XmAbCD40 enhances ADCC in multiple B-lineage tumor cell lines. ADCC was measured with an LDH release assay using purified IL-2–activated NK cells as effectors and cells from human B-lymphoma, leukemia, or MM cell lines as targets. Target cells were opsonized with antibodies and mixed with NK cells at an effector/target (E/T) ratio of 5:1; LDH release was measured 4 hours later. Percentage ADCC is shown for 4 tumor cell lines: (A) IM-9 (a lymphoblastoid line). (B) Ramos (Burkitt lymphoma), (C) MEC-1 (CLL), and (D) RPMI8266 (MM). Data were obtained in triplicate and represent the mean plus or minus SD. Cell-surface expression of CD40 and CD20 was determined by flow cytometry using QIFIKIT (Dako North America; Table 3). We demonstrate that XmAbCD40 increased ADCC relative to the IgG1 analog in all cell lines tested (potency improved up to 150-fold; maximal lysis increased 2- and 8-fold in Ramos and RPMI266 cell lines, respectively). No correlation is seen between CD40 expression levels and ADCC activity. XmAbCD40 is also superior to rituximab, exhibiting greater maximal lysis and 10- to 200-fold improved potency, although cell-surface CD40 levels are much lower than CD20 levels. No cell lysis is observed with the anti-CD40 Fc-KO or the XmAb isotype control antibodies.