A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

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 (51)Cr 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.

Figure 1. Phenotypic analyses of three human breast cancer cell lines and NK cells.

(a) Expression levels of the cell surface HER2 (black filled histogram) on human breast cancer cell lines measured using flowcytometry. The histograms in black lines represent the isotype control. (b,c) NK92MI + CD16a cells (b) or NK cells in freshly isolated healthy human PBMCs (c) were stained with anti-CD16 antibody (upper panel, black-filled histogram), anti-Tim3 antibody (middle panel, black-filled histogram) and anti-NKG2D antibody (lower panel, black-filled histogram). Respective isotype controls are shown as black lines in the histograms in both panels.

Figure 2. Staining strategy of the new flowcytometric assay.

(a) Breast cancer cell lines, BT-474, MCF-7 and Hs578T, were stained with CFSE, and the intensity was then measured by flowcytometry, soon after staining (black filled histogram), after an overnight incubation (gray-filled histogram) and after 2 days of incubation (open histogram). (b) Human PBMCs were stained with FVD (1:1000 dilution, at RT for 20 min.), PI (2 μg/mL, at RT for 10 min.), or 7-AAD (5 μg/mL, at RT for 10 min.), and the dead cells (%) were then measured by flowcytometry. (c) Data obtained with the new flowcytometric analysis method. CFSE⁺cells are target cells, and FVD⁺cells are dead cells, such that dead target cells are represented by the CFSE⁺FVD⁺area.

Figure 3. Assessment of the ADCC bioassay using the NK cell line.

(a) The BT-474 as target cells were incubated overnight with NK92MI + CD16a as effector cells at various E:T ratios without (white bar) or with 10 μg/mL of Trastuzumab (black bar). (b,c) The three human breast cancer cell lines, expressing different HER2 levels, serving as target cells were incubated overnight with the NK92 + CD16a as effector cells, at an E:T ratio of 1:1, without (white bar) or with 10 μg/mL of Trastuzumab (black bar) or Bevacizumab (dot bar) using the flowcytometric assay (b) or calcein-release assay (c). The results of the analyses of dead cells (%) and ADCC (%) are presented as mean values ± SD of at least three experiments.

Figure 4. Comparison of the new flowcytometric and calcein-release assay.

The flowcytometric (a,c) and calcein-release (b,d) assays were performed on different days, independently, to assess their reliability and reproducibility for evaluating ADCC, using NK92MI + CD16a (a,b) or PBMCs freshly isolated from the same healthy donor on different days (c,d). The results of the analyses of dead target cells (%) and ADCC (%) are presented as mean values ± SD for each of the experiments.

Figure 5. Evaluation of healthy-donor specific ADCC.

(a) BT-474 serving as target cells were incubated overnight with PBMCs, serving as effector cells, freshly isolated from healthy volunteers. Various E:T ratios, without (white bar) or with 10 μg/mL of Trastuzumab (black bar), were employed. (b) PBMCs, freshly isolated from four healthy volunteers, were used as effector cells and were found to be capable of inducing individual ADCC activities against BT-474 as target cells, when incubated overnight without (white bar) or with 10 μg/mL of Trastuzumab (black bar) or Bevacizumab (dot bar). (c) The individual ADCC activities, determined every 1~2 months using freshly isolated PBMCs from healthy volunteers, were measured employing our flowcytometric assay with (black circles) or without (white circles) Trastuzumab. The results of the analyses of dead target cells (%) are presented as mean values ± SD for each of the experiments.

Figure 6. Assessment of the ADCC bioassay using frozen NK cell lines and human PBMCs.

(a,b) BT-474 serving as target cells were incubated with frozen NK92MI + CD16a serving as effector cells at an E:T ratio of 4:1 without (white circles) or with Trastuzumab (black circles). ADCC activity was then detected using the flowcytometric (a) or the calcein-release (b) assay. (c) The stability of frozen human PBMCs was assessed using the flowcytometric assay. PBMCs were isolated and cryopreserved at –80˚C using CellBanker I. Portions of the PBMCs were then thawed every 3 days after initially being frozen. ADCC activity was detected, using BT-474 as target cells at an E:T ratio of 4:1, without (white circles) or with 10 μg/mL of Trastuzumab (black circles). The results of the analyses of dead target cells (%) are presented as mean values ± SD. CV values were calculated using the following formula: SD/mean×100 (%).