NMDA receptor subunit-dependent modulation by conantokin-G and Ala7-conantokin-G.

The modulation of recombinant NMDA receptors by conantokin-G (con-G) and Ala7-conantokin-G (Ala7-Con-G) was investigated in Xenopus oocytes injected with capped RNA coding for NR1 splice variants and NR2 subunits using the two-electrode voltage clamp technique. Glutamate exhibited a marginally higher apparent affinity for NR2A-containing receptors than NR2B-containing receptors, regardless of the NR1 subunit present. Conantokins were bath applied to give cumulative concentration responses in the presence of 3 and 30 mum glutamate. Both contantokins exhibited biphasic concentration-response relationships at NR2A-containing NMDA receptors, producing potentiation at low conantokin concentrations and inhibition at high concentrations. These effects were stronger with glutamate concentrations near its EC50, and less marked at saturating concentrations. In contrast, the conantokin concentration-response relation was monophasic and inhibitory at NR2B-containing receptors. We conclude that the combinations of subunits that comprise the NMDA receptor complex influence conantokin and glutamate affinities and the nature of the responses to conantokins.

Multiple myeloma cells are killed by syndecan-1-directed superantigen-activated T cells.

Multiple myeloma (MM) is an incurable plasma cell/plasmablast malignancy with a great need for innovative treatment strategies. Since experimental immunotherapy with targeted superantigens (SAg) proved to be effective in other haematopoietic tumours, we investigated whether this would also hold true for MM. We used the bacterial SAg Staphylococcus enterotoxin A (SEA), a potent activator of T cell cytotoxicity by means of its binding to particular T cell receptor Vbeta sequences on effector cells and MHC class II molecules on target cells. To eliminate potentially unspecific binding via MHC class II, SEA was point mutated (SEAm). In a second step SEAm was genetically fused to protein A (PA), resulting in a fusion protein (PA-SEAm). This fusion protein was used together with four different plasma-cell-specific/associated mAbs to direct T cells towards 10 MM target cell lines. Three of these mAbs were directed against syndecan-1/CD138, known to be highly expressed on MM and plasma cells, but absent on other haematopoietic cells. All MM cell lines proved to be sensitive to SAg-activated T cell killing (15-50% lysis), as measured in a 51Cr-release assay. This effect was clearly mediated via the plasma-cell-reactive antibodies, as control antibodies only conferred a low background lysis. MM therapy based on targeted SAgs could in theory be hampered by dysfunctional T cells in MM patients. However, we show that T cells from MM patients and healthy controls responded equally well to activation by SAg.

Targeted superantigens for immunotherapy of haematopoietic tumours.

With the exception of childhood common acute lymphoblastic leukaemia (cALL), treatment of other hematopoietic B cell lineage tumours such as non-Hodgkin's lymphoma (B-NHL), adult ALL and multiple myeloma (MM) is unsatisfactory. Similarly, the therapeutic outcome of acute and chronic myeloid leukaemia (AML, CML) is frequently dismal. At the same time, leukaemia/lymphoma cells represent ideal targets for immunotherapy. The present review summarizes our preclinical experience with a novel type of cytotoxic T cell based immunotherapy for B-lineage and myeloid tumours. Staphylococcal enterotoxin-derived superantigens (SAgs) are among the most potent T cell activators known, linking the T cell receptor to HLA-DR on natural target cells. SAgs were genetically engineered to reduce DR binding and were then fused to Fab parts of tumour-directed monoclonal antibodies (mAbs). Using these "targeted" SAgs, highly efficient lysis of B-lineage (B-NHL, B-CLL, ALL, MM) and myeloid (AML, CML) tumour cells by T-cells was achieved in vitro and in an animal model. We are entering an interesting era of innovative cancer therapy based on novel man-made biotherapeutic agents.