Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody

Schneeweis, Lumelle A.; Obenauer-Kutner, Linda; Kaur, Parminder; Yamniuk, Aaron P.; Tamura, James; Jaffe, Neil; O’Mara, Brian W.; Lindsay, Stuart; Doyle, Michael L.; Bryson, James W.

doi:10.1002/jps.24624

Cited by 4 publications

(6 citation statements)

References 33 publications

(86 reference statements)

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“…2 d, e). The epitope binning test of the C44 was performed against the positive control evinacumab by a dual antibody sandwich ELISA [ 48 ]. Evinacumab coated on a plate was employed to capture hANGPTL3 (S17-K170)-Fc protein, the C44-His6 could bind other epitopes on hANGPTL3 (S17-K170)-Fc (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The VHHs have prominently short circulation half-life due to their small size of 15 kDa. Multiple methods have been applied to increase their half-life period in vivo, such as binding to human serum albumin, fused to IgG-Fc and PEGylated [ 47 , 48 ]. Meanwhile, previous studies revealed that bivalent nanobody had better affinity than monovalent modality.…”

Section: Discussionmentioning

confidence: 99%

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A novel nanobody-heavy chain antibody against Angiopoietin-like protein 3 reduces plasma lipids and relieves nonalcoholic fatty liver disease

Fan

et al. 2022

J Nanobiotechnol

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Background Nonalcoholic fatty liver disease (NAFLD) is a metabolic disease mainly on account of hypercholesterolemia and may progress to cirrhosis and hepatocellular carcinoma. The discovery of effective therapy for NAFLD is an essential unmet need. Angiopoietin-like protein 3 (ANGPTL3), a critical lipid metabolism regulator, resulted in increased blood lipids and was elevated in NAFLD. Here, we developed a nanobody-heavy chain antibody (VHH-Fc) to inhibit ANGPTL3 for NAFLD treatment. Results In this study, we retrieved an anti-ANGPTL3 VHH and Fc fusion protein, C44-Fc, which exhibited high affinities to ANGPTL3 proteins and rescued ANGPLT3-mediated inhibition of lipoprotein lipase (LPL) activity. The C44-Fc bound a distinctive epitope within ANGPTL3 when compared with the approved evinacumab, and showed higher expression yield. Meanwhile, C44-Fc had significant reduction of the triglyceride (~ 44.2%), total cholesterol (~ 36.6%) and LDL-cholesterol (~ 54.4%) in hypercholesterolemic mice and ameliorated hepatic lipid accumulation and liver injury in NAFLD mice model. Conclusions We discovered a VHH-Fc fusion protein with high affinity to ANGPTL3, strong stability and also alleviated the progression of NAFLD, which might offer a promising therapy for NAFLD. Graphical Abstract

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A novel nanobody-heavy chain antibody against Angiopoietin-like protein 3 reduces plasma lipids and relieves nonalcoholic fatty liver disease

Fan

et al. 2022

J Nanobiotechnol

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“…Multiple methods have been applied to bene t its half-life period in vivo, such as VHHs can be conjugated with human serum albumin, fused to IgG-Fc and PEGylated. [46][47][48] Meanwhile, previous studies revealed that bivalent nanobody had better a nity than monovalent modality. In particular, the bivalent VHHs fused with Fc showed above 10 times higher a nity and stronger therapeutic effects than monovalent in vitro and in vivo, which also emerged longer half-life of about 15 days when compared to that of monovalent (~30 min) and tandem bivalent VHHs (~60 min).…”

Section: Discussionmentioning

confidence: 99%

A Novel Nanobody-Heavy Chain Antibody Against Angiopoietin-Like Protein 3 Reduces Plasma Lipids and Relieves Nonalcoholic Fatty Liver Disease

Fan

et al. 2022

Preprint

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Background Nonalcoholic fatty liver disease (NAFLD) is a metabolic disease mainly on account of hypercholesterolemia and may progresses to cirrhosis and hepatocellular carcinoma. The discovery of effective therapies for NAFLD is a critical unmet need. Angiopoietin-like protein 3 (ANGPTL3), a critical lipid metabolism regulator, cause elevated blood lipids and are elevated in NAFLD. Here, we develop a nanobody-heavy chain antibody (VHH-Fc) to inhibit ANGPTL3 for NAFLD treatment. Results In this study, we screened out a anti-ANGPTL3 VHH and Fc fusion protein, C44-Fc, which exhibited a high affinity to human and mouse ANGPTL3 proteins at 0.1402~0.2088 nM and rescued ANGPLT3-mediated inhibition of lipoprotein lipase (LPL) activity. The C44-Fc had different ANGPTL3 binding epitope, a double expression yield and better thermostability when compared with the evinacumab, maintaining no obvious degradation or aggregation at low and high pH, 40°C for 28 days, and freeze-thaw. Furthermore, C44-Fc significantly ameliorated the triglyceride (~44.2%), total cholesterol (~36.6%) and LDL-cholesterol (~54.4%) levels in hypercholesterolemic mice and ameliorated hepatic lipid accumulation and liver injury in NAFLD mice. Conclusions We discover a VHH-Fc fusion protein with high affinity to ANGPTL3 and alleviates the progression of NAFLD, which offers a prospective therapy for NAFLD.

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“…Overall, the use of single molecule methods for particle characterization and receptor binding characterization offers the ability to investigate particle structure and heterogeneity that complements the mass, kinetics, and affinity measured by ensemble [1]. PEGylation of protein therapeutic molecules is a common method for prolonging serum half-life.…”

mentioning

confidence: 99%

“…AFM recognition imaging efficiency (Figure 1) and unbinding force curves (Figure 2) provided an orthogonal method to SPR and ITC solution ensemble methods that measure the single molecule binding interaction of the dAb-1-PEG conjugate with its target antigen. Ultimately, the comparison of ensemble methods (SEC-MALS, SPR, ITC) with AFM single molecule topography and recognition imaging methods provided an improved understanding of the dAb-1-PEG conjugate particle properties and how the PEG impacts the interaction of dAb-1with the target antigen.Overall, the use of single molecule methods for particle characterization and receptor binding characterization offers the ability to investigate particle structure and heterogeneity that complements the mass, kinetics, and affinity measured by ensemble [1]. PEGylation of protein therapeutic molecules is a common method for prolonging serum half-life.…”

mentioning

confidence: 99%

Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single Domain Antibody

Obenauer-Kutner

Schneeweis²

2018

Microsc Microanal

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Domain antibodies (dAb) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigated the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of this small protein. The effect of the 40kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution methods and single molecule atomic force microscopy methods. AFM offered a unique strength for characterizing protein-protein interactions and complemented many of the techniques used routinely in the Biotech and Pharmaceutical industries. SEC-MALS, dynamic light scattering (DLS) and AFM topography measurements provided orthogonal methods to characterize the hydrodynamic radius and particle properties of the dAb-1-PEG conjugate in comparison to the non-PEGylated dAb-1. Though the PEG is conjugated at a site distant from the paratope, given that the PEG is four times the size of the dAb-1 protein, the PEG moiety has the potential to change the functional characteristics of dAb-1 such as its binding kinetics and affinity, and its fractional activity. AFM recognition imaging efficiency (Figure 1) and unbinding force curves (Figure 2) provided an orthogonal method to SPR and ITC solution ensemble methods that measure the single molecule binding interaction of the dAb-1-PEG conjugate with its target antigen. Ultimately, the comparison of ensemble methods (SEC-MALS, SPR, ITC) with AFM single molecule topography and recognition imaging methods provided an improved understanding of the dAb-1-PEG conjugate particle properties and how the PEG impacts the interaction of dAb-1with the target antigen.Overall, the use of single molecule methods for particle characterization and receptor binding characterization offers the ability to investigate particle structure and heterogeneity that complements the mass, kinetics, and affinity measured by ensemble [1]. PEGylation of protein therapeutic molecules is a common method for prolonging serum half-life. In the case of small, single domain protein therapeutics such as domain antibodies, the PEG contribute significantly to the particle characteristics and hydrodynamic properties with very minimal impact on the thermodynamics of the protein-protein interaction with its target antigen.

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Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody

Cited by 4 publications

References 33 publications

A novel nanobody-heavy chain antibody against Angiopoietin-like protein 3 reduces plasma lipids and relieves nonalcoholic fatty liver disease

A novel nanobody-heavy chain antibody against Angiopoietin-like protein 3 reduces plasma lipids and relieves nonalcoholic fatty liver disease

A Novel Nanobody-Heavy Chain Antibody Against Angiopoietin-Like Protein 3 Reduces Plasma Lipids and Relieves Nonalcoholic Fatty Liver Disease

Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single Domain Antibody

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