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A Potential Mechanism for ADC-Induced Neutropenia: Role of Neutrophils in Their Own Demise
Molecular Cancer Therapeutics ( IF 5.3 ) Pub Date : 2017-09-01 00:00:00 , DOI: 10.1158/1535-7163.mct-17-0133
Hui Zhao 1 , Sara Gulesserian 1 , Maria Christina Malinao 1 , Sathish Kumar Ganesan 1 , James Song 1 , Mi Sook Chang 1 , Melissa M. Williams 1 , Zhilan Zeng 1 , Michael Mattie 1 , Brian A. Mendelsohn 1 , David R. Stover 1 , Fernando Doñate 1
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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][1] This article is featured in Highlights of This Issue, [p. 1727][2] [1]: /lookup/volpage/16/1877?iss=9 [2]: /lookup/volpage/16/1727?iss=9

中文翻译:

ADC诱导的中性粒细胞减少症的潜在机制:中性粒细胞在自身消沉中的作用

中性粒细胞减少症是接受抗体-药物偶联物(ADC)治疗的癌症患者的常见不良事件,我们旨在阐明这种毒性的潜在机制。为了研究ADC是否影响骨髓中性粒细胞的产生,开发了一种体外测定方法,其中将造血干细胞(HSC)分化为中性粒细胞。将几种针对HSC和嗜中性粒细胞中不存在的靶标的抗体通过可裂解的缬氨酸-瓜氨酸接头(vcMMAE-ADC)或MMAF通过不可裂解的马来酰亚胺基丙烯酰基接头(mcMMAF-ADC)偶联至MMAE,并在中性粒细胞分化测定中测试了它们的细胞毒性。结果表明,HSC对vcMMAE-ADC和mcMMAF-ADC具有相似的敏感性。但是,vcMMAE-ADCs对分化中性粒细胞的细胞毒性比与mcMMAF偶联的相同抗体更高。这种抑制作用不是通过巨胞饮作用或FcγRs通过ADC的内在化而介导的。我们的结果表明,可裂解的缬氨酸-瓜氨酸接头的细胞外蛋白水解作用是对分化中性粒细胞的细胞毒性作用。质谱分析表明,将游离的MMAE与分化的嗜中性粒细胞或嗜中性粒细胞条件培养基一起孵育,而不与HSC条件培养基一起孵育时,会从细胞外区室的vcMMAE-ADC中释放出游离MMAE。使用不同的蛋白酶抑制剂,我们的数据表明丝氨酸而不是半胱氨酸蛋白酶是裂解的原因。体外实验表明,纯化的丝氨酸蛋白酶弹性蛋白酶能够从vcMMAE-ADC释放游离的MMAE。在这里,我们提出了含有蛋白酶可裂解的连接子的ADC可以通过分化骨髓中嗜中性粒细胞所分泌的丝氨酸蛋白酶介导的细胞外裂解来促进嗜中性白血球减少症。分子癌疗法;16(9); 1866–76。©2017 AACR。参见Zhao等人的相关文章,[p。[1877] [1]本文在本期要闻中精选,[p.1]。1727] [2] [1]:/ lookup / volpage / 16/1877?iss = 9 [2]:/ lookup / volpage / 16/1727?iss = 9
更新日期:2017-09-05
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