The modeled structure of the A2 domain also revealed two clusters of amino acids V 388 G 389 P 390 E 392 and I 408 N 410 in segment C that were spatially close to the already identified critical residue N 333 in segment A ( Figure 5B ). This observation suggested that these amino acids could contribute to the binding of MEDI-565 in concert with N 333 . Knocking-out amino acids V 388 G 389 P 390 E 392 (KO_VGPE) or I 408 N 410 (KO_IN) separately ( Figure 5A ), or knocking-in the entire segment C which includes both clusters of V 388 G 389 P 390 E 392 and I 408 N 410 (KI_C) ( Figure 4C ) had no effect in abolishing or restoring MEDI-565 binding, respectively. However, knocking-out these amino acids together reduced (but did not completely abolish) MEDI-565 binding (KO_VGPE+IN, Figure 5C ). Thus, amino acids V 388 , G 389 , P 390 , E 392 , I 408 , and N 410 in segment C of CEA could also be involved in MEDI-565 binding.