Design of Bispecific scFv with Improved Stability

Abstract

Bispecific antibodies represent a promising modality in cancer immunotherapy by simultaneously engaging two distinct targets for synergistic effects. While conventional IgG-based formats have shown potential, their large size and manufacturing complexity pose limitations. Single-chain variable fragment (scFv)-based bispecific antibodies offer enhanced tumor penetration and cost-effective production. However, extremely short serum half-life and severe aggregation propensity have hindered clinical translation of bispecific scFv therapeutics. This study presents an integrated engineering approach combining albumin-binding domain (ABD) for half-life extension and charged elastin-like polypeptide (ELP)-tag for aggregation prevention. We developed a HER2×PD-L1 bispecific scFv incorporating both modules, targeting clinically validated antigens with complementary anti-tumor mechanisms. The optimized HER2ABD × PDL1 candidate demonstrated nanomolar binding affinities for both targets. However, accelerated stability testing revealed substantial aggregation common to scFv formats. Incorporation of negatively charged ELP-tag (E9 variant) dramatically improved stability, reducing aggregation by over 90% at physiological temperature while increasing bacterial expression yields 3.2-fold. The resulting candidate preserved binding functionality throughout accelerated storage, successfully integrating tumor targeting, immune checkpoint blockade, extended circulation, and superior stability in a compact format. This integrated approach, combining ABD for half-life extension and ELP- tag for aggregation prevention, establishes a comprehensive engineering platform that addresses fundamental scFv limitations with minimal size compromise. The modular strategy offers broad applicability to various bispecific formats, providing a practical framework for developing next-generation antibody fragments with enhanced stability, manufacturability, and clinical application.