Background Trifunctional antibodies, such as catumaxomab (anti-EpCAManti-CD3) and ertumaxomab (anti-HER-2/neuanti-CD3), transiently link immune effector cells to tumour cells, which results in cellular cytotoxicity towards the tumour cells. A functional immune system is therefore essential for effective anti-tumour activity. However, the commonly observed haematotoxicity of chemotherapeutics and radiation therapy may be associated with some degree of immunosuppression. Combining chemotherapy and trifunctional antibodies in cancer treatment requires understanding of the impact of chemotherapeutics on immune cell function and, thus, on the activity of trifunctional antibodies. Methods The effect of chemotherapeutic treatment on trifunctional antibody-mediated anti-tumour activity was assessed in vitro. Blood samples were collected from 12 head and neck squamous cell carcinoma patients after chemotherapy (5-fl uorouracil, cisplatin) and radiotherapy, and from one healthy control donor. The immune cell status was analysed and mononuclear cells (MNC) were isolated. The potency of catumaxomab and ertumaxomab was assessed in a cytotoxicity assay using MNC isolated from each patient sample in co-culture with a tumour target cell line. The release of infl ammatory cytokines was also monitored in the cell culture supernatant. Results Most patients included in this study had decreased immune cell counts during the course of chemotherapy. Nonetheless, an effective and concentration-dependent anti-tumour activity mediated by trifunctional antibodies was demonstrated using these patient immune effector cells. The immune response activity of the patient immune cells was not impaired one week after cisplatin administration or even three days after the last 5-fl uorouracil treatment. Conclusion This study shows for the fi rst time that immune effector cells from cancer patients undergoing standard chemotherapy and radiotherapy can be activated by trifunctional antibodies for effi cient killing of tumour cells.
Introduction In patients, a transient decrease in peripheral blood lymphocyte counts was observed following intraperitoneal administration of the trifunctional monoclonal antibody catumaxomab (anti-human EpCAM x anti-human CD3). The aim of this study was to clarify the observed effect in a preclinical mouse model and to analyse the related mechanism of action in vitro. Materials and methods A related antibody, BiLu (antihuman EpCAM x anti-mouse CD3), was administered to mice and blood leukocytes were analysed. In vitro studies measured activation and cytokine secretion from human peripheral blood mononuclear cells (PBMC). For the analysis of T cell adhesion, PBMC were preincubated with catumaxomab and then co-cultured with human endothelial cells (HUVEC); T cell adhesion was assessed in the presence or absence of endothelial cell preactivation by TNF. Adherent T cells were determined by fl ow cytometry. Results Treatment of mice with BiLu resulted in a dosedependent transient decrease in CD3+ T cells (both CD4+ and CD8+) that returned to the normal range within 48 h. Catumaxomab physiologically activated T cells in vitro (increased CD69 expression) and induced cytokine release (TNF, IFN). TNF increased expression of adhesion molecules CD54 and CD62E on endothelial cells. Furthermore, catumaxomab dose-dependently enhanced adhesion of T cells to endothelial cells. Adhesion was further increased when endothelial cells were preactivated with TNF.Conclusions Catumaxomab increases adhesion of T cells to endothelial cells due to antibody-mediated activation of T cells and production of T cell cytokines that up-regulate endothelial cell adhesion molecules. These results provide a mechanistic rationale for the transient, reversible decrease in lymphocyte counts observed following catumaxomab administration in patients, which is likely to be due to redistribution of lymphocytes.
The trifunctional bispecific antibodies catumaxomab (anti-EpCAM x anti-CD3) and ertumaxomab (anti-HER-2/neu x anti-CD3) exert their mechanism of action by simultaneous recruitment and activation of two different types of immune effector cells at the tumor site. The interaction of different immune effector cells results in efficient elimination of tumor cells by several killing mechanisms including T-cell cytotoxic responses and direct effects mediated by accessory cells. Effective treatment of cancer is essentially based on cytotoxic drugs that kill tumor cells or inhibit their proliferation. However, the commonly observed hematotoxicity of chemotherapeutics is regularly associated with some degree of immunosuppression. On the other hand, some of these chemotherapeutic agents are reported to support or even enhance the anti-tumoral effect of immunotherapeutic drugs in certain doses. A largely intact immune system is the key for trifunctional antibodies to exhibit their full mode of action. Since chemotherapy might impact the number and function of immune effector cells, we evaluated in different preclinical in vitro models whether the efficacy of trifunctional bispecific antibodies would be influenced when combined with chemotherapeutic drugs (5-FU and cisplatin). The preclinical studies performed included in vitro cytotoxicity assays with established tumor cell lines evaluating potential synergy by using the method of Chou and Talalay. Furthermore, results from 3D tumor spheroids and from an autologous human ex vivo setting are presented. In addition, immune cells from cancer patients were obtained at different time points during and after chemotherapy and were assessed for their in vitro cytotoxicity mediated by trifunctional antibodies. So far the results from the different in vitro assay systems used indicate synergy for the combination of the trifunctional antibodies with 5-FU and cisplatin. Furthermore, no negative influence of these chemotherapeutic drugs was observed on the trifunctional mode of action in vitro. These results provide a basis for the possible combination of these drugs with trifunctional antibodies in the clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3481.
Catumaxomab is a trifunctional, bispecific antibody targeting EpCAM and CD3 which redirects T-cells to EpCAM expressing tumor cells and is able to evoke T-cell cytotoxic responses. The hybrid Fc region of catumaxomab provides a third functional binding site, which is able to bind and activate Fcγ receptor (FcγR)-positive accessory cells. In clinical trials a transient decrease in peripheral blood lymphocyte counts was observed following i.p. administration of catumaxomab. Lymphocyte decrease may be a result of either decreased production or increased destruction of lymphocytes, redistribution of lymphocytes or various unknown / multifactorial pathogeneses. We determined whether the observed transient decrease in patients might be due to antibody induced adhesion of lymphocytes to endothelial cells or to migration of lymphocytes into the tissue. Therefore, we investigated the influence of catumaxomab on adhesion of human T cells to endothelial cells in vitro. Catumaxomab physiologically activated T-cells by increasing expression of the activation marker CD69 and induced the release of cytokines, including TNFα and IFNγ. TNFα increased expression of adhesion molecules CD54 and CD62E on endothelial cells. Furthermore, catumaxomab dose-dependently increased adhesion of T cells to endothelial cells, and the adhesion was further increased when the endothelial cells were preactivated with TNFα. Thus, catumaxomab increases adhesion of T-cells to endothelial cells, which is due to antibody mediated upregulation of adhesion molecules on T-cells and production of cytokines that upregulate endothelial cell adhesion molecules. Furthermore, we showed that treatment of BALB/c mice with the related trifunctional antibody BiLu (anti-human EpCAM x anti-mouse CD3), that binds to mouse CD3, also resulted in a dose-dependent transient decrease in CD3+ T-cells (both CD4+ and CD8+) that returned to the normal range within 48 h. These results provide a rationale for the transient and reversible decrease in peripheral lymphocyte counts observed following catumaxomab administration in clinical studies, which is likely to be due to redistribution of lymphocytes and not due to depletion of circulating lymphocytes or stem cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2447.
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