Table 2.

Epitope mapping using mutagenesis scanning of phage-displayed EGF-R domain III.

	R3 (parental mouse antibody of nimotuzumab)			K4 mAb			K5 mAb
aa in EGF-R	NT	PT	Tol	NT	PT	Tol	NT	PT	Tol
L325	—	—	A, K, P, R, S, V	—	—	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	—	C	D, L, Q, S, W
K333	C	V	I, L, M, Q, R, S	C	—	I, L, M, Q, R, S, V	—	—	C, I, L, M, Q, R, S, V
R353	H, P, Q, S, T	—	K, L, M, W	H, P, Q, T	S	K, L, M, W	P	H, S, T	K, L, M, Q, W
S356	I, L, P, Q, R, T	—	—	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	C, K, R, S, T, V	—	L, M, Q, Y
T358	F, G, P	E, H, Q, R, W	S	P	E, F, G, Q, R, W	H, S	P	E, F, G, Q, R, W	H, S
H359	L, P, Q, R, S, T, Y	—	—	L, P, Q, R, S, T, Y	—	—	L, P, Q, R, S, T, Y	—	—

Phage-displayed single-mutated variants of domain III were evaluated by ELISA on microtiter plates coated with the different anti-EGF-R mAbs and with the anti-c-myc tag 9E10 mAb. Normalized reactivities were obtained by dividing the signal obtained with a given antibody by the reference signal (measured with the anti-tag mAb). Relative reactivities (%) were calculated using the ratio between normalized reactivity of each mutated variant and the normalized reactivity of the non-mutated phage-displayed domain III. Those replacements leading to a relative reactivity below 50% were considered to be non-tolerated (NT), while mutations producing a relative reactivity in the range 50–75% were classified as partially tolerated (PT). Tolerated substitutions (Tol) were those present in variants keeping more than 75% relative reactivity. Rows corresponding to amino acids belonging to the functional nimotuzumab epitope are shaded, with the two crucial residues S356 and H359 highlighted in dark grey.