CD33 Directed Chimeric Antigen Receptor T Cell Therapy As a Novel Preparative Regimen Prior to Allogeneic Stem Cell Transplantation in Acute Myeloid Leukemia

Allogeneic stem cell transplantation (allo-SCT) is the only potentially curative option in relapsed or high risk acute myeloid leukemia (AML). However, patients are often excluded due to refractory disease. Chimeric Antigen Receptor (CAR) T cell therapy has shown significant efficacy in acute lymphoid leukemia and its successful translation to AML would constitute a vertical advance in the field. 

CD33 is known to be expressed on AML blasts. We developed a 2^nd generation CAR from the anti-CD33 scFv of Gemtuzumab ozogamicin, 41BB costimulation, CD3ζ signaling domain, and a lentiviral (LV) vector. T cells were transduced with this construct and expanded in culture using anti CD3/CD28 magnetic beads (CART33 cells).

In vitro, CART33 cells resulted in robust functions when incubated with the CD33+ cell line MOLM14 (Table 1). In NSG-S mice (NOD-SCID-γc^-/- (NSG), additionally transgenic for human stem cell factor, IL3 and GM-CSF) engrafted with primary AML blasts, CART33 treatment eradicated AML within 4 weeks and led to a long term disease free survival (Figure 1).

CD33 is also expressed on normal myeloid progenitors. Therefore, treatment with CART33 can potentially result in myelotoxicity. We assessed the potential for myeloablation of CART33 in humanized immune system (HIS) mice (NSG mice engrafted with human fetal liver CD34+ cells). These mice were treated with CART33 cells or control T cells. CART33 treatment resulted in human lineage cytopenias and in a significant reduction of CD34+CD38- hematopoietic stem cells and CD34+CD38+ myeloid progenitors in the marrow.

Both the anti-leukemic activity and myeloablative potential of CART33 could be used as a novel cellular conditioning regimen in refractory AML. However, the effect of the CAR T cells would have to be terminated prior to infusion of donor stem cells in order to avoid engraftment failure. We therefore designed a transiently expressed “biodegradable” mRNA modified CAR based on CART33. T cells were electroporated with this construct and expressed CAR for up to six days. When compared with LV transduced CART33, RNA-modified CART33 cells resulted in comparable in vitro effector functions that declined over time post electroporation, a desirable property in the setting of the myeloablative potential that was seen in the LV-CART33. We then tested the activity of RNA-CART33 in combination with chemotherapy against leukemia in AML xenografts. NSG mice were engrafted with MOLM14 and treated either with cyclophosphamide plus RNA-CART33 or with cyclophosphamide plus control T cells. The combination of RNA-CART33 and chemotherapy resulted in deeper and longer leukemic response (figure 2).

In conclusion, our preclinical studies show potent activity CART33 against AML. Biodegradable CART33 could potentially be used as a novel anti-leukemic cellular conditioning regimen in patients with relapsed AML without rejection of the donor graft.