GlycoTAIL and FlexiTAIL as Half-Life Extension Modules for Recombinant Antibody Fragments

Abstract

Many therapeutic proteins are small in size and are rapidly cleared from circulation. Consequently, half-life extension strategies have emerged to improve pharmacokinetic properties, including fusion or binding to long-lasting serum proteins, chemical modifications with hydrophilic polymers such as PEGylation, or, more recently, fusion to PEG mimetic polypeptides. In the present study, two different PEG mimetic approaches, the GlycoTAIL and the FlexiTAIL, were applied to increase the hydrodynamic radius of antibody fragments of different sizes and valencies, including scFv, diabody, and scFv-EHD2 fusion proteins. The GlycoTAIL and FlexiTAIL sequences of varying lengths are composed of aliphatic and hydrophilic residues, with the GlycoTAIL furthermore comprising N-glycosylation sites. All modified proteins could be produced in a mammalian expression system without reducing stability and antigen binding, and all modified proteins exhibited a prolonged half-life and increased drug disposition in mice. The strongest effects were observed for proteins comprising a FlexiTAIL of 248 residues. Thus, the GlycoTAIL and FlexiTAIL sequences represent a flexible and modular system to improve the pharmacokinetic properties of proteins.

Isolation of SARS-CoV-2-blocking recombinant antibody fragments and characterisation of their binding to variant spike proteins

“…While scFvs retain the intramolecular disulfide bonds of whole antibody V H and V L regions, they are nevertheless more prone to thermally-induced aggregation (Jager and Plückthun, 1999), which can be reduced using directed evolution or rational design approaches (Kang and Seong, 2020). Similarly, while their smaller size leads to faster clearance than whole antibody molecules in vivo, protein engineering can be used to extend their in vivo half-life and increase their pharmaceutical efficacy (Esquerda-Canals et al, 2019;Seifert and Kontermann, 2022).…”

Proteomics and Metabolomics in Biomedicine

“…To improve pharmacokinetic properties, the modification of molecules, such as PEGylation or fusion to PEG mimetic polypeptides, is important. Two PEG mimetic approaches, GlycoTAIL and FlexiTAIL, were applied to increase the hydrodynamic radius of antibody fragments of different sizes and valencies, with them exhibiting a prolonged half-life and increased drug disposition in mice [51]. One paper proposed three diverse techniques to isolate starch grain proteins, demonstrating how different sample preparation processes influence the respective proteomic results [52].…”