In vivo perturbomics identifies CALHM2 as a checkpoint of NK cell therapy [AAV-CRISPR screen]

Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential; However, multiple current limitations exist that naturally hinder the successful implementation of NK cell therapy against cancer, including their effector function, persistence, and tumor infiltration. To unbiasedly identify genes underlying critical NK cell characteristics against cancer, we perform functional mapping of tumor infiltrating NK (TINK) cells by both in vivo AAV-CRISPR screens and single-cell sequencing. We establish a strategy with AAV-SleepingBeauty(SB)-CRISPR screening leveraging a focused high-density sgRNA library, and perform four independent in vivo tumor infiltration screens of primary NK cells in mouse models of melanoma, breast cancer, pancreatic cancer, and glioblastoma. In parallel, we characterize single-cell transcriptomic landscapes of tumor-infiltrating NK cells, which identifies previously unexplored sub-populations of NK cells with distinct expression profiles, a shift from immature to mature NK (mNK) cells in the tumor microenvironment (TME), and decreased expression of mature marker genes in mNK cells. CALHM2, a calcium homeostasis modulator previously not linked to NK function, emerges as a convergent hit from both CRISPR screen and single-cell data. CALHM2 knockout NK cells show enhancement of in vitro cytotoxicity and in vivo tumor infiltration in both mouse primary NK and human chimeric antigen receptor (CAR)-NK cells. Re-introduction of CALHM2 mRNA reverses CALHM2 knockout phenotype of NK cytotoxicity and degranulation. Importantly, in a solid tumor model that is completely resistant to adoptive cell therapy of unmodified CAR-NK cells, CALHM2 knockout CAR-NK cells show potent in vivo anti-tumor efficacy. Human primary NK cell study with multiple donors reveals that CALHM2 knockout enhances cytotoxicity, degranulation and cytokine production of NK cells. Transcriptomics profiling of human primary NK cells reveal multiple enriched pathways of downstream genes upon CALHM2 knockout in both baseline and stimulated conditions. These data identify endogenous cellular genetic checkpoints that naturally limit NK cell function, and pinpoint CALHM2 as a key factor in which genetic modification can be engineered to enhance NK cell-based immunotherapies. Overall design: The AAV-CRISPR screen was performed with >400x coverage, in which > 5e7 Cas9+ NK cells were transduced with an approximate ~50% infectivity rate, using the AAV-Surf-v2 viral library (0.5 infectivity * 5e7 cells / 61,911 sgRNAs > 400-fold coverage). Naïve NK cells were isolated from the spleens of Rsky/Cas9? mice. Syngeneic mouse models of melanoma, GBM, and pancreatic cancers were setup with subcutaneous injections of 2e6 B16F10, 5e6 GL261, or 4e6 Pan02 cells, respectively. Syngeneic mouse models of breast cancer were established by fat-pad injections of 2e6 E0771 cells into C57BL/6J mice. AAV-Surf-v2-infected NK cells were adoptively transferred into tumor burden mice via i.v. tail vein injections. Four screen models were used with different endpoints: B16F10 melanoma and E0771 breast cancer models were euthanized by 20 days post tumor implantation (dpi), while GL261 GBM and Pan02 pancreatic cancer models were euthanized at 27 dpi and 24 dpi, respectively. For B16F10 melanoma and E0771 breast cancer models, 4e6 AAV-Surf-v2 infected NK cells were injected into 9 and 10 tumor-burden mice, respectively; 2e6 AAV-Surf-v2 infected NK cells were injected into 7 Pan02 pancreatic cancer and 11 GL261 GBM mouse models.