The trifunctional antibodies catumaxomab and ertumaxomab demonstrate efficacy in co-cultures of human multicellular tumor spheroids (MCTS) and peripheral blood mononuclear cells (PBMC)

399 Catumaxomab and ertumaxomab are intact, trifunctional monoclonal antibodies consisting of heavy chains of mouse IgG2a and rat IgG2b. The antibodies possess three different binding sites: the mouse Fab fragment binds to the tumor associated antigens human EpCAM or HER-2/neu, respectively; the rat Fab fragment targets human CD3 on T-cells; and a third functional binding site within the hybrid Fc-region selectively binds to and activates Fcγ-receptor-positive accessory cells.u2028 The mode of action is based on the formation of a postulated tricell complex. The simultaneous recruitment and activation of T-cells and accessory cells leads to physiological co-stimulation of T cells, improved tumor cell elimination by different killing mechanisms and phagocytosis as well as processing and presentation of tumor material by bound and activated accessory cells.u2028 We have used a 3D tumor model well-established in our laboratory, i.e. multicellular tumor spheroids (MCTS). MCTS were cultured using the EpCAM and HER-2/neu expressing cell line FaDu (human HNSCC) under standardized conditions. Studies were done in miniaturized spinner flasks in suspension culture. We focused on dose response and immune effector cell functions. Spheroids were co-cultured in spinner flasks under constant conditions in 20 ml medium with PBMC (1x106/ml) and different concentrations of antibody (0-10 ng/ml). The effect of combined therapy was quantified by the reduction of spheroid volume and clonogenicity. In histological sections the infiltration of CD45+ cells and the proportions of proliferating and apoptotic cells within the spheroid were assessed by immunostaining (Ki-67 and FragEL, respectively). Furthermore, different cytokines were analyzed using cytokine bead arrays (CBA). In addition, PCR with different PBMC relevant molecules was done to distinguish subsets of infiltrating leukocytes.u2028 Here, we demonstrate the feasibility of co-culturing MCTS, as an adequate model for micro-metastases, with PBMC in suspension under well-defined conditions. Furthermore, we show that therapeutic efficacy of trifunctional antibodies can be quantified by various endpoints.u2028 Our studies revealed that both antibodies caused a significant reduction or rather destruction in spheroid volume. Tumor cell clonogenicity was reduced as well; clinically relevant concentrations decreased colony formation to non-detectable low levels. In this set of in vitro experiments apoptosis was one of the major causes of therapeutic spheroid shrinkage. Immunohistochemical staining suggests that apoptosis was caused by infiltrating leukocytes. Measurement of cytokine concentrations and PCR results suggest that these PBMC are mostly identified as NK and T cells.u2028 Supported by Fresenius Biotech GmbH and TRION Pharma GmbH (Munich, Germany)