{
  "abstract": "Background CD93 chimeric antigen receptor (CAR) T cells efficiently kill acute myeloid leukemia (AML) but risk endothelial cell (EC) toxicity due to CD93 expression on ECs. One solution for this on-target, off-tumor (OTOT) toxicity is ‘NOT-gating,’ where a second inhibitory receptor (iCAR) that inactivates CAR T cells upon binding healthy cells is added to increase CAR T-cell precision. Here we describe a novel VE-cadherin iCAR that protects ECs from CAR T-cell killing without impairing antileukemic activity.Methods We compared the transcriptional signatures of AML and ECs both at rest and in cytokine-activated states (IFNγ, TNFα) using RNA-seq. Protein-level expression for iCAR targets was confirmed with flow cytometry. Single-chain fragment variable sequences recognizing VE-cadherin were inserted into a bicistronic viral vector to generate CAR T cells expressing both CD93 CAR and VE-cadherin iCAR constructs. Controls included CAR T cells lacking a functional inhibitory intracellular domain with the VE-cadherin iCAR (Pdel). Incucyte killing assays compared viability of AML cells and ECs following exposure to CAR T cells over 72 hours at various effector-to-target (E:T) ratios (1:2, 1:4, 1:8). Additional Incucyte assays were performed in which CAR T cells were co-cultured with either AML or ECs for 48 hours and then added to the alternate cell type (AML to EC; EC to AML) for 48 hours.Results We identified VE-cadherin as a potential iCAR ligand that was stably upregulated on the mRNA level in ECs but not AML, both at rest and in cytokine-activated conditions ( figure 1A). As predicted by RNA-seq, protein-level expression of VE-cadherin was high on ECs and not detected on AML (figure 1B-C). Using the bicistronic vector, we successfully generated NOT-gated CAR T cells that killed AML but spared ECs over multiple E:T ratios (figure 2A-B). By contrast, control cells that lacked the inhibitory iCAR domain (Pdel) failed to protect ECs from CAR T cell-directed killing, indicating that the AML specificity was conferred by inhibitory signaling through the iCAR construct. Cytokine production also increased with exposure to AML but not ECs (figure 2C). Importantly, EC protection was sustained even with prior exposure of CAR T cells to activating AML cell targets (figure 2D).Conclusions NOT-gated CAR T cells killed AML while sparing ECs at risk for OTOT toxicity, including after prior CAR T cell activation. This strategy supports the development of CD93 CAR T cells for AML and may apply more generally to other cancers lacking cell therapy options.Acknowledgements We thank Dr. Barry Gumbiner for providing VE-cadherin antibody sequences to generate the scFv for our iCAR.Abstract 294 Figure 1A. Gene expression of VE-cadherin, at rest and with cytokine activation. B. VE-cadherin protein expression by flow cytometry. C. VE-cadherin and CD93 with exposure to IFNγ or supernatant (SN) from 24-hour AML/CD93 CAR T-cell co-cultureAbstract 294 Figure 2A. CAR and iCAR constructs. B. EC or AML survival in 48-hour co-culture with CAR T cells, averaged over 3 replicates (1:4 E:T). C. IFNγ and IL2 in supernatant from a killing assay. D. EC or AML survival after CAR T-cell exposure to opposite cell type",
  "authors": [
    {
      "affiliations": [
        "University of Wisconsin-Madison, Madison, WI, USA"
      ],
      "name": "Therese Woodring"
    },
    {
      "affiliations": [
        "University of Wisconsin-Madison, Madison, WI, USA"
      ],
      "name": "Otto Kletzien"
    },
    {
      "affiliations": [
        "University of Wisconsin-Madison, Madison, WI, USA"
      ],
      "name": "Rebecca M Richards"
    }
  ],
  "title": "294 NOT-gated chimeric antigen receptor (CAR) T cells kill acute myeloid leukemia and spare endothelial cells via VE-cadherin inhibitory CAR",
  "uid": "6c8a92b5-8ecb-51b7-9698-0528a3076830"
}