Abstract
Natural killer (NK) cells have clinical potential against cancer; however, multiple limitations hinder the success of NK cell therapy. Here, we performed unbiased functional mapping of tumor-infiltrating NK (TINK) cells using in vivo adeno-associated virus (AAV)–SB (Sleeping Beauty)-CRISPR (clustered regularly interspaced short palindromic repeats) screens in four solid tumor mouse models. In parallel, we characterized single-cell transcriptomic landscapes of TINK cells, which identified previously unexplored subpopulations of NK cells and differentially expressed TINK genes. As a convergent hit, CALHM2-knockout (KO) NK cells showed enhanced cytotoxicity and tumor infiltration in mouse primary NK cells and human chimeric antigen receptor (CAR)-NK cells. CALHM2 mRNA reversed the CALHM2-KO phenotype. CALHM2 KO in human primary NK cells enhanced their cytotoxicity, degranulation and cytokine production. Transcriptomics profiling revealed CALHM2-KO-altered genes and pathways in both baseline and stimulated conditions. In a solid tumor model resistant to unmodified CAR-NK cells, CALHM2-KO CAR-NK cells showed potent in vivo antitumor efficacy. These data identify endogenous genetic checkpoints that naturally limit NK cell function and demonstrate the use of CALHM2 KO for engineering enhanced NK cell-based immunotherapies.
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Data availability
All generated data and analysis results for this study are included in this article and its supplementary information files. Specifically, source data and statistics for non-high-throughput experiments are provided in Supplementary Datasets and Source Data. Processed data and raw sequencing data are available from the GEO under accession numbers GSE262707, GSE262708 and GSE262760, all under the GSE262709 super-series (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262709). Original cell lines are available from the commercial sources listed in the Supplementary Information. Source data are provided with this paper. Other relevant information or data are available from the corresponding authors upon reasonable request.
Code availability
The code used for data analysis and the generation of figures related to this study is available from the Supplementary Dataset 6 and GitHub (https://github.com/Prenauer/TumorInfiltrating_CAR-NK)59.
Change history
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Acknowledgements
We thank all members of the Chen laboratory, as well as our colleagues in the Department of Genetics, Systems Biology Institute, Cancer Systems Biology Center, MCGD Program, Immunobiology Program, BBS Program, Yale Cancer Center (YCC), Yale Stem Cell Center, RNA Center and Center for Biomedical Data Sciences at Yale for assistance and/or discussion. We thank the YCGA, Yale Center for Molecular Discovery, High-Performance Computing Center, West Campus Analytical Chemistry Core, West Campus Mass Spec Core and Keck Biotechnology Resource Laboratory at Yale for technical support. S.C. is supported by a Yale SBI/Genetics Startup Fund, a Cancer Research Institute Lloyd J. Old STAR Award (CRI4964), the National Institutes of Health (NIH) National Cancer Institute (NCI) (DP2CA238295, R01CA231112 and 1R33CA281702), the Department of Defense (W81XWH-20-1-0072, W81XWH-21-1-0514, HT9425-23-1-0472 and HT9425-23-1-0860), the Alliance for Cancer Gene Therapy (ACGT), the Pershing Square Sohn Cancer Research Alliance, the Sontag Foundation, Dexter Lu, the Ludwig Family Foundation, the Blavatnik Family Foundation and the Chenevert Family Foundation. P.A.R. is supported by a Yale PhD training grant from NIH (T32GM007499), the Lo Fellowship of Excellence of Stem Cell Research, the NCI diversity supplement and the YCC T32 fellowship program. G.S. is supported by a Vita-Salute San Raffaele University fellowship. J.J.P. is supported by an NIH Medical Scientist Training Program (MSTP) training grant (T32GM007205). R.D.C. is supported by an NIH MSTP training grant (T32GM007205) and National Research Service Award fellowship (F30CA250249).
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Contributions
S.C. conceptualized the study and designed it with L.P., P.A.R. and L. Ye. L.P. performed most experiments. P.A.R. performed most NGS analyses. L. Ye coperformed the screen and single-cell experiments and supported or supervised certain validation experiments. G.S., L. Yang, Y. Zou, Z.F., Q.L., M.B., A.S., Yueqi Zhang and S.Z.L. assisted with experiments. J.J.P. and Yongzhan Zhang performed certain NGS analyses. R.D.C. designed the Surf-v2 library. L.P., P.A.R. and S.C. prepared the manuscript with input from all authors. S.C. secured funding and supervised the work.
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A patent was filed by Yale University regarding the data in this study, which was licensed to Cellinfinity Bio, a Yale biotech startup founded by S.C. S.C. is also a founder or cofounder of EvolveImmune, NumericGlobal, MagicTime Med and Chen Consulting, all unrelated to this study. The other authors declare no competing interests.
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Supplementary information
Supplementary Information
Supplementary discussion, methods, datasets, figures, legends and references.
Supplementary Dataset 1
Screen analysis.
Supplementary Dataset 2
Single-cell analysis.
Supplementary Dataset 3
Single-cell DE analysis.
Supplementary Dataset 4
Single-cell pathway analysis.
Supplementary Dataset 5
mRNA-seq analysis.
Supplementary Dataset 6
Analytic codes.
Source data
Source Data Figs. 2–4 and Supplementary Figs. 9–12
Source data, statistical details and reagent info for non-NGS experiments.
Source Data Figs. 2–4 and Supplementary Fig. 9
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Peng, L., Renauer, P.A., Sferruzza, G. et al. In vivo AAV–SB-CRISPR screens of tumor-infiltrating primary NK cells identify genetic checkpoints of CAR-NK therapy. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02282-4
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DOI: https://doi.org/10.1038/s41587-024-02282-4
