CITE-seq of CAR-NK cells

Primary chimeric antigen receptor (CAR) natural killer (NK) cells show strong cytotoxic efficacy against acute myeloid leukemia (AML) in vivo. However, NK cell-mediated tumor killing is often impaired by tumor-mediated immune cell inactivation. Here, we report a novel strategy to overcome NK cell inhibition caused by the immune checkpoint NKG2A, which interacts with HLA-E expressed on AML blasts. We generated AML-specific CD33-directed CAR (CAR33)-KLRC1ko-NK cells with CRISPR/Cas9-based gene editing of the NKG2A-encoding KLRC1 gene. Single-cell multi-omic analyses revealed a higher proportion of activated cells in CAR33-NK- and CAR33-KLRC1ko-NK pools, which were preserved following AML-cell contact. This activated state of the CAR33-KLRC1ko-NK cells has been translated into improved antileukemic activity in vitro and in vivo against AML cell lines and primary blasts. This dual modification of primary NK cells has the potential to bypass the suppressive effect not only of AML but also in a broad range of other cancer identities. For single cell sequencing analysis, the cryopreserved (genetically modified) NK cells from three different donors were thawed and seeded at 2x106 cells/ml in a 48-well plate in 750µl in NK-MACS® medium supplemented with 1% NK-MACS® Supplements, 5% human plasma, 1% Pen/Strep, 500 U/ml IL-2 (Miltenyi Biotec or Novartis) as well as 50 ng/ml IL-15 (CellGenix) per well. Three days later cells were harvested and co-cultivated with OCI-AML2 cells in 6cm dishes at an E:T ratio of 1:1. OCI-AML2 cells were previously stained with Cell Trace CFSE proliferation kit (Invitrogen) according to manufacturer’s protocol. As a control, NK cells were cultured without contact to AML cells. After two hours the plated cells were harvested, and an Fc-block was performed using hIgG antibodies (University Hospital Frankfurt). Subsequently cells were stained with an anti-NKG2A-PE antibody (Clone Z199, Beckman Coulter, Brea, CA, USA), CD33-CAR-detection reagent (CD33-protein-biotin + anti-biotin-APC (Clone RE746, Miltenyi Biotec) depending on the cell type and each condition was labeled with a different SampleTag from a human Single Cell Sample Multiplexing Kit (BD Biosciences, Franklin Lakes, NJ, USA). Subsequently, cells were stained with 7-AAD (BD Biosciences) to exclude dead cells and were sorted using a BD FACSAria™ machine. NK cells were sorted as follows: NT-NK as CFSE-, KLRC1ko-NK as CFSE-/NKG2A-, CAR33-NK as CFSE-/CAR+ and CAR33-KLRC1ko-NK cells as CFSE-/NKG2A-/CAR+ cells. Afterwards, single cell sequencing was performed using a BD Rhapsody™ Single-cell analysis system (BD Biosciences). **RAW data not provided due to patient privacy**