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Abstract
In this report we describe the role of apoptosis in the process of tumour cell killing by bispecific monoclonal antibody (BsMAb)-redirected cytolytic T cells. The BsMAb used, BIS-1, has dual specificity for the CD3 complex on T cells and the pancarcinoma-associated 38 kDa transmembrane antigen EGP-2. BIS-1 allows activated T cells to specifically recognise and kill EGP-2-positive but not EGP-2-negative target cells. An assay was developed to quantify apoptosis in cells by separation of 3H-thymidine-labelled low-molecular, i.e. fragmented, from high-molecular, i.e. non-fragmented DNA. The presence of low molecular weight DNA was measured both within the target cells and in the cell-free supernatant. After exposure to BIS-1-redirected, -activated T cells, apoptosis was observed in EGP-2-positive target cells but not in EGP-2-negative target cells. Also no DNA fragmentation proved to be induced in the activated effector cells during assay. The degree of EGP-2-positive target DNA fragmentation depended on the concentration of BsMAb, the E/T ratio and the incubation time. Using a low E/T ratio (1/1), DNA fragmentation in and 51Cr release from target cells showed similar characteristics and kinetics. At higher E/T ratio (20/1), the 51Cr release from the target cells increased to a greater extent than the percentage fragmented target cell DNA. Inhibitors of DNA fragmentation added to the cytotoxicity assay inhibited not only DNA fragmentation, but also the release of chromium-51 from the target cells, suggesting that apoptosis and cell lysis are closely related in BsMAb-mediated cell killing.
Highlights
Our results show that the different mechanisms of BIS-1-mediated target cell lysis are closely linked
50 pl of culture medium containing various amounts of BIS-1, interleukin 2 (IL-2) (400 IU ml-') and 100 pl of cytotoxic T lymphocytes (CTLs) effector cells were added to each well to give the desired final BIS-1 concentration and effector to target ratio in a final volume of 200 Ml per well
DNA fragmentation can be detected in EGP-'-positive targzet cells upon specific BIS-1 bispecific monoclonal antibody (BsMAb)-mediated CTL cytotoxicitv (Figures 1 -4)
Summary
GLC-1 (EGP-2 negative) and a clonal derivative thereof, GLC-IM13 (EGP-2 positive), are human small-cell lung cancer (SCLC)-derived cel lines (De Leij et al, 1985). The cell lines were cultured according to routine procedures in culture medium, i.e. RPMI-1640 (Gibco/BRL, Paisley, UK) supplemented with 14% heat-inactivated fetal calf serum (Gibco/ BRL), 2 mM glutamine (Gibco/BRL), 60 Mg ml-1 gentamicin (Schering, Kenilworth, USA), 0.05 mM fl-mercaptoethanol (Merck, Darmstadt, Germany) and 1 mm sodium pyruvate (Gibco/BRL) at 37°C in a humidified atmosphere containing 5% carbon dioxide
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