Cytotoxic effects of the trifunctional bispecific antibody FBTA05 in ex-vivo cells of chronic lymphocytic leukaemia depend on immune-mediated mechanism.

Monoclonal antibodies such as rituximab and alemtuzumab show considerable therapeutic efficacy in chronic lymphocytic leukaemia (CLL). Aiming to further improve antineoplastic efficacy, the trifunctional bispecific antibody FBTA05 was developed. FBTA05 is thought to function by simultaneously binding B cells and T cells by its variable regions and by recruiting FcγR-positive accessory immune cells by its intact Fc region. As it was previously shown that this antibody shows considerable cytotoxicity towards a spectrum of B-cell lymphoma cell lines, we here tested its potential efficacy ex vivo against malignant B-CLL cells. Therefore, we assessed the capacity of increasing concentrations of FBTA05 to bind to neoplastic cells, to induce cytotoxicity (comparing it with rituximab and alemtuzumab) and cytokine release. We evaluated the results with respect to the extent of CD20 expression, the effector:target cell ratio as well as with the patients' overall effector cell status. Thus, we show that, although FBTA05-elicited cytotoxicity was comparable with that induced by alemtuzumab, it considerably exceeded the antineoplastic effects of rituximab. Noteworthy, FBTA05 shows effective elimination of malignant B cells even if CD20 surface expression is low. Importantly, a high grade of cytotoxicity was associated with the induction of T-cell proliferation and the concomittant release of interferon-γ and interleukin-6, thus overcoming the detrimental effects of an unfavourable effector:target cell ratio. In conclusion, we here present novel evidence for the therapeutic efficacy of the trifunctional, bispecific antibody FBTA05 in CLL and provide evidence for the importance of immune-mediated mechanisms conveying the cytotoxic effects against malignant B lymphocytes.

Vascular endothelial growth factor and basic fibroblast growth factor are released by squamous cell carcinoma cells after irradiation and increase resistance to subsequent irradiation.

We analysed the release of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) from squamous cell carcinoma (SCC) after irradiation and their potential contribution to radiation resistance. Three SCC cell lines were irradiated, and VEGF- and bFGF-release was quantified by Elisa-assay. Conditioned media (CM) were used in clonogenic assays for analysis of tumor cell survival. To evaluate the effect of tumor released VEGF and bFGF on survival, experiments with neutralizing monoclonal anti-VEGF and anti-bFGF antibodies were conducted in parallel. Cell cultures were irradiated with 2 Gy to analyse the effects of CM on tumor cell escape from radiation-induced death. We observed a marked increase in VEGF- and bFGF-release after irradiation by the surviving cells. Using these conditioned media, subsequently we observed an up to 10-fold increase in colony formation. The addition of anti-VEGF- and anti-bFGF-antibodies reduced colony formation, indicating that irradiation stimulates the release of growth promoting substances including VEGF and bFGF by the surviving cells. Additionally, irradiation of cells cultured with CM decreased colony formation about 50%, however, it was still increased 5-fold compared to the cultures without CM. The addition of VEGF- and/or bFGF-antibodies led to an additional 20% reduction of this radioprotective effect of the CM. This means, bFGF and VEGF contribute to a significant proportion of the survival stimulating activity. We thus show that irradiation might result in autologous protection of tumor cells from irradiation-induced cell death by the release of growth factors. These observations suggest that radiation might lead to unsuspected and undesired effects in tumorous tissue with possible clinical impact.

VEGF-subtype specific protection of SCC and HUVECs from radiation induced cell death.

The contribution of VEGF (vascular endothelial growth factor) to angiogenesis and tumor aggressiveness has been shown in several tumor types, but no comparative data regarding the impact of the VEGF-subtypes on endothelial and tumor cell protection are available. Therefore, we analysed the cytoprotective effects of the two major soluble VEGF-subtypes, VEGF-121 and -165, on HUVEC cell cultures (human umbilical vein endothelial cells) and squamous cell carcinoma (SCC) cell lines after ionizing radiation. We performed clonogenic analyses and proliferation assays for evaluation of cell survival and proliferative activity. Experiments were performed employing different intensities up to 8 Gy with or without added recombinant VEGF-121 or -165 and compared to non-irradiated cultures. To evaluate a possible contribution of the VEGF-receptors, we performed immunohistochemical stainings of VEGF-R1 (flt), -R2 (KDR,flk), and Neuropilin-1. In the SCC cell lines and HUVEC cultures, cell survival and proliferative activity were reduced in a dosage-dependent manner. The addition of VEGF-121 yielded a significant increase of resistance from 1.2 to 2.7 Gy (+125%) for killing 50% of subjected cells, while VEGF-165 was less effective in this regard (+83%). Conversely, in HUVEC cultures, 50%-survival was increased more strongly by VEGF-165 compared to VEGF-121 (+100% vs. +43%). Accordingly, proliferation was more intensely stimulated in HUVECs by VEGF-165 than by VEGF-121. VEGF-receptors were expressed in all cell cultures analysed at comparable levels. We conclude that the two VEGF-subtypes differentially increase the survival of tumor and endothelial cells after irradiation in vitro. We hypothesise, that the release of VEGF by the tumor protects tumor cells and endothelium resulting in increased radiation resistance.

The cyclooxygenase inhibitor flurbiprofen reduces radiation-induced angiogenic growth factor secretion of squamous cell carcinoma cell lines.

Surgical intervention and radiotherapy still represent the gold standard in the therapy of head and neck squamous cell carcinoma (SCC), although often with unsatisfactory results. Radiation might induce the expression and secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) by unknown mechanisms. These two highly active proangiogenic and cytoprotective factors might contribute to a limited therapeutic success by promoting revascularization and cytoprotection of the radiated tumor. The aim of the present study was to analyze the potential of the cyclooxygenase inhibitor flurbiprofen to reduce radiation-induced increase of VEGF and bFGF secretion of tumor cells. We analyzed the expression of VEGF and bFGF at 72 h after radiation with 30 Gy in four SCC cell lines (De-pt, Hun, Lau, and A549) in cell culture with or without added flurbiprofen. Controls were not exposed to radiation and were analyzed at the same time after culture in the same media. We observed increased VEGF levels in all and increased bFGF levels in three of four lines after radiation. In irradiated cultures with flurbiprofen, VEGF was reduced between 13% and 26% and bFGF was reduced between 84% and 93% compared with radiated cultures without flurbiprofen. We found no reduction of VEGF and bFGF secretion in the unirradiated cultures despite added flurbiprofen. We conclude that flurbiprofen is able to alter the radiation-induced secretion of these two growth factors and might be useful in decreasing the resistance of SCC to radiation.

Transient lymphocyte decrease due to adhesion and migration following catumaxomab (anti-EpCAM x anti-CD3) treatment in vivo.

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 TNFα. Adherent T cells were determined by flow 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 (TNFα, IFNγ). TNFα 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 TNFα. 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.