Institute of Virology, Saarland University Medical Center, 66421 Homburg, Germany.
Department of Biophysics, Center of Integrative Physiology and Molecular Medicine (CIPMM), Saarland University, 66421 Homburg, Germany.
J Immunol Methods. 2019 Oct;473:112644. doi: 10.1016/j.jim.2019.112644. Epub 2019 Aug 9.
Human IgA antibodies effectively engage myeloid cells for the FcαRI-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells. Established methods to investigate ADCC are the chromium and Calcein release assays. Their critical limitations are the end-point measurement, the unspecific release of the probes, the requirement of target cells in suspension and thus do not reflect physiologic conditions of adherently growing cells. Here we report the label-free real-time monitoring of granulocyte-mediated ADCC using an impedance-based method. We investigated the efficacy of an engineered epidermal growth factor receptor (EGFR)-directed IgA2 antibody to engage neutrophils for ADCC against a panel of adherently growing EGFR-expressing cancer cell lines majorly head and neck squamous cell carcinoma (HNSCC). The impedance assay allowed the documentation of the IgA-neutrophil-and FcαRI-signaling dependent ADCC of adherently growing target cells. While at a short-term it provided comparable results to release assays, in the long run real time monitoring also revealed cell-line specific kinetics and long-term efficacy. Although short-term results may depend on EGFR expression, long-term efficacy did not correlate with the surface level of EGFR nor of the myeloid checkpoint CD47 pointing to additional critical parameters to predict the treatment efficacy. Real-time monitoring of neutrophil-mediated ADCC allowed documenting effector cell activity and exhaustion. Along with excess expression of Mac-1 ligands, which may explain the target cell resistance, this eventually leads to tumor cell outgrowth at later time points. In conclusion, the impedance assay provides valuable information on the kinetics, effector cell performance, efficacy and critical parameters of IgA-dependent granulocyte-mediated cytotoxicity and is expected to become an important tool in its evaluation.
人类 IgA 抗体可有效地与髓样细胞结合，从而实现 FcαRI 依赖性抗体依赖的细胞介导的细胞毒性 (ADCC)，杀伤肿瘤细胞。目前用于研究 ADCC 的方法主要有铬释放和 Calcein 释放实验。这些方法存在的关键局限性在于终点测量、探针的非特异性释放、需要悬浮状态的靶细胞，因此无法反映贴壁生长细胞的生理条件。在此，我们报告了一种基于阻抗的无标记实时监测粒细胞介导的 ADCC 的方法。我们用一种工程化的表皮生长因子受体 (EGFR) 导向的 IgA2 抗体来研究其招募中性粒细胞，进行 ADCC 杀伤一组贴壁生长的、表达 EGFR 的肿瘤细胞系，主要是头颈部鳞状细胞癌 (HNSCC)，以评估该方法的效果。该阻抗检测法可记录 IgA-中性粒细胞-FcαRI 信号依赖性 ADCC 对贴壁生长靶细胞的杀伤作用。虽然在短期内，它与释放实验提供的结果相当，但实时监测还揭示了细胞系特异性动力学和长期疗效。虽然短期结果可能取决于 EGFR 表达，但长期疗效与 EGFR 的表面水平或髓系检查点 CD47 无关，这表明存在其他预测治疗效果的关键参数。实时监测中性粒细胞介导的 ADCC 可以记录效应细胞的活性和耗竭情况。除了过量表达 Mac-1 配体可能导致靶细胞耐药外，这最终会导致肿瘤细胞在稍后时间点生长。总之，阻抗检测法提供了有关 IgA 依赖性粒细胞介导的细胞毒性的动力学、效应细胞性能、疗效和关键参数的有价值信息，有望成为其评估的重要工具。
