Abstract
Identifying suitable therapeutic targets in childhood B cell precursor acute lymphoblastic leukaemia (BCP ALL) to develop more specific, less toxic, therapies is an ongoing challenge. CD200 (OX-2) is a type-1 membrane glycoprotein that is overexpressed in many haematological malignancies, including ALL. We have previously shown that CD200 antigen levels were significantly higher (~60%) in BCP ALL compared to normal bone marrow cells (0.1%). In addition, in low risk minimal residual disease (MRD) samples, only CD200+ cells could initiate leukaemia in NOD.Cg-Prkdcscid Il2rgtm1Wjl/Sz (NSG) mice, suggesting that CD200 may have a role in ALL initiation and progression in these cases. CD200 is also a key immunosuppressive molecule. When bound to its receptor (CD200R), found on monocytes/macrophages and some T cells, it suppresses the immune system by inhibiting the action of the CD200R-bearing cells. Anti-CD200 antibodies (Ab) exert their effect by binding to CD200 antigen and blocking its interaction with CD200R and not by directly inducing cell death. Treating chronic lymphocytic leukaemia cells with anti-CD200 Abs has been shown to prevent engraftment in NSG mice, demonstrating their potential for therapy in haematological cancers that overexpress CD200. The aim of this study was to investigate the effects of monoclonal anti-CD200 Abs on the viability and functional capacity of childhood BCP ALL cells. Primary BCP ALL samples, expressing varying levels of CD200, were randomly selected and the effects of anti-CD200 Abs were assessed in a mixed lymphocyte reaction (MLR). In parallel, the production of interleukin (IL)-2 was measured using ELISA, as an indicator of immune system activation. Monocytes were isolated from blood of healthy donors and cultured for 7 days with 50ng/ml macrophage-colony stimulating factor. At day 7, macrophages were mixed 1:1 with BCP ALL cells with or without anti-CD200 Ab. CD4+ T cells from the same normal donor were added to the plates after 2 hours in a 1:5 ratio (macrophages : T cells). MLR plates were incubated for 72 hours at 37°C, then supernatants analysed by ELISA for IL-2 production and cells were stained with propidium iodide for flow cytometric analyses. When anti-CD200 was added to BCP ALL cells, the amount of IL-2 produced increased significantly. In cases with high CD200 expression (87 - 93.4%) a 16-18.4 fold increase in IL-2 was observed. In cases with very low expression (<0.9% CD200+) only a 3.2 fold increase was observed, as might be expected. Antibody treatment reduced cell viability by 5-7% in cases with high CD200 expression and by 5% in those with low levels. As a more relevant measure of toxicity, the effects of anti-CD200 Ab were investigated in vivo using 2 MRD low and 2 MRD risk cases. Unsorted cells and both CD200+ and CD200- subpopulations were inoculated into NSG mice and once human leukaemia levels in PB were ≥0.1%, animals received 4 doses of anti-CD200 (20mg/kg i.v.) over 10 days. In low risk cases, a 13.5±24.6 fold reduction in leukaemia burden was observed after only 2 doses, while leukaemia levels in the placebo treated group increased by 5.2±9 fold. Similar effects were observed in mice engrafted with CD200+ cells, with a 1.5±16 fold reduction in leukaemia burden in the Ab treated mice while leukaemia levels increased 4.8±3.8 fold in the placebo group. NSG mice engrafted with MRD risk cases were also treated to assess whether this approach would only apply to low risk cases, which our previous functional analyses had indicated. Engraftment with all inoculated populations was rapid and Ab treatment did not delay progression or reduce disease burden, confirming other approaches will be required for MRD risk cases. Since many therapeutics have adverse side effects, including cardiac toxicity, we investigated the effects of anti-CD200 Abs on primary cardiac myocytes in vitro. The antibodies had no effect on viability or IL-2 production compared to controls. In conclusion, these are very promising results for use of anti-CD200 Abs to treat low risk BCP ALL and further in vivo investigation in a larger cohort of patients is warranted.
Disclosures
Petrova: Trillium Therapeutics Inc: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Uger:Trillium Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
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