OR7A10 GPCR engineering boosts CAR-NK therapy against solid tumors [mini screen]
Chimeric antigen receptor (CAR)-natural killer (NK) cell therapies hold promise for solid tumors but remain limited by poor tumor infiltration, persistence, and resistance within the tumor microenvironment (TME). To identify gain-of-function (GOF) targets that enhance CAR-NK efficacy, we performed an unbiased in vivo Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) activation (CRISPRa) screen, followed by a barcoded targeted in vivo open reading frame (ORF) screen in primary human CAR-NK cells. We identified, and robustly validated OR7A10, a G protein-coupled receptor (GPCR), as the top candidate. Engineering CAR-NKs with OR7A10 cDNA, a CRISPR-independent method with simple manufacturing strategy, enhanced proliferation, activation, degranulation, cytokine production, death ligand expression, chemokine receptor expression, cytotoxicity, persistence, metabolic fitness, and TME resistance, while reducing exhaustion in primary human NK cells derived from multiple peripheral blood and cord blood donors. OR7A10-GOF CAR-NKs displayed robust in vivo efficacy across multiple solid tumor models, achieving a 100% complete response in an orthotopic breast cancer model with long term tumor control and survival benefit. These findings establish OR7A10-engineered CAR-NKs as a highly potent and scalable off-the-shelf therapeutic for solid tumors. Overall design: This experimental data is a primary human CAR-NK barcoded ORF-UMI mini-screen. An ORF library was generated for the top 35 gene targets (cDNA length = 1 kb) from the initial CRISPRa screen, along with five fluorescent reporter genes as null controls. The ORF library was synthesized as individual eBlocks (IDT), pooled at equimolar ratios, and amplified with primers containing four-nucleotide random barcodes (UMIs; NNNN) to introduce molecular redundancy targeting the same gene. The ORF expression vector plasmid was bought from Addgene (#145026). The barcoded ORF library was pool-cloned into the expression vector using Gibson assembly.  Primary human peripheral blood NK cells from two healthy donors were first transduced with HER2-CAR lentivirus and then with the ORF library lentivirus at an MOI of ~0.2. A total of 20 million library-transduced CAR-NK cells were injected into each HT29 tumor-bearing mouse (n = 5 mice for Donor 0957 and n = 3 for Donor 0958 for each time point). Genomic DNA was extracted from tumors at day 3 and 6 post-treatment, as well as from pre-injection cells, and subjected to NGS to quantify ORF and UMI barcode representations.  Note that each set of fastq files is demultiplexed by the reverse