Inhibition of Megakaryocyte Differentiation by Antibody-Drug Conjugates (ADCs) is Mediated by Macropinocytosis: Implications for ADC-induced Thrombocytopenia.

Hui Zhao,Hector Aviña,Josh Snyder,Sara Gulesserian,David R Stover,Jimmy Ou,Sher Karki,Fernando Doñate,Karen Morrison,Veronica Robles,Sathish Kumar Ganesan,Zhilan Zeng,Lisa Do

doi:10.1158/1535-7163.mct-16-0710

Abstract

Thrombocytopenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC), including AGS-16C3F, an ADC targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase-3) and trastuzumab emtansine (T-DM1). This study aims to elucidate the mechanism of action of ADC-induced thrombocytopenia. ENPP3 expression in platelets and megakaryocytes (MK) was investigated and shown to be negative. The direct effect of AGS-16C3F on platelets was evaluated using platelet rich plasma following the expression of platelet activation markers. Effects of AGS-16C3F, T-DM1, and control ADCs on maturing megakaryocytes were evaluated in an in vitro system in which human hematopoietic stem cells (HSC) were differentiated into MKs. AGS-16C3F, like T-DM1, did not affect platelets directly, but inhibited MK differentiation by the activity of Cys-mcMMAF, its active metabolite. FcγRIIA did not appear to play an important role in ADC cytotoxicity to differentiating MKs. AGS-16C3F, cytotoxic to MKs, did not bind to FcγRIIA on MKs. Blocking the interaction of T-DM1 with FcγRIIA did not prevent the inhibition of MK differentiation and IgG1-mcMMAF was not as cytotoxic to MKs despite binding to FcγRIIA. Several lines of evidence suggest that internalization of AGS-16C3F into MKs is mediated by macropinocytosis. Macropinocytosis activity of differentiating HSCs correlated with cell sensitivity to AGS-16C3F. AGS-16C3F was colocalized with a macropinocytosis marker, dextran-Texas Red in differentiating MKs. Ethyl isopropyl amiloride (EIPA), a macropinocytosis inhibitor, blocked internalization of dextran-Texas Red and AGS-16C3F. These data support the notion that inhibition of MK differentiation via macropinocytosis-mediated internalization plays a role in ADC-induced thrombocytopenia. Mol Cancer Ther; 16(9); 1877-86. ©2017 AACRSee related article by Zhao et al., p. 1866.

Highlights

Antibody–drug conjugates (ADC) are targeting antibodies conjugated to a cytotoxic agent
Mature platelets can be activated by thrombin receptor-activating peptide (TRAP) or collagen, which can be measured by expression of glycoprotein IIb/IIIa (PAC-1) and P-selectin (CD62P)
Thrombocytopenia has been reported in cancer patients treated with antibody–drug conjugates such as AGS-16C3F and T-DM1

Summary

Introduction

Antibody–drug conjugates (ADC) are targeting antibodies conjugated to a cytotoxic agent. Brentuximab vedotin (ADCETRISÒ), approved for the treatment of Hodgkin lymphoma and anaplastic large cell lymphomas, is an anti-CD30 antibody conjugated to MMAE, a potent microtubuledisrupting agent via a protease cleavable linker [1, 2]. T-DM1 (KADCYLAÒ), approved for treatment of HER2-positive metastatic breast cancer, is an IgG1 mAb against HER2 (trastuzumab) conjugated to maytansine derivative DM1 via a stable thioether linker [3,4,5]. There are many ADCs in clinical development with some showing promising benefit [6,7,8]. AGS-16C3F is an ADC currently being investigated in a phase II study in metastatic renal cell carcinoma AGS-16C3F is comprised of a fully human IgG2 monoclonal ENPP3-targeting

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