citations

                    CRISPR/Cas9 editing of NKG2A improves the efficacy of primary CD33-directed chimeric antigen receptor natural killer cells
                

Authors:

Bexte T, Albinger N, Al Ajami A, Wendel P, Buchinger L, Gessner A, Alzubi J, Särchen V, Vogler M, Rasheed HM, Jung BA, Wolf S, Bhayadia R, Oellerich T, Klusmann JH, Penack O, Möker N, Cathomen T, Rieger MA, Imkeller K, Ullrich E

In:

Source: Nat Commun.
Publication Date: (2024)
Issue: 15(1): 8439

Cells used in publication:

Natural killer Cells (NK), human: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® X-Unit

Experiment

KLRC1-targeting CRISPR/Cas9 nucleases
Primary NK cells were edited using the non-viral CRISPR/Cas9 system as recently described 25. Briefly, 1 × 10^6 pre-cultured (IL-2 + IL-15) nontransduced (NT)-NK or CD33-directed CAR (CAR33)-NK cells were nucleofected (4D-Nucleofector; Lonza) after one week posttransduction or additional culture using amounts of Cas9:gRNA according to the previously optimized NK cell nucleofection protocol25,53. Cells were seeded with 1 × 10^6 cells/ml in 500 µL culture medium. CAR33-KLRC1KO-NK cells and control cells were cultivated for at least 10 days following nucleofection before being deployed in functional assays in vitro or in vivo.

Abstract

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells show antileukemic activity against acute myeloid leukemia (AML) in vivo. However, NK cell-mediated tumor killing is often impaired by the interaction between human leukocyte antigen (HLA)-E and the inhibitory receptor, NKG2A. Here, we describe a strategy that overcomes CAR-NK cell inhibition mediated by the HLA-E-NKG2A immune checkpoint. We generate CD33-specific, AML-targeted CAR-NK cells (CAR33) combined with CRISPR/Cas9-based gene disruption of the NKG2A-encoding KLRC1 gene. Using single-cell multi-omics analyses, we identified transcriptional features of activation and maturation in CAR33-KLRC1^ko-NK cells, which are preserved following exposure to AML cells. Moreover, CAR33-KLRC1^ko-NK cells demonstrate potent antileukemic killing activity against AML cell lines and primary blasts in vitro and in vivo. We thus conclude that NKG2A-deficient CAR-NK cells have the potential to bypass immune suppression in AML.

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