It is possible that some knock-out CEA variants exhibited an incorrect fold in or near the MEDI-565 epitope region, thereby losing their binding capacity. Therefore, we further confirmed the potential epitope regions identified with knock-out variants by using gain of function (knock-in) mutants. Indeed, ''knocking in'' segments A (326-349) and C (388-410) of the A2 domain into the A3 domain (KO_B/KI_A+C, KO_B encodes the same amino acids as KI_A+C) resulted in MEDI-565 binding comparable to full length CEA ( Figure 4C, 5C) . Furthermore, only grafting the three amino acids F 326 T 328 N 333 of segment A with the six amino acids V 388 G 389 P 390 E 392 and I 408 N 410 of segment C into the A3 domain (KI_FTN+VGPE+IN) also resulted in MEDI-565 binding comparable to full length CEA ( Figure 5C ). In summary, these results demonstrate that the epitope of CEA bound by MEDI-565 is a nonlinear, conformational epitope located in the A2 domain of CEA; it is comprised of two segments of amino acids 326 to 349 and 388 to 410 with critical amino acids F 326 , T 328 , N 333 , V 388 , G 389 , P 390 , E 392 , I 408 , and N 410 . The residue N 333 may contribute more to the binding of MEDI-565, since mutating it alone completely disrupted the interaction ( Figure 5C ).