CAR therapy is efficacious in relapsed/refractory CD19-positive malignancies including B-ALL. In AML, conventional 2nd-generation CAR therapies are being evaluated clinically. Some of the potential obstacles are antigen-low leukemia escape and on-target toxicities due to widespread CAR target expression in normal hematopoietic cells. We hypothesized that novel targets and fine-tuned combinatorial CAR approaches may enable distinctive target elimination based on differential antigen co-expression patterns in AML vs normal cells, thereby maintaining anti-leukemic efficacy while limiting toxicity on normal cells. Here, we present our target phenotypic profiling of primary AML (n>30 r/r AML patients) and normal hematopoietic cells, and link it with CAR efficacy and changes in normal hematopoiesis. Using spectral flow cytometry on bone marrow and peripheral blood from AML patients and healthy controls, we quantified differential target co-expression of several AML targets, including CD33, CD123, CLEC12A and the novel target ADGRE2. We estimated the numbers of surface molecules per cell and created two-dimensional maps of combinatorial target densities in AML vs normal. In this analysis, the combination of ADGRE2+CLEC12A offered the best therapeutic window. We developed a combinatorial CAR+CCR platform, ADCLEC.syn1, consisting of an ADGRE2-targeting 28z1XX-CAR and a CLEC12A-targeting chimeric costimulatory receptor (CCR) providing additional 4-1BB costimulation. This CAR+CCR configuration triggers killing of cells with high CAR target density alone, whereas cells with low CAR target density are only killed if the CCR target is co-expressed (IF-BETTER gate). Both in vitro and in vivo, we used MOLM13 AML cell line variants matching a variety of different ADGRE2 and CLEC12A target density combinations found in AML or normal hematopoietic cells. The observed target density thresholds for ADCLEC.syn1-mediated cell lysis demonstrated potential for sparing vital normal hematopoietic cells. In addition, we evaluated ADCLEC.syn1 in several molecularly distinct AML PDX models with clinically representative target phenotypes, achieving long-term AML remissions in all studied PDX models. In a humanized mouse model co-engrafted with MOLM13 AML cells, ADCLEC.syn1 T cells rapidly induced complete and durable AML remissions in all mice, with minimal toxicity of normal hematopoietic cells compared to control CARs. Overall, we profiled antigen densities of several CAR target combinations in AML and normal hematopoietic cells, which informed the development of the ADCLEC.syn1 platform. Pre-clinical in vivo models based on AML with clinically representative target densities demonstrated high anti-leukemic efficacy of ADCLEC.syn1 and established killing thresholds with potential for sparing normal hematopoietic cells. A first-in-human phase 1 clinical trial evaluating ADCLEC.syn1 T cells is anticipated to be initiated at MSKCC in 2023.
Citation Format: Sascha Haubner, Jorge Mansilla-Soto, Sarah Nataraj, Friederike Kogel, Qing Chang, Elisa De Stanchina, Kathryn Fraser, Jae H. Park, Xiuyan Wang, Isabelle Rivière, Michel Sadelain. ADCLEC.syn1 CAR T cells detect combinatorial target signatures to safely prevent antigen-low AML escape [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr PR03.