Final result in high-risk patients with refractory or relapsed germ cell tumours (GCT) remains poor. cell engagement via the Fc domain promotes profound cytotoxicity across a broad range of antibody dilutions. In contrast, tumor cell lysis mediated by either immune cell subset alone is influenced by surface density of the target antigen. In the CHC line JAR, NK cell-dependent cytotoxicity dominates, which may be attributed to differential surface expression of immunomodulatory proteins such as MHC-I, CD24, and Fas receptors on CHC and EC. In view of redirecting T cell therapy mediated by bispecific antibodies, such differences in GCT immunophenotype potentially favoring immune escape are worth further investigation. expression analysis (Figure 1b), high levels of mRNA are found in TCam-2, JAR, and 2102Ep, while mRNA expression is low in the EC cell line NCCIT and negligible in nonmalignant Sertoli cells (FS1) and fibroblasts (MPAF). CD133, which combined with EpCAM can be indicative for cancer stem cells, is expressed to high levels on the seminoma cell line TCam-2 and the EC lines GCT27 and NCCIT. CD133 is detected only on half of the cells in the nullipotent EC line 2102Ep and is absent on the CHC line JAR (Figure 1a). 2.2. Marked Cytotoxicity in the EC Line 2102Ep Mediated by the Bispecific EpCAM/CD3 Antibody in the Presence of Peripheral Blood Mononuclear Cells Persists Across a Broad Range of Antibody Dilutions Cytotoxicity was assessed by europium release assay after treatment of the highly EpCAM-positive EC cell line 2102Ep for 4 h with different concentrations of peripheral blood mononuclear cells (PBMC; 25:1/50:1) including T, NK, and Triciribine B cells as well as monocytes and either the bispecific trifunctional EpCAM antibody Catumaxomab (bAb) or the monoclonal EpCAM antibody Vu1D9 (mAb; Figure 2a,b). Open in a separate window Figure 2 EpCAM/CD3-bispecific antibody mediates time-dependent strong cytotoxicity with stable activity at decreasing drug concentrations in the embryonal carcinoma cell line 2102Ep. 2102Ep cells were incubated for 4 h (a,b) or 8 h (c) with peripheral blood mononuclear cells (PBMC) at an effector:target cell ratio of 25:1 (a) or 50:1 (b,c) and stated concentrations of the monoclonal EpCAM-Ab Vu1D9 (mAB) or the bispecific trifunctional EpCAM/CD3-Ab Catumaxomab (bAb). Antibody-dependent cytotoxicity was assessed by europium release assay in triplicates and indicated in percentage of deceased cells. Data are shown as mean SD of 2C3 3rd party experiments. Statistically factor between mAb- and bAb-mediated cell loss of life can be designated by an asterisk (* 0.001). PBMC only got no cytotoxic influence on 2102Ep cells. On the other hand, at an effector-to-target (E:T) percentage of Triciribine 25:1, bAb-induced tumor cell lysis can be 44.9 2.5% at 1 g/mL and 44.2 5.4% at 0.01 g/mL bAb. With further reduced amount of bAb concentration right down to 0 Actually.0001 g/mL, tumor cell lysis is 35 even now.8 6.9% (Figure 2a). In the current presence of the mAb, cytotoxicity can be much less pronounced across all medication concentrations ( 0.001) and lowers with each dilution stage. Thus, cell loss of life can be 18.4 7.4% at 1 g/mL in support of 3.1 2.1% at 0.01 g/mL mAb. Raising the E:T percentage to 50:1 enhances both bAb- and mAb-mediated mobile kill (Shape 2b). Once again, the EpCAM/Compact disc3-bAb exhibits a lot more powerful cytotoxicity compared to the mAb for many concentrations right down to the lowest medication level ( 0.001). Furthermore, cytolytic activity of the bAb persists at high amounts across the whole medication concentration range, with 55.1% 5.7% at 1 g/mL bAb and with 57.7 6.0% and 53.6 7.4% when treated with 0.01 g/mL and 0.0001 g/mL bAb, respectively. Upon incubation with the mAb in the presence of PBMC, only 34.7 10.6% of 2102Ep cells die at 1 g/mL and 10.7 2.2% die at 0.01 g/mL. Prolongation of the incubation period further improves the cytotoxic effect of both the bAb and mAb (Figure 2c). Again, bAb-mediated cell death is marked and remains high despite decreasing drug concentrations. After incubation for 8 h in the presence of PBMC at an E:T ratio of 50:1, cell death is 83.3 9.2% at 1 g/mL bAb, 85.3 6.8% at 0.01 g/mL, and 70.7 Triciribine 8.2% at 0.0001 g/mL bAb. In contrast, cytotoxicity mediated by the mAb is significantly less pronounced across all drug concentrations ( 0.001) and successively declines with each dilution step from 63.0 3.4% at HVH3 1 g/mL to 33.9 6.4% at 0.01 g/mL and only 4.0 3.3% at 0.0001 g/mL. 2.3. The EpCAM/CD3-Binding Bispecific Antibody Exerts Potent Cytotoxic Activity in GCT Cell Lines of Different Histologies Next, three additional histologically different GCT cell lines were incubated with EpCAM-recognizing bAb or mAb in the presence Triciribine of PBMC at an E:T ratio of 50:1 (Figure 3aCc). Cytotoxicity was assessed.