Metabolic reprogramming of Trimeric APRIL-CAR-engineered Natural Killer (NK) cells to enhance tumor-cell recognition, in vivo persistence and anti-tumor potency in Multiple myeloma
Final Report Abstract
CAR-NK cell immunotherapy has emerged as a promising treatment option for patients with relapsed and refractory cancers. First clinical trials have demonstrated the safety and efficacy of genetically engineered allogeneic natural killer cells to induce remissions in heavily pretreated patients with lymphoid malignancies. Despite these successes, eventual functional exhaustion and tumor relapse remain a pre-eminent concern. Engineering next-generation NK cell therapies which are more potent, can resist functional exhaustion and can overcome immunosuppressive cues, hence, promises to address the clinical demand for better therapies. Therefore, understanding the immune cell-intrinsic checkpoints and signaling mechanisms which ultimately restrict NK cell anti-tumor immunity is imperative. Genome-wide forward genetic studies including loss-of-function CRISPR screening can help interrogate cellular dependencies in an unbiased and massively parallel manner. In this study, we performed the first genome-wide loss-of-function CRISPR screen in primary human NK cells testing multiple biologically relevant selective pressures including different tumor co-culture conditions as well as a spectrum of TME (tumor microenvironment) -associated immunosuppressive factors. Cord-blood derived NK cells were transduced with a genome-wide sgRNA library and transfected with recombinant Cas9 to investigate genotype-phenotype correlations in a pooled format. We found multiple enriched sgRNA, indicating improved NK cell fitness under different selective conditions. The identified hits were functionally validated against multiple human cancer cell lines to prioritize the most promising candidates for further characterization. After CRISPR/Cas9-RNP-mediated deletion of the identified checkpoints, we observed significant increases in NK cell-mediated anti-tumor immunity. We corroborated our findings in the context of NK cells transduced with two different CAR constructs, demonstrating that our identified top hit was able to augment NK cell killing both in an antigen-unspecific as well as antigen-specific manner. We complemented our studies by testing CRISPR-engineered CAR- NK cells in vivo in two different xenograft mouse models. In conclusion, we leveraged for the first time unbiased genome-wide forward genetic screening of primary human NK cells to identify functional checkpoints which can enhance NK cell killing of human cancers when deleted. Our findings have high translational relevance as they can inform the design of next-generation NK cell-based immunotherapies with increased anti-cancer potency.
Publications
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Engineering the next generation of CAR-NK immunotherapies. International Journal of Hematology, 114(5), 554-571.
Biederstädt, Alexander & Rezvani, Katayoun
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KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape. Nature Medicine, 28(10), 2133-2144.
Li, Ye; Basar, Rafet; Wang, Guohui; Liu, Enli; Moyes, Judy S.; Li, Li; Kerbauy, Lucila N.; Uprety, Nadima; Fathi, Mohsen; Rezvan, Ali; Banerjee, Pinaki P.; Muniz-Feliciano, Luis; Laskowski, Tamara J.; Ensley, Emily; Daher, May; Shanley, Mayra; Mendt, Mayela; Acharya, Sunil; Liu, Bin ... & Rezvani, Katayoun
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Multiplexed engineering and precision gene editing in cellular immunotherapy. Frontiers in Immunology, 13.
Biederstädt, Alexander; Manzar, Gohar Shahwar & Daher, May
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Natural killer cells in antitumour adoptive cell immunotherapy. Nature Reviews Cancer, 22(10), 557-575.
Laskowski, Tamara J.; Biederstädt, Alexander & Rezvani, Katayoun
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How I treat high-risk acute myeloid leukemia using preemptive adoptive cellular immunotherapy. Blood, 141(1), 22-38.
Biederstädt, Alexander & Rezvani, Katayoun
