Abstract 3853: Potential mechanisms for thrombocytopenia and neutropenia induced by antibody-drug conjugates
Authors: Zhao Hui , Gulesserian Sara , Ganesan Sathish K. , Zeng Zhilan , Ou Jimmy , Robles Veronica , Snyder Josh , Stover David R. , Donate Fernando 
Published: July 2016

Abstract
Thrombocytopenia and neutropenia are common adverse events in cancer patients treated with antibody-drug conjugates (ADCs). We used human hematopoietic stem cells (HSC) in vitro and various ADCs to investigate the potential mechanisms of thrombocytopenia and neutropenia.
Two ADCs, AGS-16C3F and T-DM1, were selected for the investigation of megakaryocytes (MKs) as they both induced thrombocytopenia in patients. AGS-16C3F is an ENPP3-targeting antibody conjugated to microtubule-disrupting agent MMAF (licensed from Seattle Genetics) via a non-cleavable linker (mcMMAF) tested in a phase 1 trial in kidney cancer patients. T-DM1 (KADCYLA) is an anti-HER2 antibody conjugated to the maytansine derivative DM1, approved for the treatment of advanced Her-2 positive breast cancer. HSCs were differentiated to MKs and percentage of CD41 positivity from FACS analysis was used to measure the effect of ADCs on MK differentiation. Results showed that while MKs do not express ENPP3 or Her-2, both AGS-16C3F (IC50 = 40nM) and T-DM1 inhibited MK differentiation. MKs express FcγRIIA to which T-DM1 (IgG1) and, to lesser degree, AGS-16C3F (IgG2) can bind; however, blockage of FcγRIIA failed to rescue MKs from cytotoxicity induced by either T-DM1 or AGS-16C3F suggesting that binding to FcγRIIA is not critical for MK cytotoxicity. AGS-16C3F is internalized into MKs via macropinocytosis and causes significant disorganization of tubulin structure as observed by confocal microscopy.
HSCs were also differentiated to neutrophils which were characterized by the percentage of CD66b positivity in FACS analysis. Several antibodies to targets absent in neutrophils were conjugated to both MMAE and MMAF and were tested for cytotoxicity during neutrophil differentiation. Our results showed that ADCs containing MMAE via a cleavable linker were more cytotoxic to neutrophil differentiation than the same ADC containing MMAF via a non-cleavable linker, in agreement with clinical results. Different from MKs, results showed that neutrophils have low macropinocytosis activity. Secretion of cathepsins increased during neutrophil differentiation, and a membrane impermeable cathepsin inhibitor blocked ADC cytotoxicity to neutrophils, suggesting that extracellular protease cleavage is critical for neutrophil cytotoxicity in vitro. Neutrophils express high levels of FcγRIIA and saturation of this receptor by control IgG inhibited ADC binding to neutrophils and at least partially rescued neutrophils from ADC cytotoxicity, suggesting that FcγRIIA can play a role in neutropenia induced by ADCs, particularly with IgG1 isotype.
In conclusion, applying these in vitro data to clinical experience suggests that thrombocytopenia may be mediated by macropinocytosis of ADC in MK and neutropenia by secreted proteases and interactions with their FcγRs. Understanding these toxicities may help design new ADCs with improved toxicity profile.
Citation Format: Hui Zhao, Sara Gulesserian, Sathish K. Ganesan, Zhilan Zeng, Jimmy Ou, Veronica Robles, Josh Snyder, David R. Stover, Fernando Donate. Potential mechanisms for thrombocytopenia and neutropenia induced by antibody-drug conjugates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3853.