Non-viral genome engineered CAR-NK cells for enhanced acute myeloid leukaemia targeting

Gurney, Mark
Rapid developments in adoptive cell transfer (ACT) immunotherapy have led to the widespread use of chimeric antigen receptor (CAR)-T cell therapies in B-cell malignancies and multiple myeloma. Aiming to meet the need for tolerable and curative therapies for acute myeloid leukaemia (AML), investigational CAR-T cell approaches must overcome the challenging antigenic profile of this disease. An ACT approach based on the innate anti-leukemic potential of natural killer (NK) cells provides an alternative solution. Optimization of genome editing technologies and investigation of genome editing targets in NK cells is of therapeutic interest. Herein, complementary technologies are combined and optimized to conduct a pre-clinical investigation of novel CAR-NK cell approaches to AML therapy. Initially, a viable approach to a CD38 CAR-NK cell approach is developed, overcoming the phenomena of NK cell fratricide through CRISPR/Cas9 genome editing with enhanced targeting of primary AML patient blast cells confirmed. Next, a non-viral approach to stable CAR transgene expression is optimized in donor-derived NK cells, using the TcBuster transposon system. The characteristics of the resultant production process are described and shown to be suited to clinical scale development. This system is used to investigate a potential CAR-NK cell therapy for AML targeting the leukemia stem cell (LSC) antigen CLL-1 (C-type lectin like molecule 1/CLEC12A). Enhanced targeting of a primary AML blast cell population that is enriched for LSCs is demonstrated. Finally, anticipating the limitations of a single antigen targeted approach, two adaptations to the process are used to investigate multiplex genome edits. Simultaneous knockout of the intracellular checkpoint molecule cytokine inducible SH2 containing protein (CISH), enhanced the cytotoxicity and metabolic phenotype of CLL-1 CAR-NK cells. Concurrent introduction of a variant of TNF-related apoptosis inducing ligand (TRAIL) selective for the DR4 death-inducing receptor enhanced innate NK cell anti-leukemic activity. Together the work herein describes a versatile optimization of transposon-based NK cell genome editing, contributes to the investigation of the targets CD38 and CLL-1 in AML immunotherapy, and the potential role of specific secondary genome edits on CAR-NK cell activity.
NUI Galway
Publisher DOI
Attribution-NonCommercial-NoDerivs 3.0 Ireland