

Section: Introductionmentioning

confidence: 99%

Accelerated Clearance of Ultrasound Contrast Agents Containing Polyethylene Glycol is Associated with the Generation of Anti-Polyethylene Glycol Antibodies

Fix

Nyankima

McSweeney

et al. 2018

Ultrasound in Medicine & Biology

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Emerging evidence suggests that the immune system can recognize polyethylene glycol (PEG), leading to the accelerated blood clearance (ABC) of PEGylated particles. Our aim here was to study the generation of anti-PEG immunity and changes in PEGylated microbubble pharmacokinetics during repeated contrast-enhanced ultrasound imaging in rats. We administered homemade PEGylated microbubbles multiple times over a 28-d period and observed dramatically accelerated clearance (4.2 × reduction in half-life), which was associated with robust anti-PEG IgM and anti-PEG IgG antibody production. Dosing animals with free PEG as a competition agent before homemade PEGylated microbubble administration significantly prolonged microbubble circulation, suggesting that ABC was largely driven by circulating anti-PEG antibodies. Experiments with U.S. Food and Drug Administration-approved Definity microbubbles similarly resulted in ABC and the generation of anti-PEG antibodies. Experiments repeated with non-PEGylated Optison microbubbles revealed a slight shift in clearance, indicating that immunologic factors beyond anti-PEG immunity may play a role in ABC, especially of non-PEGylated agents.

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“…It is widely stated that PEG increases the half-life by increasing the hydrodynamic volume, and it has been shown increasing the size of PEG attached to mmTrail increases serum half-life (23). Because a change in PEG size does not account for the different biological properties of our proteins, we employed dynamic light scattering to assess the hydrodynamic volume of the proteins and found for the first time in our knowledge that the hydrodynamic size of protein can be fine-tuned depending on the position of the PEG.…”

Section: Discussionmentioning

confidence: 99%

Site-Specific PEGylation of Anti-Mesothelin Recombinant Immunotoxins Increases Half-life and Antitumor Activity

Zheng

Okada

Kobayashi

et al. 2020

Molecular Cancer Therapeutics

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Recombinant immunotoxins (RIT) are chimeric proteins containing an Fv that binds to tumor cells, fused to a fragment of Pseudomonas exotoxin (PE) that kills the cell. Their efficacy is limited by their short half-life in the circulation. Chemical modification with polyethylene glycol (PEG) is a well-established method to extend the half-lives of biologics. Our goal was to engineer RITs with an increase in half-life and high cytotoxic activity. We introduced single cysteines at different locations in five anti-mesothelin RITs and employed site-specific PEGylation to conjugate them to 20-kDa PEG. Because our previous PEGylation method using b-mercaptoethanol reduction gave poor yields of PEG-modified protein, we employed a new method using tris(2-carboxyethyl) phosphine to reduce the protein and could PEGylate RITs at approximately 90% efficiency. The new proteins retained 19% to 65% of cytotoxic activity. Although all proteins are modified with the same PEG, the radius of hydration varies from 5.2 to 7.1, showing PEG location has a large effect on protein shape. The RIT with the smallest radius of hydration has the highest cytotoxic activity. The PEGylated RITs have a 10-to 30-fold increase in halflife that is related to the increase in hydrodynamic size. Biodistribution experiments indicate that the long half-life is due to delayed uptake by the kidney. Antitumor experiments show that several PEG-RITs are much more active than unmodified RIT, and the PEG location greatly affects antitumor activity. We conclude that PEGylation is a useful approach to improve the half-life and antitumor activity of RITs.

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Conjugation to 10 kDa Linear PEG Extends Serum Half-Life and Preserves the Receptor-Binding Ability of mmTRAIL with Minimal Stimulation of PEG-Specific Antibodies

Cited by 11 publications

References 29 publications

Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs

Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs

Accelerated Clearance of Ultrasound Contrast Agents Containing Polyethylene Glycol is Associated with the Generation of Anti-Polyethylene Glycol Antibodies

Site-Specific PEGylation of Anti-Mesothelin Recombinant Immunotoxins Increases Half-life and Antitumor Activity

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