Rational design, expression and refolding of a novel bispecific antibody to simultaneously target VEGF-A and Ang-2...

Bispecific antibodies simultaneously targeting vascular endothelial growth factor A (VEGF-A) and angiopoietin-2 (Ang-2) represent a promising anti-angiogenic strategy in cancer therapy. Tandem single-chain bispecific antibodies (scBsAbs) offer modular, Fc-free formats; however, their structural complexity and multiple disulfide bonds often promote aggregation and misfolding during bacterial expression. Here, based on current knowledge, we report the first systematic design of multiple VEGF-A/Ang-2 tandem scFv constructs derived from brolucizumab and faricimab, respectively, with variations in V_H/V_L orientation and linker architecture. Then, we compared their folding compatibility and aggregation propensity using an integrated computational workflow including physicochemical profiling, homology modeling (AlphaFold and Robetta), structural validation, and 3D aggregation propensity analysis. The scBsAb_{VEGF-A/LL/Ang-2}, which contain long linker between anti-VEGF-A and anti-Ang-2 scFvs exhibited superior structural agreement and the lowest predicted aggregation propensity and was selected for experimental validation. Following expression in Escherichia coli, we systematically screened refolding conditions to evaluate the effects of dilution strategy, protein concentration, redox environment, and additives on functional recovery. Slow dilution in a glutathione redox system (GSH/GSSG 5:1) at lower initial protein concentration (250 µg/mL) significantly enhanced binding activity toward both VEGF-A and Ang-2, whereas arginine impaired recovery and glycerol provided no additional benefit. Differential scanning calorimetry confirmed improved conformational stability after optimized redox-assisted refolding (T_m ≈ 59.8 °C). Collectively, these findings demonstrate that integrating structure-guided construct selection with systematic refolding screening enables rational optimization and improved developability of Fc-free tandem scFv bispecific antibodies.