A Potential Mechanism for ADC-Induced Neutropenia: Role of Neutrophils in Their Own Demise.

Hui Zhao,Brian A Mendelsohn,Melissa M Williams,Sathish Kumar Ganesan,Sara Gulesserian,David R Stover,Michael Mattie,Mi Sook Chang,James Song,Maria Christina Malinao,Fernando Doñate,Zhilan Zeng

doi:10.1158/1535-7163.mct-17-0133

Abstract

Neutropenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC) and we aimed to elucidate the potential mechanism of this toxicity. To investigate whether ADCs affect neutrophil production from bone marrow, an in vitro assay was developed in which hematopoietic stem cells (HSC) were differentiated to neutrophils. Several antibodies against targets absent in HSCs and neutrophils were conjugated to MMAE via a cleavable valine-citrulline linker (vcMMAE-ADC) or MMAF via a noncleavable maleimidocaproyl linker (mcMMAF-ADC), and their cytotoxicity was tested in the neutrophil differentiation assay. Results showed that HSCs had similar sensitivity to vcMMAE-ADCs and mcMMAF-ADCs; however, vcMMAE-ADCs were more cytotoxic to differentiating neutrophils than the same antibody conjugated to mcMMAF. This inhibitory effect was not mediated by internalization of ADC either by macropinocytosis or FcγRs. Our results suggested that extracellular proteolysis of the cleavable valine-citrulline linker is responsible for the cytotoxicity to differentiating neutrophils. Mass spectrometry analyses indicated that free MMAE was released from vcMMAE-ADCs in the extracellular compartment when they were incubated with differentiating neutrophils or neutrophil conditioned medium, but not with HSC-conditioned medium. Using different protease inhibitors, our data suggested that serine, but not cysteine proteases, were responsible for the cleavage. In vitro experiments demonstrated that the purified serine protease, elastase, was capable of releasing free MMAE from a vcMMAE-ADC. Here we propose that ADCs containing protease cleavable linkers can contribute to neutropenia via extracellular cleavage mediated by serine proteases secreted by differentiating neutrophils in bone marrow. Mol Cancer Ther; 16(9); 1866-76. ©2017 AACRSee related article by Zhao et al., p. 1877.

Highlights

Antibody–drug conjugates (ADC) are targeting antibodies conjugated to a cytotoxic agent
To determine whether ADCs could have a direct effect on neutrophils, the viability of mature neutrophils treated with ADCs was investigated
IgG1-vcMMAE bound to neutrophils, which was inhibited by the presence of an Fc blocker reagent, suggesting this binding is mediated by Fcg receptors (Fig. 1C)

Summary

Introduction

Antibody–drug conjugates (ADC) are targeting antibodies conjugated to a cytotoxic agent. Two ADCs, brentuximab vedotin (ADCETRIS) and T-DM1 (KADCYLA), have been approved by the FDA, whereas about 50 ADCs are in clinical development [1, 2]. ADCs are designed to selectively deliver highly cytotoxic payloads to tumor cells and to minimize systemic toxicity; several common, off-target toxicities have been observed, one of which is neutropenia. Patients treated with ADCs conjugated to MMAE via a protease cleavable valine-citrulline linker [3, 4], that is, vcMMAE-containing ADCs, are likely to experience neutropenia independent of the target to which the ADC is directed against [5]. Neutropenia was one of most common events leading to dose delays in brentuximab vedotin phase II trials [6]. ASG-5ME is an ADC comprised of Agensys Inc., Santa Monica, California

Objectives

Methods

Results

Conclusion