Acute myeloid leukemia (AML) is attractive for the development of CAR T-cell immunotherapy because AML blasts are susceptible to T-cell-mediated elimination. Here, we introduce sialic-acid-binding immunoglobulin-like lectin (Siglec)-6 as a novel target for CAR T-cells in AML. We designed a Siglec-6-specific CAR with a targeting-domain derived from a human monoclonal antibody JML‑1. We found that Siglec-6 is prevalently expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6-CAR T-cells confers specific anti-leukemia reactivity that correlates with Siglec-6-expression in pre-clinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6-expression on transformed B-cells in chronic lymphocytic leukemia (CLL) and show specific anti-CLL-reactivity of Siglec-6-CAR T-cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSC/P) and that treatment with Siglec-6-CAR T-cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6-CAR T-cell therapy may be used to effectively treat AML without a need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B-cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lacking expression of Siglec-6 on normal HSC/P is a key differentiator from other Siglec-family members (e.g. Siglec-3=CD33) and other CAR target antigens, e.g. CD123, that are under investigation in AML and warrants the clinical investigation of Siglec-6-CAR T-cell therapy.

Chimeric antigen receptors (CARs) are synthetic immune receptors that are expressed in T cells through genetic engineering. CAR-T cells have been successfully used to eradicate very advanced leukemias and lymphomas and their functional properties have been intensively studied. However, relatively little is known about the spatiotemporal expression and organization of CARs on the T-cell membrane and how this influences their efficacy. Here, we applied super-resolution microscopy to visualize CD19-, ROR1-, and ROR2-specific CARs in human CD4+ and CD8+ T cells that were engineered with lentiviral and transposon-mediated gene transfer. Our data show that the majority of CARs is organized in nanodomains virtually independent of the T cell type, CAR construct and expression level. Quantitative analyses revealed a slightly higher CAR density in transposon-engineered T cells correlating with higher antigen sensitivity and faster resolution of anti-tumor functions compared to lentivirally-engineered T cells. Live-cell fluorescence imaging revealed that both, CAR nanodomains and CAR monomers accumulate at tumor contact sites and form multifocal immunological synapses. Our study provides novel insights into the membrane organization of CARs with single-molecule resolution and illustrates the potential of advanced microscopy to inform the rational design of synthetic immune receptors for applications in immune cell therapy.