Rabbit Anti-Thymocyte Globulin Kills Leukemic Stem Cells to a Greater Degree Than Healthy Hematopoietic Stem Cells

Abstract

Background: Rabbit anti-thymocyte globulin (ATG, Thymoglobulin) is given with hematopoietic cell transplant (HCT) conditioning to prevent graft-versus-host disease (GvHD). Recently we showed that in vitro ATG at clinically relevant concentrations kills not only T cells but also leukemic blasts (Dabas et al., BBMT 2016). This might explain the non-increase in relapse incidence in patients given ATG vs no ATG (Walker et al., Lancet Oncol 2016). Nevertheless, relapse of leukemia is still the most frequent cause of HCT failure. After chemotherapy (without HCT), non-relapse is associated with eradication of leukemic stem cells (LSCs). By extrapolation, non-relapse after HCT may also require the eradication of LSCs. We have shown that ATG kills LSCs, however, at a relatively high concentration of 50 mg/L. We next studied whether at that high concentration ATG kills also healthy hematopoietic stem cell (HSCs), as attempts to achieve the high concentration clinically would be indicated only if ATG killed LSCs but not HSCs.Study design: To study the cytotoxic effect of ATG on LSCs and HSCs, we used cryopreserved blood mononuclear cells (MNCs) from 8 newly diagnosed acute myeloid leukemia (AML) patients as a source of LSCs. The median percentage of blasts in the blood of the AML patients was 53% (range 24% to 90%). Healthy volunteers' cryopreserved blood MNCs (n=8) or cryopreserved filgrastim-mobilized apheresed blood MNC grafts from healthy donors (n=17) were used as the source of HSCs. The MNCs from the blood of AML patients and healthy volunteers were magnetically enriched for CD34+ cells (Miltenyi Biotech). This was not needed for the grafts as they contained a sufficient number of CD34+ cells for the assay of ATG killing. By flow cytometry, we measured stem cell killing induced by 15 min incubation with ATG (50 mg/L) and active complement (human serum). For negative control, cells were incubated with ATG in the presence of no active complement (heat-inactivated human serum). The LSCs and HSCs were phenotypically defined as CD45dim/neg, side scatterlow, CD34+, CD38neg and negative for the lineage markers (CD14, CD16, CD19, CD56, CD3, CD235a and CD41a). Dead cells were identified as 7-amino-actinomycin D positive (7AAD+).Results: Treatment of LSCs with 50 mg/L ATG in the presence of complement resulted in median 43% dead (7AAD+) cells (background subtracted). In contrast, treatment of blood HSCs and graft HSCs resulted in 3% and 13% dead cells, respectively (Figure 1).Conclusion: Here we show that in vitro LSCs are more sensitive to ATG mediated killing than healthy HSCs. However, the killing of LSCs occurs only at a relatively high ATG concentration (50 mg/L) which is ~5-fold higher than the maximum serum concentration achieved with a typical ATG dose (4.5 mg/kg). If ATG had the anti-LSCs activity also in vivo, using high dose ATG (leading to serum concentration of 50 mg/L) in patients could result in simultaneous decrease of both GVHD and relapse. Given the minimal effect on HSCs, the high dose ATG might not jeopardize engraftment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.