Design of more stable scFv variants using AGGless tags

Abstract

Single-chain variable fragment (scFv) is an antibody fragment known for their small size (approximately 25kDa), excellent tumor tissue penetration, and lack of Fc domain-mediated side effects, making them promising therapeutic agents. However, their structural instability and exposed hydrophobic regions often lead to aggregation, limiting their therapeutic potential. To address this, we developed a strategy to prevent protein aggregation using elastin-like polypeptides (ELP), biopolymers characterized by their flexible, disordered structure and tunable properties. The ELP, incorporating charged amino acids at guest residue, was fused to the C-terminus of scFv. This strategy was applied to PDL1Albubody, PDL1scFv derived from atezolizumab with albumin-binding domain and 4D5Albubody. The AGGless tag fused scFv constructs were expressed in E. coli and purified via affinity chromatography. The PDL1Albu-AGGless(E9) variants also doubled the protein yield, highlighting its potential to enhance production efficiency. The aggregation indices measured at 4°C and 37°C over 7 days demonstrated that the AGGless-tagged constructs, particularly PDL1Albu-AGGless(E9) and 4D5Albu-AGGless(R9), effectively minimized aggregation while antigen-binding efficacy was preserved in PDL1Albu-AGGless(E9). Additionally, thermal stability assays demonstrated comparable melting temperatures between tagged and untagged constructs. These results suggest that AGGless tag variants effectively inhibit scFv aggregation and could be broadly applicable for improving the stability of other therapeutic proteins, including antibody-drug conjugates.