Fig. 4.

In vitro binding of dbBiTEs to CEA positive target cells and to human T-cells. a Flow analysis of anti-CEA antibody M5A and dbBiTE to CEA positive MDA-MB-231 cells. b Flow analysis of anti-CD3 antibody OKT3 and dbBiTE to CD3 positive human T-cells. c Activated human T-cells coated with dbBiTE were incubated with target cells that expressed CEA (MB231CEA) or not (MB231) at indicated E:T ratios and cytotoxicity measured by an LDH release assay. Controls are uncoated or anti-CD3 antibody coated T-cells. The results show that only dbBiTE coated T-cells exhibit significant cytotoxity towards CEA+ and not CEA- targets, p < 0.001. Antibody concentrations were 1 μg/mL per ten million cells. d IFNγ secretion was measured in the media at 24 h at an E:T ratio of 10:1. Controls include uncoated T-cells or T-cells coated with anti-CD3. The assay was performed in triplicate, p < 0.01. e Binding of dbBiTE to human T-cells was measured at concentrations ranging from 1 μg/mL to 1 ng/mL. f Cytoxicity was measured for T-cells coated at 1 μg/mL to 1 ng/mL against MDA-MB231-CEA target cells at two E:T ratios