319 Transcriptome-guided design of logic-gated CAR T cells for treatment of acute myeloid leukemia

BackgroundIn acute myelogenous leukemia (AML), CD93 is an appealing target for chimeric antigen receptor (CAR) T-cell therapy, offering leukemic killing without myelosuppression in preclinical studies.1 However, CD93 CAR T-cell development is hampered by CD93 expression on healthy endothelium. Our group previously demonstrated the efficacy of an inhibitory CAR (iCAR) to counteract off-tumor CD93-directed activating CAR (aCAR) signaling in a model system.1 Here, we identify endothelial-specific iCAR targets in resting and cytokine-activated states to minimize off-tumor toxicity for logic-gated (a NOT b) CD93 CAR T cells.MethodsWe analyzed differentially expressed genes (DEGs) from RNA-Seq of 3 AML (Kasumi-1, THP-1, NOMO-1) and 2 endothelial (iHUVEC, TIME) cell lines, incubated with and without proinflammatory cytokines (IFNγ, TNFα). We applied a statistical filter (FDR10) to identify genes specific to endothelial cells and selected surface-expressed genes using annotations from a surfaceome database (figure 1).2 DEGs present in both resting and cytokine-activated conditions were reviewed as potential iCAR targets and cross-referenced with CD93 in a healthy transcriptome atlas (Tabula Sapiens) and 3,225 pediatric AML samples (TARGET-AML).3 4 ResultsFrom the thousands of DEGs in resting and cytokine-activated conditions, statistical and surfaceome filters reduced DEGs by 99%, with only half of remaining genes (N=16) upregulated in both conditions. Among these, we identified 3 potential iCAR targets (CHD5, KDR, TEK) encoding well characterized, single-pass receptors with strong endothelial cell expression (figure 2A). Validating our cell line data, we found iCAR targets expressed along with CD93 in all 21 organ-specific endothelial cell subpopulations in Tabula Sapiens (figure 2B). By contrast, median expression of iCAR targets was extremely low in TARGET-AML samples (1), with median expression twice that of existing AML immunotherapy target CD33 (CD93, 36.7 TPM; CD33, 17.2 TPM).ConclusionsOur findings suggest that VE-cadherin (CDH5), VEGFR-2 (KDR), and Tie2 (TEK) could function as iCAR targets in NOT-gated CD93 CAR T cells for AML, predicted to protect endothelium without sheltering leukemic cells. Limited overlap of endothelial-specific DEGs in resting