Direct and complement dependent cytotoxicity in CLL cells from patients with high-risk early-intermediate stage chronic lymphocytic leukemia (CLL) treated with alemtuzumab and rituximab

Abstract

The mechanism of cytotoxicity of alemtuzumab and rituximab in chronic lymphocytic leukemia (CLL) is not well understood. We obtained fresh CLL cells from early-intermediate stage high-risk patients just prior to treatment with alemtuzumab and rituximab to study mechanisms of action and resistance. Alemtuzumab had minimal direct cytotoxicity but caused significant complement dependent cytotoxicity (CDC) although a subpopulation of CLL cells had intrinsic resistance. Rituximab had no direct cytotoxicity and caused minimal CDC in cells from most patients. These data suggest that CDC has a therapeutic role in patients treated with alemtuzumab and that measures to decrease resistance to CDC could increase efficacy.

1. Alemtuzumab and rituximab are not toxic to CLL cells in culture

CLL cells at 2 × 10⁶/ml were cultured with either alemtuzumab (10 µg/ml)(A), rituximab (20 µg/ml)(R), or alemtuzumab and rituximab (A&R) at the same concentrations. Viable cell (trypan blue negative) counts were evaluated every 24 hours for 72 hours. These data demonstrate that cells cultured with rituximab have identical survival to control cells (CON) cultured in AIM V medium. Cells cultured with alemtuzumab or alemtuzumab and rituximab have slightly lower survival.

2. Alemtuzumab complement dependent cytotoxicity (CDC) induces rapid cytotoxicity of CLL cells

CLL cells at a concentration of 2 × 10⁶/ml were treated with alemtuzumab (10µg/ml) and 10% human serum as a source of complement. Cytotoxicity was determined by counting the number of viable cells (trypan blue negative) 1 hour after treatment. These values were normalized using the counts in control cell cultures treated with complement only. These studies showed that alemtuzumab CDC (A&C) killed a median of 67% (range 15 – 100%) of the CLL cells from 27 patients within 1 hour. The cells surviving CDC did not have an increased rate of death during the subsequent 23 hours. Addition of rituximab to alemtuzumab (A&R&C) did not significantly increase CDC (p = 0.20).

3. The subpopulation of CLL cells that survive alemtuzumab CDC remains viable for 72 hours

CLL cells at 2 × 10⁶/ml were treated with alemtuzumab (10µg/ml) and 10% human serum as a source of complement and cultured for 72 hours. These assays showed that cells surviving initial CDC caused by alemtuzumab (A&C) or the combination of alemtuzumab and rituximab (A&R&C) had the same survival as cells treated with complement alone (C).

4. Cells treated with MoAb and complement had increased membrane iC3 binding

Membrane bound iC3b was measured in CLL cells by flow cytometry with an anti-iC3b antibody. Control CLL cells in AIM V medium stained with anti-iC3b antibody are shown in panel a. Addition of 10% serum (4U/ml complement) resulted in a modest increase in MFI (from 20 to 36)(panel b). Addition of both rituximab and complement (panel c) resulted in an increase in MFI to 201. In this experiment, the highest level of iC3b was measured in cells surviving treatment with alemtuzumab and complement (panel d) which had a MFI of 1129.