OR7A10 GPCR engineering boosts CAR-NK therapy against solid tumors [Primary 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: a-HER2-CAR-NK92 cell were transduced with dCas9-VP64-Blast and MS2-P65-HSF1-Hygro (Addgene) at MOI < 1 and selected with Hygromycin and Blastcidin for 5-7 days. After complete selection, 1.8e8 CAR-NK92 cells were transduced with the sgRNA library (Addgene) at MOI = 0.2 (0.2 infectivity * 1.8e8 cells / 70,290 sgRNAs = 512x coverage). After 7 days of selection with Zeocin, > 4e7 Library-infected CAR-NK92 cells were maintained in culture (4e7 cells / 70,290 sgRNAs = 569x, although the coverage was limited by the bottleneck of the smaller in the process, e.g. 512x coverage). Eight-to-twelve-week-old female NSG mice were inoculated with 1e6 HT29-GL cells through subcutaneous injection. On day 8, 5e6 library-infected CAR-NK92 cells were adoptively transferred into each tumor-bearing mouse via intravenous injection (5e6 / 70,290 sgRNAs = 71x coverage). To support CAR-NK92 cell activity, mice received daily intraperitoneal injections of 2.5 µg human IL-2 beginning on the day of NK cell transfer. On 13-day post-transfer, mice were euthanized, and tumors were harvested for the subsequent experiments.