Antigen specificity is important for optimal antitumor activity. The role of tumor cell targeting was tested by comparing the activity of DI-Leu16-IL-2 and another immunocytokine with binding specificity for EGFR, a molecule expressed at only low levels on Daudi lymphoma cells. Experimental conditions were the same as in Figures 4 and 5 except for the indicated changes in dosing. (A) Treatments included PBS only (×; on days 7-11); rituximab (♦; 25 mg/kg on days 7, 9, and 11); DI-Leu16 antibody (⋄; 25 mg/kg on days 7, 9, and 11); medium-dose DI-Leu16-IL-2 (▪; 1 mg/kg on days 7-11); reduced-dose DI-Leu16-IL-2 (○; 1 mg/kg on days 7 and 10); low-dose DI-Leu16-IL-2 (□; 0.25 mg/kg on days 7-11); and medium-dose anti-EGFR–IL-2 (▴; 1 mg/kg on days 7-11). Results were scored as disease-free survival. (B) FACS analysis of Daudi lymphoma cells using either anti-EGFR antibody or anti-CD20 as the primary unlabeled antibodies. Labeled antihuman Fc antibody was used for detection. The low level of EGFR expression was confirmed by PCR analysis (data not shown). All treatment groups were significantly different from the PBS control (P < .005 or less) but the difference between the groups treated with either rituximab or DI-Leu16 antibody, or the groups treated for different numbers of days with the higher dose of DI-Leu16-IL-2 were not. The group treated with the anti-EGFR–IL-2 immunocytokine was significantly different from the antibody treatment groups (P < .005) as well as the low dose of DI-Leu16-IL-2 (P < .05).