TissueScan TM Disease Tissue quantitative polymerase chain reaction (qPCR) arrays (Origene Technologies, Rockville, MD) were employed to determine the expression of full-length and CEA splice variant (SV) transcripts in normal and cancerous tissues of various stages and grades. For each tissue cDNA array, lyophilized cDNA was resuspended in 2.5 mL of ribonuclease-free water. Plates were sealed, vortexed, and centrifuged (1200 g for 1 min) to ensure resuspension of the full cDNA sample. A 0.2X mixture of the TaqMan assays (Applied Biosystems) was then created according to protocols supplied by the manufacturer for pre-amplification (one cycle of 95uC, 10 min followed by 14 cycles of 95uC 15 sec/60uC 4 min). Subsequent amplification and transcript detection (one cycle of 95uC, 20 sec followed by 40 cycles of 95uC, 1 sec/60uC, 20 sec) of the specific target transcripts was carried out using an ABI 7900HT Fast Real Time PCR Instrument (Applied Biosystems). Upon completion of the qPCR, SDS 2.2 Software (Ericsson, Stockholm, Sweden) was used to generate threshold cycle (Ct) values. A Ct cutoff value of 30, below which the samples were considered to be positive and above which the samples were considered to be negative for transcript expression, was empirically selected. As positive controls, cDNAs from CHO cells expressing either full-length CEA (CHO FL CEA) or the CEA splice variant (CHO SV CEA) were used. Complementary DNA isolated from DHFR-deficient CHO cells lacking CEA expression was used as a negative control for qPCR. To measure the killing of human tumor cell lines by MEDI-565 or the control BiTE, cell lines were co-cultured with enriched CD3+ T cells and BiTE antibody as described above, and cell lysis was measured by the release of caspase-3 into the tissue culture media via a caspase-3 specific electrochemiluminescence assay (Meso Scale Discovery, Gaithersburg, MD).