Engineering multi-specific and multi-payload ADCs to address tumor heterogeneity and drug resistance

Antibody-drug conjugates (ADCs) are advancing beyond single-target, single-payload formats to address the challenges of heterogeneity and resistance to treatment. We previously generated ADC's based on monospecific, bispecific, biparatopic, and tri-specific antibodies, as well as dual-payload conjugates. In vitro and in vivo evaluation of these constructs demonstrated enhanced internalization and cytotoxicity. In this study, we combine these design strategies to generate next generation ADCs that incorporate multi-specific antigen recognition and multi-payload delivery, aiming to further improve therapeutic efficacy. A biparatopic anti-PD-L1 antibody was produced by combining two humanized parental antibodies via scFv integration. The antibody was expanded into tri-specific formats by incorporating additional binding domains targeting VEGF, HER2 and TROP2 tumor associated targets, enabling differential tumor engagement. Three cytotoxic payloads with distinct mechanisms of action (DNA damaging, microtubule inhibition, and topoisomerase I inhibition) were individually conjugated to generate monospecific single-payload ADCs. Based on the relative potency of these conjugates, dual and tri-payload ADCs were developed using the tri-specific antibodies with each payload conjugated at a Drug -to-Antibody Ratio (DAR) calibrated to achieve comparable cytotoxic effects. In vitro cytotoxicity was assessed across a panel of tumor cell lines with heterogenous target expression to evaluate the therapeutic potential of combining multi-specific antigen recognition with multi-payload delivery. The biparatopic anti-PD-L1 construct showed enhanced binding and internalization compared to its parental antibodies. Tri-specific formats retained cooperative engagement of multiple-antigens, supporting their potential for broader tumor targeting. Among these, the HER2/TROP2 targeting tri-payload ADC maintained molecular integrity and exhibited enhanced potency in mixed-resistance models, outperforming corresponding monospecific or single-payload ADCs. All three PD-L1-based tri-specific tri-payload ADC's exhibited stronger and more widespread cytotoxic activity across diverse cell lines. In vivo studies confirmed that ADC's combining multi-specificity with multi-payload delivery achieved better tumor growth inhibition relative to monospecific or single-payload controls. Multi-specific antibody mechanisms and multi-payload delivery are complementary strategies to improve ADC performance by promoting efficient internalization, expanding the range of cytotoxic mechanisms, and addressing tumor resistance. These modular approaches offer a flexible framework for next-generation ADCs targeting heterogeneous solid tumors.