Researchers Use In Vivo AAV–SB-CRISPR Screening Technology to Discover New NK Cell Cancer Immunotherapy Targets

Recently, researchers from Yale University and other institutions published a research paper titled "In vivo AAV-SB-CRISPR screens of tumor-infiltrating primary NK cells identify genetic checkpoints of CAR-NK therapy" in the journal Nature Biotechnology. After years of technical research, the research team used the AAV-transposon system to integrate the CRISPR library into the genome, overcoming the obstacles of low efficiency of primary NK cell transduction and gene editing. Through four parallel CRISPR screening experiments and single-cell sequencing data, the study discovered a new target, CALHM2, which provides a new therapeutic target for NK cell tumor immunity.

Natural killer (NK) cells have strong potential as cancer immunotherapy. Therapeutic NK cells can be made into allogeneic or off-the-shelf products, and recent clinical trials have shown that CAR-NK cells have similar efficacy but much lower toxicity compared to CAR-T cell therapy. However, NK cell-based immunotherapy also has its own challenges, in which NK cell function is naturally limited by endogenous genetic inhibitors. This requires modifying endogenous genes to enhance the effectiveness of NK cells against cancer. Several existing approaches, including overexpression of cytokines ("armored" fourth-generation CAR-NK cells) (e.g., IL-15) and KO of negative regulators (e.g., CISH), have shown significant functional enhancement of cord blood-derived CAR-NK cells. However, the number of NK cell checkpoints that are currently known or identified is small.

To systematically identify genes that inhibit NK function and, in turn, CAR-NK therapy, the researchers performed in vivo pooled adeno-associated virus (AAV)-SB (Sleeping Beauty)-CRISPR KO screening directly in primary NK cells of mice introduced into a fully immune-competent syngeneic tumor model. They also used an orthogonal unbiased method, scRNA-seq, to identify subsets and gene signatures of TINK cells in the TME. CALHM2/Calhm2 was identified as a convergent hit from both datasets. CALHM2 KO significantly increased cytotoxicity, degranulation, cytokine production, and tumor infiltration of primary NK cells and CAR-NK cells. Targeting CALHM2 makes human anti-HER2-CAR-NK92 cells more capable of overcoming solid tumor resistance and substantially improves in vivo efficacy.

Figure 1. In vivo AAV-SB-CRIPSR NK cell screen and scRNA-seq of TINK cells jointly identified Calhm2 as a convergent gene for NK cell engineering. (Peng L, et al., 2024)

Of note, unlike simple cytokine overexpression, approaches to knockout endogenous checkpoints such as CALHM2 require gene editing, which may involve a more complex manufacturing process for CAR-NK therapies. In the future, gain-of-function screening based on CRISPR activation or open reading frames can be used to identify functional enhancers in CAR-NK cells that are more easily engineered into therapeutic cell products. In summary, the in vivo AAV-SB-CRISPR screening and TINK scRNA-seq of this study identified endogenous genes that may serve as genetic checkpoints that naturally inhibit NK function. Targeting these inhibitory genes such as CALHM2 provides a promising approach to design new enhanced anti-cancer NK cell therapies.

Reference

Peng L, et al. In vivo AAV-SB-CRISPR screens of tumor-infiltrating primary NK cells identify genetic checkpoints of CAR-NK therapy. Nature Biotechnology, 2024: 1-10.