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

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.