CD19 Chimeric Antigen Receptor Engineered NK92 Natural Killer Cells: Novel “Off the Shelf” Immunotherapy in Non-Hodgkin Lymphoma II

NK-92 a continuously growing cell line bioengineered to express human anti-CD19 chimeric antigen receptor (CAR) CD19.TaNK recognizing CD19+ B cells represents as potential “off the shelf” therapy candidate for B cell malignancies. The goal of this study was to establish the mechanistic rationale for CD19.TaNK therapy in B-cell NHL (bNHL) and to determine the therapeutic potency in vitro against a host of bNHL cell lines, patient derived primary cell lines (including anti-CD20 antibody resistant cell lines), and in in vivo mouse models. We utilized bNHL cell lines SU-DHL10, SU-DHL4, SU-DHL2 (DLBCL), HF-1 (follicular) and Raji (Burkitt’s) and Rituximab- (RR) and obinutuzumab (OR)-resistant bNHL cell lines (SU-DHL2, SU-DHL4, SU-DHL10), and patient derived primary cells (EL-5 and KSC) were investigated the cytolytic activity of CD19.TaNK. We observed significantly increased CD19.TaNK mediated cytolytic activity at E:T ratios (1:1-10:1) via LDH release in all bNHL cell lines and CD20 resistant bHNL cells. Further, the dynamic efficacy of CD19.TaNK determined using droplet based single cell microfluidics analysis of cell interactions (1:1) between CD19.TaNK and anti-CD20 sensitive or resistant bNHL cell showed that vast majority of the cells were killed by single contact >80% (SU-DHL 4, SUD-HL 4 -OR), 40% (SU-DHL10-RR), >60% (SU-DHL10, SUD-HL-10-OR) and 40% (SU-DHL10-RR) within first 40 minutes, while the remainder were killed through events requiring multiple contacts. Thus, suggesting that CD19.TaNK indiscriminately kills both anti-CD20 sensitive and resistant cells. Global transcriptome analysis performed using flow sorted bNHL co-cultured with CD19.TaNK at 1:1 ratio for two hours, revealed conserved activation of IFNγ signaling, execution of apoptosis, ligand binding, immunoregulatory or chemokine signaling pathways in these bNHL cells. Using proximity extension assay based 92-plex cytokine panel we observed increased secretion of various cytokines, granzymes and decreased secretions of ADA, HO-1, CD5, CD28, CD70, CD244, IFN and TNF consistently with anti-CD20 sensitive and resistant cells. Altogether these results demonstrate that CD19.TaNK inflicts mechanistically conserved killing activity against different bNHL cell lines, including in anti-CD20 refractory bNHL. Finally, in SCID mice experiments we observed marked reduction in the volume of SU-DHL10 derived tumor xenografts with infusion of CD19.TaNK compared to control. Overall, we observed potent anti-lymphoma activity with CD19.TaNK involving biologically conserved mechanisms indicating that CD19.TaNK could be equally active under untreated or refractory bNHL in the clinical settings. For genome-wide expression profiling of SUDHL4, SUDHL4 rituximab resistant (RR), SUDHL4 obinutuzumab resistant (OR), SUDHL10, SUDHL10 rituximab resistant, SUDHL4 obinutuzumab resistant, Raji, EL-2, and EL-5 cells were co-cultured with CD19.TaNK cells, Human HT-12 BeadArray chips (Illumina, San Diego, CA) were used. RNA was isolated using RNeasy Minikit (Qiagen), following instructions recommended supplied by the manufacturer. Purity and the yield of isolated RNA was determined using Bioanalyzer. These experiments were performed in biological triplicates. All replicate samples were run individually. Total RNA of 500ng per sample was amplified using AmbionTotalPrep, and 1.5ug of the product was loaded onto the chips. Following hybridization at 55C, the chips were washed and then scanned using the Illumina iScan System. The data was checked with GenomeStudio (Illumina) for quality control. Data were corrected through COMBAT correction, quantile normalized, collapsed to genes from probes, then imported into MultiExperiment Viewer, MeV for analysis. Statistically significant genes were determined by applying a one-way ANOVA with an adjusted Bonferroni correction and false discovery rate (FDR) < 0.05 that resulted in a list of significant genes.