Mechanism of Alemtuzumab Depletion of T Cells in Stem Cell Products.

Abstract

Alemtuzumab (a humanized murine monoclonal antibody [MAb] against CD52) is an effective drug for in vitro T cell purging of allogeneic stem cell products when a product is incubated with 10mg of antibody for 30 minutes prior to infusion. Recovery of CD34+ cells has been excellent and T cell purging with alemtuzumab has resulted in a decreased incidence of grade 2–4 GVHD and a reduced incidence of extensive GVHD and a low overall risk of graft failure. However, a higher incidence of cytomegalovirus (CMV) reactivation, respiratory and polyoma virus infections have been observed as has an increased relapse rate in patients with chronic myelogenous leukemia. In vitro purging, using alemtuzumab and infusion of unwashed products, has been associated with a reduced incidence of clinically significant GVHD, an increase in infectious complications and a possible increase in relapse rates. The present studies optimize the purging protocol and examine the mechanism of alemtuzumab purging to identify the role of antibody dependant cell mediated cytotoxicity (ADCC) versus antibody dependant complement mediated cytotoxicity (Ab–C'). Both ADCC and Ab–C' mediated cytotoxicity were active, although ADCC was noticeably more effective. However, cellular viability was improved by incubation with 10% autologous serum. A two hour incubation (1–2×01e8 cells/ml) resulted in a 73% reduction in CD3+ cells, which was increased to 85% after a 16 hour co-incubation. In addition, natural killer (NK) and B cells were purged with alemtuzumab; as were lineage negative (Lin−)-HLA-DR+CD123+ dendritic cells (DCs) but not Lin-HLA-DR+CD11c+ DCs. Immature myeloid suppressor cells (IMSCs), which are Lin−DR−CD33+ or DR−CD14−CD11b+, have a low sensitivity to alemtuzumab cytotoxicity as did hematopoietic progenitor cells (CFU-c function or CD34+ number). This is an exciting observation as IMSCs have the potential to induce T cell tolerance. Optimal T cell purging, with retention of CD34+ hematopoietic stem cell number and activity, requires a 4–6 hr co-incubation of 2×10e8/ml hematopoietic stem cells, 100 ug/ml alemtuzumab in 10% autologous serum at 4C and removal of alemtuzumab by washing in Plasma-Lyte. Co-incubation at 4C and the presence of 10% autologous serum, was required to minimize the toxicity of a 24 hr co-incubation using a high frequency (1–2×10e8/ml) of hematopoietic cells. The selective depletion of T cells, with a retention of IMSCs, suggests that such a purging protocol has potential to not only reduce acute and chronic GVHD but to also induce major histocompatibility complex tolerance. However, our clinical studies need to be completed in order to assess the impact on leukemic relapse.