MEDI-565 recognizes full length CEA; however, the corresponding epitope is unknown. CEA deletion mutants were generated to identify the domain of CEA to which MEDI-565 binds. The full-length CEA transcript (NCBI accession number NM_002483) contains 10 exons and 9 introns encoding a 702 amino acid (aa) protein composed of a 34 aa processed leader sequence, one IgV-like N-terminal domain, six immunoglobulin constant (IgC)-like domains denoted A1, B1, A2, B2, A3 and B3, and a C-terminal 17 aa peptide which is removed during GPI linkage [1, 2, 45, 46] (Figure 1, 2) . Five deletion mutants were constructed by removing the following IgC-like domains: A1, B1 and A2 domains (DelA1-A2), A1 domain (DelA1), B1 domain (DelB1), A2 domain (DelA2), and B2 and A3 domains (DelB2-A3) as shown in Figure 3A . The mutants were transiently expressed as GPI-anchored proteins on HEK293 F cells. The binding of MEDI-565 or the anti-CEA polyclonal control antibody to each of the deletion mutants was analyzed using flow cytometry. MEDI-565 did not recognize any of the deletion mutants which lack the A2 domain (DelA1-A2 and DelA2, Figure 3B ), but bound well to all mutants that contained it (DelA1, DelB1 and DelB2-A3, Figure 3B ). Additionally, when all but the A2 IgC-like domains were removed (A2; Figure 4B ), MEDI-565 still recognized the corresponding truncated CEA protein at a level similar to that measured for full-length CEA protein ( Figure 4C ). These results suggested that the epitope of MEDI-565 is localized in the A2 domain of CEA.