Abstract 1730: Guide-effector bsADCs: Driving co-endocytosis for enhanced payload delivery

Abstract Bispecific antibodies (BsAbs) are engineered to simultaneously engage two distinct receptors, offering a powerful approach to enhance receptor internalization and intracellular payload delivery. By exploiting differences in endocytic capacities between receptors, dual targets can convert poorly internalizing target into efficiently internalized complexes—a mechanism particularly advantageous for bispecific antibody-drug conjugates (BsADCs). BsAbs targeting HER2×TROP2 and cMET×HER3 were engineered, expressed in ExpiCHO cells, and purified via Protein A affinity chromatography. BsADCs were conjugated by the glycan-based site-specific platform via a cleavable linker-payload system. The drug-antibody ratio (DAR) was determined per candidate. Internalization kinetics of BsAbs and their monospecific counterparts were assessed in receptor-coexpressing tumor cell lines (HER2/TROP2+ and cMET/HER3+) using a pH sensor dye. Upon antibody binding and internalization, the dye transitions from the neutral extracellular pH (7.4) to the acidic lysosomal environment (pH 4.5-5.5), resulting in increased fluorescence. Fluorescence signals were monitored in real time at 37 °C using a Cytation 5 imaging system. In this study, the internalization of two BsAb pairs—HER2 × TROP2 and c-MET × HER3, was compared with their respective monospecific antibodies. Using a guide-effector mechanism, BsAbs pair a rapidly internalizing receptor (e.g., HER2 or c-MET) with a poorly internalizing one (e.g., TROP2 or HER3), leveraging the guide receptor’s endocytic capacity to drive co-endocytosis and lysosomal trafficking. HER2 × TROP2 BsADC (OBI-201) illustrates this concept: HER2 facilitates co-internalization of TROP2 (1.7-fold increase compared to the mono-ADC), enhancing lysosomal trafficking and intracellular payload delivery in tumors co-expressing both receptors, overcoming resistance seen with monospecific antibodies and improving cytotoxic efficacy. Similarly, c-MET × HER3 BsAbs exploit c-MET’s rapid internalization to drive HER3 uptake (5.5-fold increase compared to the HER3 mono-Ab), inducing receptor clustering, coordinated lysosomal trafficking, and increased intracellular payload delivery, thereby enhancing antitumor activity. These findings show that BsAbs enhance receptor internalization via co-endocytosis, using a guide-effector mechanism to improve intracellular payload delivery and therapeutic potency. This strategy offers a generalizable approach for BsADC development, enabling targeting of low-expression or non-internalizing antigens and addressing resistant or heterogeneous tumors. Citation Format: Wei-Jhen Huang, Woan Eng Chan, Meng-Hsin Liu, Yueh Chin Wu, Ya-Chi Chen. Guide-effector bsADCs: Driving co-endocytosis for enhanced payload delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1730.