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

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

doi:10.1158/1535-7163.22508311.v1

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

https://doi.org/10.1158/1535-7163.22508311

journal contribution

posted on 2023-04-03, 15:46 authored by Hui Zhao, Sara Gulesserian, Maria Christina Malinao, Sathish Kumar Ganesan, James Song, Mi Sook Chang, Melissa M. Williams, Zhilan Zeng, Michael Mattie, Brian A. Mendelsohn, David R. Stover, Fernando Doñate

<p>Figure S1. Chemical structures of MMAF, MMAE and 13C5-MMAE. The asterisks indicate where Carbon-13 isotope has replaced Carbon-12; Figure S2. Expression of CD32 on mature neutrophils. Circulating neutrophils (AllCell) were incubated with anti-CD32 antibody conjugated with FITC (CD32-FITC) or control IgG1-FITC, and were subjected to FACS analysis. Typical results from one experiment were shown; Figure S3. Little expression of SLC44A4 and ENPP3 on neutrophils at different differentiation stages. Microarray analyses were done for SLC44A4 (AGS5 target) or ENPP3 (AGS16 target) in HSCs, differentiating neutrophils at Day 8 or Day 14, or circulating neutrophils (mature); Figure S4. AGS5-vcMMAF conjugation did not affect intracellular cleavage. PC3 cells overexpressing SLC44A4, PC3-SLC44A4 were incubated with indicated concentrations of AGS5-vcMMAE, AGS5-vcMMAF or AGS5-mcMMAF for 6 days; Table S1: Information about ADCs used in this report; Table S2: Chromatographic gradient; Table S3: MRM Transitions monitored; Table S4. CD66b expression during neutrophil differentiation; Table S5. Correlation between sensitivity and extracellular cleavage.</p>

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Agensys Inc.

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DOI - Is supplement to A Potential Mechanism for ADC-Induced Neutropenia: Role of Neutrophils in Their Own Demise

ARTICLE 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 AACR.See related article by Zhao et al., p. 1877