Elsevier

Immunobiology

Volume 214, Issue 6, June 2009, Pages 441-453
Immunobiology

Mode of cytotoxic action of T cell-engaging BiTE antibody MT110

https://doi.org/10.1016/j.imbio.2008.11.014Get rights and content

Abstract

MT110 is an EpCAM/CD3-bispecific antibody construct in clinical development for the treatment of patients with adenocarcinoma expressing EpCAM (CD326). Like other members of this antibody class, MT110 can engage resting, polyclonal CD8+ and CD4+ T cells for highly potent redirected lysis of target cells. Here we further explored the mechanism of this action. Complete lysis of EpCAM+ Kato III gastric cancer cells by previously unstimulated T cells was achieved within 48 h. During this period, a high percentage of CD4+ and CD8+ T cells became activated and increased expression of granzyme B. This apparently boosted the capacity for serial target cell lysis as studied at very low effector-to-target ratios. Elimination of cancer cells by MT110-redirected T cells involved membrane damage as was evident from nuclear uptake of propidium iodide and release of the cytosolic enzyme adenylate kinase. Redirected T cells also potently triggered programmed cell death in cancer cells as was evident by membrane blebbing, activation of procaspases 3 and 7, fragmentation of nuclear DNA and cleavage of the caspase substrate poly (ADP ribose) polymerase. Chelation of extracellular calcium fully protected cancer cells from lysis by MT110-redirected T cells, while the pan-caspase inhibitor Z-VAD-FMK blocked activation of procaspases, cleavage of poly (ADP ribose) polymerase and fragmentation of nuclear DNA in cancer cells, but could not prevent nuclear uptake of propidium iodide. Soluble factors did not significantly contribute to cancer cell death. Our study shows that MT110 can efficiently gear up the potential of CD8+ and CD4+ T cells for serial lysis, and mediate kill of cancer cells predominantly through poreforming and pro-apoptotic components of cytotoxic T cell granules.

Introduction

T cells have the potential to control tumor growth and patient survival (Galon et al., 2006) and, under certain circumstances, can effectively treat late-stage cancer (Dudley et al., 2005; Morgan et al., 2006). However, a multitude of immune escape mechanisms challenge the effectiveness of natural and therapeutically induced specific T cell responses (Rabinovich et al., 2007; Meidenbauer et al., 2004). One strategy to mitigate the influence of evasion mechanisms are antibodies engaging T cells via binding their monomorphic CD3 antigen, a signalling component of all T cell receptors (Staerz et al., 1985).

Data from recent clinical studies have provided proof-of-concept that this strategy has therapeutic benefit. In one case, a CD19/CD3 bispecific antibody, called blinatumomab, has shown at a dose level of 0.06 mg/m2 per day a high rate of complete and partial responses in relapsed non-Hodgkin's lymphoma patients (Bargou et al., 2008). In two other trials, so called trispecific anti-CD3 antibodies binding EpCAM (catumaxomab) or HER-2 (ertumaxomab) have shown clinical activity in ovarian and breast cancer patients, respectively (Kiewe et al., 2006; Burges et al., 2007). These kinds of antibodies can engage pre-existing effector T cells irrespective of their T cell receptor specificity, and thereby do not rely on presentation, processing and transport of tumor-associated peptide antigens, T cell co-stimulation, or the presence of MHC class I molecules on target cells. Like conventional monoclonal antibodies, BiTE and trispecific antibodies recognize on target cells a cell surface antigen, which is then transiently connected to CD3 on T cells (Baeuerle et al., 2008; Wolf et al., 2005).

BiTE antibodies specific for target antigens CD19, epithelial cell adhesion molecule (EpCAM), and receptor tyrosine kinase EphA2 have been characterized in vitro, ex vivo, and in animal models (Dreier et al., 2002, Dreier et al., 2003; Brischwein et al., 2006, Hammond et al., 2007). All three can induce potent redirected lysis of target antigen-expressing cells at pico- to femtomolar concentrations, which is accompanied by highly conditional T cell activation (Brischwein et al., 2007). Redirected lysis involves formation of a cytolytic synapse (Offner et al., 2006), allows for serial lysis at very low effector-to-target (E:T) ratios, and no longer depends on MHC class I expression or co-stimulatory molecules (Hoffmann et al., 2005). Here we have investigated in more detail by which mode of action resting peripheral T cells are redirected by EpCAM/CD3-bispecific BiTE antibody MT110 for lysis of EpCAM+ target cells. Only one previous study has thus far studied selected aspects of how CD19/CD3-bispecific BiTE antibody MT103 (blinatumomab) lysed lymphoma cells (Gruen et al., 2004).

We here found that during the first 20 h after addition of MT110 the lytic potential of resting T cells markedly increased and reached an activity level close to that of T cells pre-stimulated with immobilized αCD3/αCD28 antibodies. This was accompanied by upregulation of granzyme B, and to a lesser extent, perforin. Inhibition of perforin pore assembly in target cell membranes by a chelator of extracellular calcium strongly intercepted with nuclear uptake of propidium iodide (PI) and activation of pro-caspases. As expected, a pan-caspase inhibitor could potently inhibit only events downstream of granzyme B-activated caspases such as cleavage of poly (ADP ribose) polymerase (PARP) and DNA fragmentation, but could not affect nuclear uptake of PI. Membrane perforation and caspase activation in target cells appeared to predominantly account for the lytic activity of MT110-redirected T cells.

Section snippets

BiTE antibodies

MT110 was constructed, purified and characterized as described previously (Brischwein et al., 2006). For the experiments, clinical drug substance of lots #050224SLi03 and #070323LPa02 was used. The control BiTE antibody shared the αCD3 single-chain antibody with MT110 but recognized with the second arm a haptene (mecoprop). It was produced by the same procedure as MT110.

Cell lines

EpCAM+ Kato III were purchased from the ‘European Collection of Cell Cultures’ (ECACC, Salisbury, UK), EpCAM+ SW-480 cells and

T cells redirected through MT110 induce apoptosis of cancer cells

We have investigated by microscopy morphological changes of EpCAM-expressing lung cancer cell line A549 in response to MT110-activated cytotoxic T cells. Pre-stimulated PBMC were depleted of CD56+ and CD4+ cells and enriched CD8+ T cells seeded at a ratio of 1:2 onto A549 cells forming a confluent cell monolayer on the culture dish. No morphological changes were observed after 2 h when CD8+ T were co-cultured with cancer cells in the presence of a control BiTE antibody (100 ng/ml) sharing the

Discussion

T cells can eliminate cancer and virus-infected cells by various modes of cytotoxic action. A pivotal one is the delivery of cytotoxic granule content to target cells via a cytolytic synapse (Stinchcombe et al., 2001; Grakoui et al., 1999). In the discrete secretory domain of the cytolytic T cell synapse, subunits of the pore-forming protein perforin assemble in a calcium-dependent fashion in the target cell membrane to form 16 nm pores (Russell and Ley, 2002). Aided by the inserted perforin

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    Present address: Merck Serono, Am Feld 32, 85567 Grafing, Germany.

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