CRISPR-Cas9 based gene editing of the immune checkpoint NKG2A enhances NK cell mediated cytotoxicity against multiple myeloma

Bexte T, Alzubi J, Reindl LM, Wendel P, Schubert R, Salzmann-Manrique E, von Metzler I, Cathomen T, Ullrich E
Source: Oncol Rep
Publication Date: (2022)
Issue: 31;11(1): 2081415
Research Area:
Dermatology/Tissue Engineering
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
Natural killer Cells (NK), human
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit

Shortly, peripheral blood mononuclear cells (PBMCs) were thawed and activated using soluble anti-CD2/3/28 (Stem Cell Technology). At day 3 post-activation, 1x10^6 cells were nucleofected (4D-Nucleofector, Lonza; kit P3, program EO-115) with 20 pmol of SpCas9 protein (PNA Bio) complexed with 100 pmol of gRNA for 10 min at RT. T cells were cultured in 96 well plates (Corning, U bottom) up to 6 days before genotypic analyses. 1x10^6 pre-cultivated NK cells were nucleofected (4D-Nucleofector, Lonza; optimized NK cell nucelofection protocol recently described by Ullrich group 4) using the indicated amounts of Cas9:gRNA. NK cells were then expanded with IL-15 containing medium as recently described 3 up to two to three weeks before further analyses. MM1.S cells were cultured 2 days before genome editing. Afterward, 3 µg of SpCas9 protein (BioInc) was complexed with 100 pmol of gRNA for 10 min at RT, followed by nucleofection of 2x10^5 MM1.S cells (4D-Nucleofector, Lonza; kit SF, program CM-137).  


Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity, and the possibility to be applied as ‘off-the-shelf’ product makes them highly attractive for cell-based immunotherapies. In patients with multiple myeloma (MM), an elevated number of NK cells has been correlated with higher overall-survival rate. However, NK cell function can be impaired by upregulation of inhibitory receptors, such as the immune checkpoint NKG2A. Here, we developed a CRISPR-Cas9-based gene editing protocol that allowed us to knockout about 80% of the NKG2A-encoding killer cell lectin like receptor C1 (KLRC1) locus in primary NK cells. In-depth phenotypic analysis confirmed significant reduction in NKG2A protein expression. Importantly, the KLRC1-edited NK cells showed significantly increased cytotoxicity against primary MM cells isolated from a small cohort of patients, and maintained the NK cell-specific cytokine production. In conclusion, KLRC1-editing in primary NK cells has the prospect of overcoming immune checkpoint inhibition in clinical applications.