Abstract 5187: Development of multiomics approaches to evaluate NKG2D ligand dynamics and anti-tumor immune responses during CAR-NK treatment
Authors: Hansen Kyle , Sahu Sombeet , Duong Bao , Cho Cynthia , Zhang Qi , Trager James , Tan Joanne 
Published: June 2022

Abstract
NK cells are highly potent, fast-acting cytolytic cells capable of eradicating cancer with limited risk of adverse effects such as cytokine release syndrome or GVHD. NKX101 is a NK cell therapy product engineered to overexpress a chimeric receptor consisting of NKG2D ectodomain, costimulatory signaling motifs, and a membrane-bound form of IL-15. NKX101 is currently under clinical evaluation for treatment of relapsed/refractory AML and MDS. To better understand patterns of response to NKX101, we describe the development of (i) a single-cell (sc) RNAseq approach to assess gene expression pattern changes in NKX101 and patient cells, (ii) a multiplex IHC panel to monitor NKG2D-ligand expression by cancer cells, and (iii) an ELISA method to detect NKG2D-ligand shedding in serum.
To verify that our scRNAseq approach can distinguish the NKX101 product cells from patient cells, healthy human PBMCs were pre-mixed with NKX101 cells at set ratios. 10x Genomics hybrid capture baits were prepared to enrich for exogene reads, first via single-cell 5’ gene expression assays and then further processed for target enrichment using a human immunology gene set panel. Multiplex IHC was carried out using the Vectra Polaris Phenoptics platform. FFPE biopsy samples were stained with an 8-marker multiplex panel. Digital image analysis was conducted using the inForm analysis software. For the detection of shed NKG2D-ligands, a sandwich ELISA was employed using recombinant human NKG2D to capture the ligands, followed by detection using ligand-specific antibodies and HRP-conjugated secondary antibodies.
We can successfully separate NKX101 product cells from the host PBMC background using our scRNAseq data. NKX101 cell proportions in the transcriptome datasets correlated with the expected titration ratios against host PBMC, with samples titrated at 25% NKX101+ cells yielding approximately 25% NKX101 exogene expressing cells by scRNAseq. Our internally developed multiplex IHC methods can quantify NKG2D-ligand expression levels in AML and solid tumors. We show examples of NKG2D-ligand expression on blast cells from AML solid bone marrow biopsies. In solid tumors, we demonstrate that NKG2D-ligands are upregulated on PanCK+ cancer cells compared to PanCK- cells or healthy normal tissues. Finally, we found certain commercial kits for the detection of soluble NKG2D ligands to potentially under-report the NKG2D-ligand concentrations due to their propensity for aggregation. Using recombinant NKG2D as a capture reagent, we have developed sandwich ELISA method to detect NKG2D-ligands shed into the culture supernatant or serum. In summary, we describe here the development of IHC and ELISA methods to determine NKG2D-ligand expression and shedding as well as a scRNAseq assay to identify NKX101+ product cells and evaluate the anti-tumor immune response during CAR-NK treatment.
Citation Format: Kyle Hansen, Sombeet Sahu, Bao Duong, Cynthia Cho, Qi Zhang, James Trager, Joanne Tan. Development of multiomics approaches to evaluate NKG2D ligand dynamics and anti-tumor immune responses during CAR-NK treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5187.