OR07
CRISPR-edited allogeneic BCMA-CAR NK cells to overcome loss-of-target in multiple myeloma
T Bexte(1,2) L Krähl(1) M Baulig(1) A Moter(1) S Friedel(3) Z Ivics(4) T Cathomen(5,6) M Hudecek(3,8) E Ullrich(1,9)
1:Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy; 2:DRK Blutspendedienst Baden-Württemberg - Hessen, Institute for Transfusion Medicine and Immunohematology; 3:University Hospital Würzburg, Department of Internal Medicine II, Cellular Immunotherapy; 4:Fraunhofer Institute for Cell Therapy and Immunology Leipzig; 5:Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg; 6:Center for Chronic Immunodeficiency, Medical Faculty – University of Freiburg; 7:German Cancer Consortium (DKTK) partner site Freiburg; 8:Fraunhofer Institute for Cell Therapy and Immunology, Branch Site Würzburg - Cellular Immunotherapy; 9:German Cancer Consortium (DKTK) partner site Frankfurt/Mainz
Background: CAR immune cell therapies targeting B cell maturation antigen (BCMA) show promise for the treatment of Multiple Myeloma (MM). But tumor escape through BCMA loss limits their effectiveness. Natural killer (NK) cells offer potent killing capacity and a favorable safety profile as allogenic products in early clinical trials.
Here, we aim to overcome BCMA-target loss by enhancing the intrinsic anti-MM killing capacity of BCMA-CAR NK-cells through CRISPR-editing of inhibitory-immune-checkpoints. We present for the frist time, full non-viral protocol for CRISPR/Cas9-edited, SleepingBeauty (SB) BCMA-CAR NK-cells.
Methods: Primary NK cells were genetically modified using SB/minicircle-technology in parallel with CRISPR/Cas9-knockout of KLRC1. Genomic (TIDE) and phenotypical (flow cytometry) analysis were performed and anti-tumor functionality was addressed.
Results: BCMA-CAR NK-cells exhibit a similar expansion rate compared to non-transfected (NT) NK-cells. Long-lasting BCMA-CAR expression was monitored over four weeks. Significantly enhanced cytotoxicity of BCMA-CAR NK-cells compared to NT-NK cells was observed in end-point co-incubation and long-term live-cell imaging.
To model BCMA-target escape, we generated a MM1.S BCMA-knockout cell line, and confirmed loss of CAR-dependent anti-tumor capacity. To overcome tumor escape, NT and BCMA-CAR NK-cells were modified using CRISPR/Cas9-deletion of the KLRC1 gene (encoding for NKG2A), in an efficient one-step nucleofection process. The fully virus-free engineered NKG2A-KO-BCMA-CAR NK-cells exhibited improved cytotoxic capacity compared to NT, CAR, or NKG2A-KO NK-cells against MM.
Conclusion: Advanced engineering of CAR-NK cells has the potential to overcome limitations of current immune cell therapy in MM. Our CAR-FACTORY-consortium, funded by German-Cancer-Aid, will support GMP manufacturing for upcoming phase I/II clinical studies.