Table 1. Effects of individual replacements within phage-displayed EGFR domain III on recognition by anti-EGFR mAbs.

		R3 binding			Cetuximab binding
	Targeted residue	Non-tolerated	Partially tolerated	Tolerated	Non-tolerated	Partially tolerated	Tolerated
Nimotuzumab antigenic region	D323	I, L, M, V	P, R, S	N	I, L, M, V	P, R, S	N
	L325	-	-	A, K, P, R, S, V	-	-	A, K, P, R, S, V
	T330	-	C	D, L. Q, S, W	-	C	D, L, Q, S, W
	K333	C	I, V	L, M, Q, R, S	C	I, V	L, M, Q, R, S
	R353**	P	H, Q, S, T	K*, L, M, W	P	-	H, K*, L, M, Q, S, T, W
	S356	I, L, P, Q, R, T	-	-	-	-	I, L, P, Q, R, T
	F357	C, K, L, M, Q, R, S, T, V	-	Y	-	C	K, L, M, Q, R, S, T, V, Y
	T358	F, G, P	E, H, Q, R, W	S	-	H, P, W	E, F, G, Q, R, S
	H359	L, P, Q, R*, S, T, Y	-	-	-	L, P	Q, R*, S, T, Y
	P361	F, G, H, V	Q	A, R	F, H	G, Q, V	A, R
	P362	H, S, T	-	G	H	S, T	G
Cetuximab structural epitope	Q384	P	-	E, H, K, L, R, T	K, P, R	-	E, H, L, T
	Q408	-	-	A, F, L, P, S, T, V	F, P, S, T	-	A, L, V
	S418	C	-	A, G*, K, L, M, V	C, K, L, M, V	-	A, G*
	S440	Y	-	F, H, L, Q, R, T, V	F, H, L, Q, R, T, V, Y	-	-
	K443	-	L, W	D, E, H, N, Q, R*, S	D, E, H, L, N, Q, S, W	-	R*
	K465	-	-	E, L, Q, R, S, T, Y	E, Q, S, T	Y	L, R
	S468	-	C	A, L, N, P, R, V	C, P, R	-	A, L, N, V
	I467	-	-	K, L, M*, N, R, S	K, M*, N, R, S	L	-
	N473	-	-	E, H, K*, L, P, T, V, W	-	-	E, H, K*, L, P, T, V, W

Solvent-exposed residues (> 20% RSA) of EGFR domain III surrounding by less than 12 Å the first identified critical aa contributing to nimotuzumab epitope formation (H359), as well as residues comprised in the cetuximab structural epitope, were individually randomized. * indicate replacements by the residue found at the equivalent position in mouse EGFR. Residue R353 (**) is included in both antigenic regions. The resulting phage-displayed mutated Ag variants were tested by ELISA on microplates coated with either anti-EGFR antibodies or the anti-tag 9E10 antibody (recognizing the c-myc tag fused to every protein in our display system). Display levels assessed with the anti-tag mAb were taken as a reference to normalize the reactivity of the different variants toward anti-EGFR antibodies. Relative reactivities (%), compared with the normalized reactivity obtained for wt EGFR domain III, were calculated for each of them. Each mutated variant was evaluated twice by ELISA in two independent experiments with separately produced phage preparations. Replacements resulting in mean relative reactivities below 50% and between 50 and 75% were classified as non-tolerated and partially tolerated, respectively. Tolerated substitutions were those contained in mutated variants keeping more than 75% relative reactivitiy. Profiles of tolerated, non-tolerated and partially tolerated mutations for nimotuzumab (left panel) and cetuximab (right panel, shaded in gray) binding are shown. Residues contributing to the formation of nimotuzumab functional epitope are contained in the box.