Abstract 4829: Improved safety profile of HER2-KSPi-ADCs compared to T-DM1 in in vitro megakaryocyte assay predictive of thrombocytopenia

KSP inhibitors (KSPis) are a versatile new payload class for the generation of highly potent and selective antibody-drug conjugates (ADCs) against different targets. For HER2 (c-ERBB2)- and TWEAKR (Fn14/ TNFRSF12A)-KSPi-ADCs, we have previously shown that they have potent and selective anti-proliferative activity and induce apoptosis in HER2- or TWEAKR-positive cancer cell lines in vitro. Moreover, TWEAKR-KSPi-ADCs induced strong and long-lasting anti-tumor efficacy and complete tumor regression in cell line- and patient-derived xenograft models. Shortcomings of clinically tested and marketed ADC payload classes are the off-target / on-toxophore dose-limiting toxicities observed in the clinic, in particular neutropenia and thrombocytopenia. Thrombocytopenia is a common side effect of 3 of 4 approved ADCs (including T-DM1, Kadcyla). Thrombocytes are generated by proliferation, differentiation and fragmentation of specific megakaryocyte (MK) progenitors. As ADCs can be taken up by differentiating hematopoietic stem cells the released toxic payload can inhibit MK proliferation/differentiation and prevent generation of platelets resulting in thrombocytopenia. The potential to induce thrombocytopenia of KSPi-based ADCs with different effector chemistries (ECs) was compared with the clinically approved ADC T-DM1 (with a non-cleavable SMCC linker) by evaluating their impact on in vitro megakaryocyte differentiation. To this end, the HemaToxTM MK assay (Stemcell, Cologne, Germany) was used which allows to assess the impact of compounds on proliferation/differentiation of CD34+ stem cells into GPIIb/IIIa (CD41) and CD45 double positive megakaryocytes by flow cytometry. Whereas the anti-HER2 Ab trastuzumab had no impact on MK differentiation, T-DM1 elicited MK toxicity in vitro. HER2-targeted or isotype control KSPi-ADCs with a legumain (LGMN) cleavable EC showed a comparably benign profile in the MK assay as trastuzumab, and a further improved profile versus KSPi-ADCs with a non-cleavable EC. Both features, the LGMN specific cleavage of KSPi-ADCs after internalization and cellular trafficking to the lysosome, and the released, non-cell-permeable KSPi payload, may contribute to an improved safety profile compared to T-DM1. This is further supported by the safety profile of a KSPi-ADC with a LGMN cleavable EC was also studied in vivo: In a repeat dose Cynomolgus study with the IL3RA-KSPi-ADC (BAY-943) dosed up to 10 mg/kg (QWx3), thrombocytopenia was not observed. Th