INTRODUCTION: Myelodysplastic syndrome (MDS) is a group of heterogeneous disorders caused by ineffective hematopoiesis characterized by bone marrow dysplasia resulting in cytopenias. Currently, treatment options for MDS are limited to supportive care and hypomethylating agents with most patients eventually succumbing to the disease (anemia, infection or hemorrhage) or progressing to leukemia. Previously it have been demonstrated that CD123 is an important target for the treatment of MDS due to its aberrantly high expression in high-risk MDS cells compared to low-risk MDS cells and normal cells. As such, CD123-directed therapy may be useful in treating patients with high-risk MDS.

METHODS: Chimeric antigen receptor (CAR) vector containing a CD123-specific single-chain variable fragment in combination with a CD28 costimulatory domain, CD3ζ signaling domain and tEGFR was expressed on healthy donor and patient derived T lymphocytes utilizing lentiviral vector delivery to target high-risk MDS cells. An anti-CD19 CAR was used as a negative control. CAR expressing or control T-cells were cocultured with MDS cell line (MDS-L) or primary MDS cells from patient bone marrow to examine in vitro anti-tumor function. Eradication of MDS cells was analyzed by flow cytometry 48hr after coculture. Intracellular TNF-α and cell surface CD107A were examined in effector cells 5 hours after cocultured with MDS cells. For in vivo analysis of CD123 CAR T cells, NSG-S mice were engrafted with 1.5x106 primary high-risk MDS cells for eight weeks, and were infused with autologous anti-CD123 CAR T-cells or control. Tumor burden was then analyzed on day 28.

RESULTS: Healthy donor derived anti-CD123 CAR T-cells effectively eliminated MDS cell line (MDS-L) (>99%), and primary bone marrow derived MDS cells (>70%) in vitro . The killing was associated with increased cytokine release and degranulation by anti-CD123 CAR T-cells, which did not occur with control anti-CD19 CAR T-cells. In addition, anti-CD123 CAR T-cells generated from patients with high-risk MDS showed 50~70% transduction efficiency. These cells successfully eradicated autologous CD123+ MDS cells at >99.9% in vitro compared to untransduced control T-cells (Figure 1A). The killing was also confirmed by increased intracellular TNF-α expression (Figure 1B). Furthermore, a patient derived xenograft system (PDX) was utilized to examine the killing ability of autologous anti-CD123 CAR T-cells in vivo . High-risk MDS bone marrow cells were engrafted in NSG-S mice. Eight weeks following engraftment, 7x104 autologous EGFRt+ anti-CD123 CAR T-cells or mock T-cells were infused via tail vein. On day 28 after autologous anti-CD123 CAR T-cell infusion, we observed significant decreases in both CD34+/CD38-/CD123+ MDS cells and MDS bulk (hCD45+/EGFRt-) while mice infused with mock CAR T-cells were unable to reduce tumor burden (Figure 1C and 1D). To extend the utility of our approach, we performed drug screening to identify the agents that increase expression of CD123 on MDS cells. We hypothesize that drugs inducing increased expression of CD123 will increase efficacy of anti-CD123 CAR T cell and avoid resistance arising from CD123 low/dim MDS progenitor cells. 134 various oncologic drugs were dosed at multiple concentrations (1nM, 100nM, 1uM, 0uM) and incubated with MDS-L cell line for 6hr, 16hr or 48hr. MDS-L cells were then analyzed for CD123 expression (Mean fluorescence) and cell viability (%live out of total) by flow cytometry. Eight drugs with low cytotoxicity significantly increased CD123 expression, including trifluridine (1uM), sunitinib (100nM), metoxantrone (1nM), dasatinib (10uM), imatinib (1uM), regorafenib (10uM), dabrafenib (100nM) and DEAB (100nM). These drug candidates will be examined further in primary MDS bone marrow samples and in combination with anti-CD123 CAR T-cells.

CONCLUSION: These results demonstrate that anti-CD123 CAR T-cells exhibit activity against high-risk MDS, and represent an effective therapeutic option for patients with high-risk MDS.


Budde: Mustang Therapeutics: Research Funding; Merck: Research Funding, Speakers Bureau; Amgen: Research Funding; KITE Pharmaceutical: Speakers Bureau; Precision Biosciences: Consultancy.

Author notes


Asterisk with author names denotes non-ASH members.