Efficacy of catumaxomab in tumor spheroid killing is mediated by its trifunctional mode of action

Abstract

Catumaxomab is an intact trifunctional bispecific antibody targeting human EpCAM (epithelial cell adhesion molecule) and CD3 with further binding to Fcgamma receptor type I, IIa and III. We choose multicellular tumor spheroids (MCTS) of human EpCAM-positive FaDu tumor cells in co-culture with human peripheral blood mononuclear cells as an adequate three-dimensional in vitro model for pharmacological testing of catumaxomab. We found a strong dose-dependent antitumor response mediated by catumaxomab, with volume-decreased or completely destroyed tumor spheroids together with a massive immune cell infiltration and decreased signals for cancer cell viability and clonogenicity. In control experiments with F(ab')2 fragments of catumaxomab and the parental antibodies alone or in combination the effects in spheroid volume reduction were less than that of catumaxomab. All binding partners of the postulated tricell complex have to be present to exert catumaxomab's full mode of action. These distinct effects of catumaxomab are based on the unique composition of the trifunctional bispecific antibody. Since, in general, many cancers are treated by chemotherapy in combination with immunological tumor therapy, we additionally analyzed the effects of cisplatin alone and in combination with catumaxomab. For cisplatin alone we detected a dose-dependent response relating to decrease of spheroid volume. The combined approach resulted in a synergistic spheroid volume decrease and the colony formation was reduced to non-detectable levels.

Fig. 1

Dose-dependent growth reduction of FaDu spheroids by catumaxomab. FaDu spheroids co-cultured with PBMC were incubated with different catumaxomab concentrations. The effect of the therapeutic antibody shows a delay in time of 3 days. Therefore, this time point was chosen as reference for the growth analyses. Up to day 7 the different approaches can be classified into three effect groups: I Untreated control (spheroids + PBMC only) and lowest catumaxomab concentration (0.05 ng/ml), where the spheroids showed a continuing increase in volume. II Intermediate catumaxomab concentrations from 0.1 up to 1.0 ng/ml resulting in a cessation of further growth. III High concentrations (2.5–5.0 ng/ml) led to a volume decrease followed by complete lysis of spheroids at day 10 for 5.0 ng/ml catumaxomab. For each data point (mean ± SEM) 200 spheroids were analyzed, *p < 0.00001 compared with control

Fig. 2

Cytokine analyses of catumaxomab treated co-culture supernatants. FaDu spheroids co-cultured with PBMC were treated with different concentrations of catumaxomab. Every other day, cytokine concentrations (IL-2, IL-6, IFN-γ and TNF-α) were measured in the supernatant using CBA. The results were divided into three effect groups: I Untreated control spheroids, II median concentration (0.625 and 1.25 ng/ml), and III high concentration (2.5 and 5.0 ng/ml) of catumaxomab

Fig. 3

Immunohistological and fluorescence stainings of catumaxomab-treated FaDu spheroids. Spheroid cryosections were stained for immune cell infiltration (anti-CD45), proliferation (anti-Ki-67) and apoptosis (FragEL). After 7 days of catumaxomab and co-culture treatment there was a concentration-dependent enhancement of immune cell infiltration along with a decrease of spheroid volume. For proliferation analyses we found the common pattern of the outer viable rim for the control, but a decrement of this layer up to a diffuse pattern with very few proliferating cells for the highest concentration of catumaxomab. For the control, FragEL stainings showed most of the apoptotic cells surrounding the central necrosis (I), while catumaxomab-treated spheroids of the median dose range (II) showed more apoptotic cells within the outer rim. Due to loss of the central necrosis for the highest concentrations (III) positive stained cells were found even in the center of the spheroids

Fig. 4

The efficacy of catumaxomab is based on its unique structure and trifunctional mode of action. In this setting the spheroids were initiated with 5,000 cells/well which were cultured 4 days in 96 well plates with subsequent cultivation in a spinner flask for 4 days. Catumaxomab showed its concentration-dependent antitumor effect resulting in a volume reduction starting from day 3. On day 7 volume reduction was 90–100% in comparison to the untreated control. For the F(ab′)2 fragment lacking the intact Fc region the volume decrease reached values from 54 to 81% at day 7. The BiLu antibody binding to mouse CD3 instead of human CD3 had nearly no effect on FaDu spheroids (±10% to control volume) (n = 200 spheroids analyzed per data point mean ± SEM, *p < 0.0001 compared with control)