Abstract
Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The 51Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies.
Highlights
Breast cancers can exceed 50%5, the vast majority of patients will eventually experience disease progression, despite ongoing Trastuzumab therapy[3]
A number of flowcytometric methods for measuring cell-mediated immunity, those based on uptake of 7-amino-actinomycin D (7-AAD) or propidium iodide (PI), and Annexin V binding have been suggested as alternatives to the 51Cr release assay
We first measured the levels of HER2/neu expression on the three breast cancer cell lines
Summary
Breast cancers can exceed 50%5, the vast majority of patients will eventually experience disease progression, despite ongoing Trastuzumab therapy[3]. Complete or partial remission in patients treated with neoadjuvant Trastuzumab correlated with tumor infiltration of immune cells and higher in vitro ADCC activity in a lysis assay[8]. A number of flowcytometric methods for measuring cell-mediated immunity, those based on uptake of 7-amino-actinomycin D (7-AAD) or propidium iodide (PI), and Annexin V binding have been suggested as alternatives to the 51Cr release assay. These release assays are known to have poor reproducibility, not allowing evaluation of the lysis susceptibilities of distinct cell types within the target cell population[12,14]. This assay is capable of assessing the cytotoxicity induced by frozen peripheral blood mononuclear cells (PBMCs)
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