Site-directed mutagenesis and computational homology modeling were implemented to identify amino acids within CEA that are critical for MEDI-565 binding. The following amino acids in the epitope-containing segments A and C of the A2 domain (see Results and Discussion section) that differ from those in the A3 domain were mutated in clusters or individually to encode the corresponding amino acids of the [44] . This model was used as a template to engineer two additional mutants. These two mutants 1) substituted clusters V 388 G 389 P 390 E 392 and I 408 N 410 of the A2 domain with the corresponding clusters of the A3 domain (KO_VGPE+IN), and 2) grafted clusters F 326 T 328 N 333 , V 388 G 389 P 390 E 392 , and I 408 N 410 of the A2 domain into the A3 domain (KI_FTN+VGPE+IN), respectively. All mutants were assembled by overlapping extension PCR using the truncated mutants A2 or A3 as a template, which encodes for the N-terminal domain, the A2 or A3 domain, and the GPI region. They were cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). Expression of these mutants and binding analyses were conducted as listed above for the deletion and swap mutants.