Potent Target-Specific Efficacy of CAR T Cells Directed Toward the AML and KMT2A-Rearranged Specific Antigen CLEC2A

KMT2A rearrangements (KMT2A-r) are a frequent event in childhood acute myeloid leukemia (AML) and in treatment-related AML in adults where they are associated with a highly refractory and deadly subtype of AML. Targeted therapies for KMT2A-r AML, including menin inhibitors, have had limited success with novel therapies urgently needed. Despite successes in other leukemias, immunotherapy in AML remains difficult, in part because of significant overlap of antigens expressed in AML and normal hematopoietic cells, which limit efficacy and increase hematopoietic toxicity. To identify novel therapeutic targets in KMT2A-r AML, we interrogated the AML transcriptome compared to normal hematopoietic cells to identify tumor-specific antigens that are both highly expressed in AML and absent in normal hematopoiesis. We discovered CLEC2A to be an AML-restricted target highly enriched in KMT2A-r AML and silent in normal hematopoiesis. As we previously reported, CLEC2A is particularly enriched in the highest risk KMT2A-r AML subtypes and entirely silent in virtually all normal tissue (Kirkey, Blood 2023). Following validation of CLEC2A cell surface expression in AML, we generated a novel fully human antibody, engineered CLEC2A targeting CAR T cells and evaluated preclinical efficacy in AML models.

Novel fully human CLEC2A binders were tested against a variety of CLEC2A positive and negative AML cell lines to identify the most specific and efficacious CLEC2A binder. The top CLEC2A binder was a human VH single domain antibody, which was cloned into our CAR T cell construct backbone, which includes an IgG4 hinge with intermediate spacer and a 41BB/CD3z costimulatory domain. In vitro cytotoxicity and cytokine production of CLEC2A CAR T cells were assessed in both CLEC2A positive and negative cell lines compared to unmodified T cell controls. In vivo efficacy was evaluated in OCI-AML2 and MOLM13 ffluciferase leukemia-bearing NSG mice treated with CLEC2A CAR T cells compared to unmodified T cells. Leukemia burden was assessed by bioluminescent imaging and flow cytometry of peripheral blood, bone marrow, liver and spleen.

In vitro evaluation of our novel CLEC2A CAR T cells demonstrated robust and target-specific cytolytic activity and pro-inflammatory cytokine (IL2, IFNγ and TNFα) production when co-incubated with OCI-AML2 cells (which show high CLEC2A expression) and NOMO-1 cells (displaying low CLEC2A expression) across multiple effector:target cell ratios, but not with CLEC2A-negative MOLM13 cells (p<0.0005). Using a preclinical AML cell-derived xenograft in vivo model, we engrafted NSG mice with either CLEC2A-positive OCI-AML2 cells or CLEC2A-negative MOLM13 cells followed by treatment with 5e6 CLEC2A CAR T or unmodified T cells. All 6 control animals engrafted with OCI-AML2 cells died by Day 31 from progressive leukemia with evidence of extramedullary disease, whereas all but one of the CAR T treated mice remained disease free thru Day 66 (p<0.001). In this aggressive leukemia model, one CAR T-cell treated mouse developed extramedullary disease with no evidence of leukemia in the marrow. CLEC2A CAR T cells demonstrated robust expansion and persistence with detectable CAR T in the peripheral blood in all CAR T treated mice at Day 27. In the CLEC2A-negative MOLM13 cell-derived xenograft model, all mice rapidly developed progressive leukemia highlighting the specificity of our CLEC2A CAR T cells.

Our data indicate that CLEC2A is a novel KMT2A-r AML-specific antigen whose expression is absent in normal hematopoietic cells. CLEC2A-directed CAR T cells show potent and target-specific cell killing in both high CLEC2A antigen and low antigen density models. Given the restricted expression of CLEC2A in KMT2A-r AML and high potency of CLEC2A CAR T cells in preclinical models, we hold that CLEC2A CAR T cell therapy is an ideal therapeutic modality for this aggressive leukemia and are currently advancing to the clinical development stage.