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The evaluation of novel immunotherapeutic anti-cancer strategies requires target and effector cells from the autologous host to avoid false positive results because of allo-reactivity. Head and neck squamous cell carcinoma cells (HNSCC) do not proliferate under standard culture conditions in plastic ware. To create an autologous model, the chorioallantois membrane (CAM) of chicken embryos was employed to culture HNSCC and peripheral blood mononuclear cells (PBMC). Separated tumor cells were co-incubated with a cytostatic agent, PBMC, or mere cell culture medium as a control. Tumor cell lysis was assessed by acridine orange staining or by flow cytometry analysis of iodide marked cells after 24 and 48 h of co-incubation. Incubation with cisplatin resulted in a decrease of viable cells by 49% after 24 h and 48 h. In contrast, incubation with mere culture medium resulted in an increase of viable tumor cells by 5% after 24 h and a decrease of 4% after 48 h; the incubation of tumor cells and PBMC led to a non-significant decrease by 14% after 24 h and 16% after 48 h. Taken together, the CAM supports a favorable environment for the co-culture of HNSCC and PBMC, thus providing an approximated in vivo autologous model to assess the efficacy of new cell-therapeutical approaches.

Background. The trifunctional bispecific antibody Removab (tbAB) bridges and activates CD3 positive T cells to EpCAM on carcinoma cells and simultaneously binds to an accessory immune-cell inducing tumor cell lysis. tbAB-induced tumor cytotoxicity was assessed in an autologous human ex vivo system. Methods. One hundred forty tumor samples and autologous peripheral blood mononuclear cells from a total of 36 patients with head and neck squamous cell carcinomas (HNSCCs) were incubated on a chicken embryo chorioallantois membrane with Removab. Tumor cells coincubated with cisplatin or cell culture medium served as positive and negative controls. Tumor cell lysis was assessed by acridine orange staining or by fluorescence-activated cell sorting of propidium iodide–marked cells after 24 and 48 hours (T24/T48) coincubation. Results. Coincubation of HNSCC cells with tbAB and autologous peripheral blood mononuclear cells resulted in a 49% ± 6% decrease of viable cells at T24 (p < .005) and in a decrease of 56% ± 8% at T48 (p < .005) compared with the control. The tumor cytotoxicity was similar to that of cisplatin (49% ± 7% decrease at T24 and 49% ± 8% at T48). Conclusion. In an autologous human ex vivo system, the tbAB-induced tumor cell lysis was comparable to that by cisplatin. © 2005 Wiley Periodicals, Inc. Head Neck27: XXX–XXX, 2005

Removab is a trifunctional bispecific antibody which can bridge CD3+ T cells and epithelial cell adhesion molecule positive (EpCAM+) tumor cells, and binds with its Fc fragment to antigen presenting cells. To explore a new approach for the treatment of patients with carcinoma of the upper aerodigestive tract, we investigated whether Removab can induce specific cellular responses to the EpCAM+ carcinoma cell line BHY. Particular emphasis was put on the opsonization of peripheral blood mononuclear cells (PBMN) with respect to clinical application. Tumor cells and allogeneic PBMN of healthy volunteers were incubated with or without Removab. In a third group, PBMN were opsonized with Removab and washed before incubation with tumor cells. Inverse microscopy, ELISPOT, flow cytometry analysis and cytotoxicity assays on the chorioallantois membrane (CAM) were performed. In comparison with PBMN alone, opsonization with Removab resulted in: a) activation of CD83+ antigen presenting cells, b) secretion of interferon gamma, and c) granzyme B mediated lysis of targeted BHY cells by EpCAM specific CD8+ T cells. The secretion of tumor necrosis factor alpha, interferon gamma and interleukin-2 by opsonized PBMN was significantly reduced after 24 h. Washed opsonized PBMN maintained their lytic activity against tumor cells as tested on the CAM. Removab is an appropriate agent for the therapeutic amplification of T cell responses against EpCAM+ tumor cells by opsonization of PBMN without putting patients at risk for severe adverse events caused by a cytokine storm.