FreeStyle human embryonic kidney (HEK293) F cells (American Type Culture Collection, Manassas, VA) were cultured in FreeStyle 293 expression medium (Life Technologies/Invitrogen, Carlsbad, CA). Dihydrofolate reductase deficient (DHFR-) Chinese hamster ovary (CHO) cells or CHO DHFR-cells (American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 media (Invitrogen) containing 10% fetal bovine serum (FBS; Invitrogen) in a humidified cell culture incubator at 37uC and 5% CO 2 . CHO cells expressing the CEA splice variant (CHO SV CEA) were created by infection of CHO DHFR-cells with the pCDH1-HCS1-EF1-Puro-CEA splice variant lentiviral vector, and selected by culturing in medium containing 5 mg/mL puromycin (EMD Chemicals Group, Merck KGaA, Darmstadt, Germany) for 72 hours. CHO cells expressing the full-length CEA sequence (CHO FL CEA) were created by infecting CHO DHFRcells with the lentiviral expression vector pCDH1-CMV-MCS-EF1-Blast-CEA, and selected by culturing in medium containing 10 mg/mL blasticidin (Invitrogen) for 10 days. Likewise, cells expressing both the CEA splice variant and full-length CEA (CHO FL+SV CEA) were created by infecting CEA splice variantexpressing cells with the pCDH1-CMV-MCS-EF1-Blast-CEA lentiviral vector followed by blasticidin selection. The anti-CEACAM5 specific monoclonal antibody (mAb) clone 26/3/13 was purchased from Genovac GmbH (Aldevron, Freiburg, Germany). Propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO). Construction and production of MEDI-565 and the Control BiTE (MEC14 BiTE) has been described [17] .