Acute myeloid leukemia (AML) is a disease with high incidence of relapse that is originated and maintained from leukemia stem cells (LSCs). Hematopoietic stem cells (HSCs) can be distinguished from LSCs by an array of markers including CD123, CLL-1, amongst others. Thus, these are thought to be unique candidates to eliminate LSCs using a variety of targeted approaches, including chimeric antigen receptor (CAR) T cells. Here, we evaluated the potential of CLL-1 as a therapeutic target for LSCs using allogeneic gene-edited CAR T cells (UCARTCLL1). UCARTCLL1 cells are TCRαβneg human T cells derived from healthy donors, and expressing an anti-CLL-1 CAR under the control of the endogenous TRAC promoter. This is achieved following a 2-in-1 TRAC knock-out CAR knock-in strategy using TALEN® gene-editing technology. UCARTCLL1 cells are also knocked-out for the beta 2 microglobulin gene. We first assessed the expression of CLL-1 across samples from 41 AML patients and 4 healthy donors. The AML samples included 32 de novo and 9 relapse samples. Using multi-parameter flow cytometry, we evaluated CLL-1 expression on blasts and phenotypically defined leukemic stem cells (CD34pos CD38neg). We found that the majority of the blasts were positive for CLL-1. Furthermore, we found that 36 out of 41 primary AML samples had phenotypically defined stem cells (CD34pos CD38neg) from which 35 showed more than 0.1% CLL-1pos cells. Previous work has shown that individuals with more than 0.1% putative LSCs will have worse outcomes (Van Renhen et al). Thus, this data suggests that there is a subset of putative CLL-1 positive LSCs that if not eliminated by current therapies could lead to disease relapse. Therefore, we sought to determine the therapeutic potential of UCART cells targeting CLL-1. First, we evaluated cytotoxic efficacy of UCARTCLL1 cells at different effector to target ratios in 7 primary AML samples with different levels of CLL-1 expression. We observed a significant cytotoxic effect specific to the CLL1pos subsets. Notably, CLL-1pos cells in the AML samples treated with 5:1 and 1:1 ratios were completely eliminated. Currently, we are evaluating the in vivo activity of UCARTCLL1 cells in AML-PDX mouse models to determine their efficacy at eliminating AML blasts, progenitors and LSCs.

Citation Format: Antonio Freitas, Anne-Sophie Gautron, Mayumi Sugita, Srinjoy Goswami, Roman Galetto, Andrew Garton, Julianne Smith, Agnes Gouble, Michael W. Becker, Gail J. Roboz, Monica L. Guzman. CLL-1 specific chimeric antigen receptor T cells (CART) as a novel AML therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 541.