Der Knockout des Immun-Checkpoint-Inhibitor-Rezeptors NKG2A mit CRISPR/Cas9 verbessert die antileukämische Wirksamkeit von primären CD33-CAR NK Zellen
Tobias Bexte (Frankfurt a. M. / DE), Nawid Albinger (Frankfurt a. M. / DE), Ahmad Al-Ajami (Frankfurt a. M. / DE), Philipp Wendel (Frankfurt a. M. / DE), Leon Buchinger (Frankfurt a. M. / DE), Alec Gessner (Frankfurt a. M. / DE), Jamal Alzubi (Freiburg i. Br. / DE), Vinzenz Särchen (Frankfurt a. M. / DE), Meike Vogler (Frankfurt a. M. / DE), Hadeer Mohamed Rasheed (Berlin / DE), Beate Anahita Jung (Berlin / DE), Sebastian Wolf (Frankfurt a. M. / DE), Raj Bhayadia (Frankfurt a. M. / DE), Thomas Oellerich (Frankfurt a. M. / DE), Jan-Henning Klusmann (Frankfurt a. M. / DE), Olaf Penack (Berlin / DE), Nina Möker (Bergisch Gladbach / DE), Toni Cathomen (Freiburg i. Br. / DE), Michael A. Rieger (Frankfurt a. M. / DE), Katharina Imkeller (Frankfurt a. M. / DE), Evelyn Ullrich (Frankfurt a. M. / DE)
Chimeric antigen receptor (CAR) natural killer (NK) cells show safe and promissing clinical response and CAR33-NK cells demonstrated high antileukemic activity against acute myeloid leukemia (AML) in vivo (Albinger et al, BCJ, 2022). However, anti-tumor cytotoxicity is often impaired by tumor-mediated immune suppression (Bexte et al, OncoImmunology, 2022). Here, we report on a novel strategy to overcome CAR-NK cell inhibition by CRISPR/Cas9-based gene editing of the KLRC1-encoding gene encoding for the immune checkpoint NKG2A.
Primary CD33-CAR NK cells were lentiviral transduction. KO of the KLRC1 locus was performed by CRISPR-Cas9 nucleofection. Genotyping of KLRC1-edited NK cells was performed using Inference of CRISPR Edits (ICE) analysis. To determine gene and surface marker expression we used single-cell level using CITE-seq. The in vivo-efficacy was evaluated in OCI-AML2 (GFP+, Luc+) xenografted NSG-SGM3 mouse models.
The dual NK modification resulted in up to 60% CAR33-expressing cells, while KLRC1 gene disruption, quantified by insertion/deletion distribution profiles using Inference of CRISPR Edits (ICE) >93%, resulted in 50% reduction of NKG2A cell surface expression. Single-cell multi-omics downstream analyses revealed a distinct gene regulation pattern in CAR33- and CAR33-KLRC1ko-NK cells with more mature and activated NK cells, which was preserved following exposure to AML cells. Importantly, CAR33-KLRC1ko-NK cells showed significantly higher elimination of AML cells compared to KLRC1ko-NK or CAR33-NK cells in vitro against AML-cell lines as well as against patient derived AML samples with high-risk subsets. In a NSG-SGM3 AML-xenograft mouse model, already a low dose injection of CAR33-KLRC1ko-NK cells led to a complete elimination of AML and leukemia-initiating cells in the bone marrow in vivo. The complete deletion of bone marrow AML cells was confirmed by bone marrow re-engraftment in vivo survival analysis.
We conclude that dual modified NK cells have the potential to bypass immune suppression following contact with malignant cells not only in context of AML, but also in a broad range of other malignant diseases.
None of the authors has COIs directly related to this manuscript. T.B, P.W. and E.U. have patent applications for gene-modified NK cell immune cell therapies for AML.