Expression vectors encoding the CEA deletion and swap mutants were transiently transfected for expression of glycophosphatidyl inositol (GPI)-anchored proteins using FreeStyle HEK293 F cells (Invitrogen). One day prior to transfection, HEK293 F cells were seeded at a density of 0.7610 6 cells/mL. Three and a half micrograms of each expression vector were transfected into 5 mL of a suspension of HEK293 F cells using 5 mL of 293fectin transfection reagent (Invitrogen). Transfected cells were incubated with 5 mg/mL of MEDI-565 for 1 hour. For the detection of bound MEDI-565, which possesses a six-histidine tag at its carboxy-terminus, the cells were washed three times with PBS and incubated with 1 mg/mL of an Alexa FluorH 488conjugated anti-penta-His antibody (Qiagen, Valencia, CA) for 30 minutes, and then analyzed for binding using the LSRII flow cytometer (BD Biosciences, San Jose, CA). Expression of CEA deletion mutants was monitored with a goat anti-CEA polyclonal antibody (R&D Systems, Minneapolis, MN) that was detected by an R-phycoerythrin-conjugated anti-goat IgG (Invitrogen).