The A3 domain is not involved in MEDI-565 binding despite being highly homologous with the epitope-containing A2 domain; therefore, swap-mutants were constructed by exchanging short segments of the A2 domain with the corresponding portions of the A3 domain to further refine the MEDI-565 binding epitope. Two truncated CEA mutants (A2 and A3) were engineered and used as templates for the construction of swap mutants. The A2 or A3 mutant is comprised of the N-domain, the A2 or A3 domain, and the GPI region. An alignment of the amino acid sequences of the A2 and A3 domains identified differences in their aa composition. These regions of sequence diversity were divided into three shorter segments labeled as A (aa 326 to 349), B (aa 360 to 382) and C (aa 388 to 410) ( Figure 4A ). Six swap mutants were generated by swapping segments A, B or C of the A3 domain into the A2 domain (KO mutants), or by swapping segments of the A2 domain into the A3 domain (KI mutants) as shown in Figure 4B . MEDI-565 did not bind to any of the KO or KI mutants lacking either the A or C segment of the A2 domain (KO_A, KO_C, KI_A, KI_B and KI_C, Figure 4B , C), but bound well to the swap variant which encoded both the A and C segments of the A2 domain (KO_B, Figure 4B , C). Some residual binding of MEDI-565 to the KO_C mutant could be detected. Together with . Swap-mutants of human CEA. A, amino acid sequence alignment of the A2 and A3 domains of CEA. Sequence homology analysis revealed 21 amino acids that differed between these two domains (amino acids boxed). Three segments, A, B, and C, were defined in the A2 and A3 domains to generate swap-mutants. B, a schematic display of swap mutants that were constructed by exchanging segments A, B, or C between the A2 (open boxes) and A3 (grey boxes) domains using the truncated mutant A2 or A3 as a template which encodes the N-domain, the A2 or A3 domain, and the GPI region. C, flow cytometry analysis of binding of MEDI-565 to deletion mutants expressed on the surface of HEK293 F cells. All mutants were expressed well as monitored by anti-CEA polyclonal antibody. MEDI-565 did not bind well to any of the knock-out (KO) or knock-in (KI) mutants which lack either the A or C segment of the A2 domain (KO_A, KO_C, KI_A, KI_B and KI_C), but bound well to the one variant which encoded both the A and C segments of the A2 domain (KO_B). doi:10.1371/journal.pone.0036412.g004 MEDI-565 lack of binding to KO_A mutant, this data indicated that segment C significantly contributed to MEDI-565 binding, but to a lesser degree than segment A. Due to the high degree of identity between segment B in the A2 and the A3 domains, direct involvement of this segment in MEDI-565 binding could not be entirely ruled out using a swap mutant-based approach. It is however unlikely for the following reasons 1) MEDI-565 did not bind to the mutants encoding the B segment of A2 domain in conjunction with either A or C segment of the A2 domain (KO_A or KO_C, Figure 4 ); 2) The binding level of MEDI-565 to the mutant encoding both the A and the C segments of A2 domain (KO_B) was comparable to the signal of both the full-length CEA and the A2 deletion mutant (Figure 4) ; and 3) The modeled structure of the A2 domain using murine CEACAM1 as a template revealed that segment B was spatially distal from critical residue N 333 (see following section). Therefore, the swap-mutants revealed that MEDI-565 bound to a nonlinear epitope in the A2 domain of CEA, which is comprised of two segments, namely 326-349 (segment A) and 388-410 (segment C).