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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE120315 | GEO | 2018/12/01

REPOSITORIES: GEO

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Project description: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.

Project description:Background: Mutant BRAF targeted therapies remain a standard of care for the treatment of metastatic malignant melanoma (MM); however high initial response rates are tempered by the persistence of residual MM cells that eventually lead to disease recurrence and mortality. Since MM recurrence during targeted therapy can present with the simultaneous occurrence of multiple tumour nodules at the original body sites, we hypothesised the presence of an intrinsically resistant MM cell sub-population. Objectives: Identify an MM cell subpopulation that is intrinsically resistant to targeted therapy and so may be responsible for MM recurrence. Methods: Using melanoma cell lines, we define culture conditions for reproducible 3D growth of melanospheres to investigate putative cancer stem cell populations. We undertook RNA sequencing and bioinformatic analysis to characterise cell populations between adherent and non-adherent culture, and cells expressing or not expressing CD20. Furthermore, we define an in vitro assay to evaluate killing of melanoma cancer stem cells as a therapeutic test using combination therapies targeting driver mutation and CD20 marker. Results: We have described culture conditions that promote MM cells to form melanospheres with a reproducible colony forming efficiency of 0.3% to 1.3%. RNA sequencing of melanosphere versus conventional MM cell cultures (n=6), irrespective of the BRAF mutation status, showed that melanosphere formation was associated with growth and differentiation transcriptional signatures resembling MM tumours. Importantly, melanosphere formation also led to the emergence of a CD20+ MM cell subpopulation, similar to that observed in primary human MM tumours. CD20+ MM cells were resistant to BRAF inhibitor therapy, and consistent with this finding demonstrated a Forkhead box protein M1 (FOXM1) transcriptomic profile (n=6). Combining BRAF inhibitor and anti-CD20 antibody treatment led to the additional killing of previously resistant CD20+ BRAF mutant MM cells. Conclusions: In MM patients that harbour a CD20+ sub-population, combined therapy with BRAF inhibitor and anti-CD20 antibody could potentially kill residual MM cells and so prevent disease recurrence.

Project description:Although new therapies have doubled the survival of multiple myeloma (MM) patients, this remains an incurable disease. It has been postulated that the so-called MM Cancer Stem Cells (MM-CSC) would be responsible for tumor initiation and relapse but their unequivocal identification remains unclear. Here, we investigated in a panel of MM cell lines the presence of CD20+ cells harboring a MM-CSC phenotype. Among the multiple cell lines investigated, only a small population of CD20dim+ cells (0.3%) in the RPMI-8226 cell line was found. CD20dim+ RPMI-8226 cells expressed the plasma cell markers CD38 and CD138 and were CD19-CD27-. Additionally, CD20dim+ RPMI-8226 cells did not exhibit stem-cell markers as shown by gene expression profiling and the aldehyde dehydrogenase (ALDH) assay. Moreover, we demonstrated that CD20dim+ RPMI-8226 cells are not essential for CB17-SCID mice engraftment and show lower self-renewal potential than the CD20- RPMI-8226 cells. These results do not support CD20+ expression for the identification of MM-CSC. For detailed description of Material and Methods, see supplementary Methods. The human MM cell lines RPMI-8226, U266, MM1S, MM1R, NCI-H929, RPMI-LR5, U266-LR7 and U266-Dox4 were studied. Cells were cultured as previously described [Ocio, 2009, 3781]. MM cell lines were immunophenotyped using a 7-color immunofluorescence technique [Paiva, 2011, 697], with the following combination of monoclonal antibodies (Pacific Blue (PB)/ anemonia majano cyan (AmCyan)/ fluorescein isothiocyanate (FITC)/ peridinin chlorophyll protein-cyanin 5.5 (PerCP-Cy5.5)/ PE-cyanin 7 (PE-Cy7)/ allophycocyanin (APC)/ alexafluor 700 (AF700)): CD19/CD45/CD20/CD138/CD27/CD56/CD38. Data were stored for a minimum of 3x105 events. CD20dim+ and CD20- RPMI-8226 cells were sorted after incubation with CD20-APC/7AAD and acquisition on a FACSAria cytometer (Becton Dickison Biosciences). Sorting was performed only for viable cells (7AAD-) and debris were excluded by scatter properties. The CD20- and CD20dim+ RPMI-8266 sorted cells had a final purity of 98% and 88%, respectively. CD20dim+ and CD20- RPMI-8226 cells were extensively characterized. Morphological characterization was done with May-Grünwald-Giemsa staining. Characterization of VDJH and IGKappa rearrangements was performed as described elsewhere [BIOMED-2, BMH4-CT98-3936]. The expression of aldehyde dehydrogenase (ALDH) was assessed using the Aldefluor Kit (StemCell Technologies) following manufacturer’s instructions with further staining with a CD20-APC antibody. For gene expression profile studies, RNA was isolated, labeled and hybridized to Human Gene 1.0 ST array (Affymetrix) according to Affymetrix protocols [Gutiérrez, 2005, 402]. The arrays were analyzed using the DNA-Chip Analyzer software (DChip). Fold change ≥2 was considered significant.

Project description:Readily accessible samples such as peripheral blood or cell lines are increasingly being used in large cohorts to characterise gene expression differences between a patient group and healthy controls. However, cell and RNA isolation procedures and the variety of cell types that make up whole blood can affect gene expression measurements. We therefore systematically investigated global gene expression profiles in peripheral blood from six individuals collected during two visits by comparing five of the following cell and RNA isolation methods: whole blood (PAXgene), peripheral blood mononuclear cells (PBMCs), lymphoblastoid cell lines (LCLs), CD19 and CD20 specific B-cell subsets. Gene expression measurements were clearly discriminated by isolation method although the reproducibility was high for all methods (range ρ = 0.90-1.00). The PAXgene samples showed a decrease in the number of expressed genes (P<1*10-16) with higher variability (P<1*10-16) compared to the other methods. Differentially expressed probes between PAXgene and PBMCs were correlated with the number of monocytes, lymphocytes, neutrophils or erythrocytes. The correlations (ρ =0.83; ρ =0.79) between the expression levels of detected probes were much lower compared to the two B-cell isolation methods (ρ =0.98). Gene ontology analysis of detected genes showed that genes involved in inflammatory responses are enriched in B-cells CD19 and CD20 whereas genes involved in alcohol metabolic process and the cell cycle were enriched in LCLs. Gene expression profiles in blood-based samples are strongly dependent on the predominant constituent cell type(s) and RNA isolation method. It is crucial to understand the differences and variability of gene expression measurements between cell and RNA isolation procedures, and their relevance to disease processes before application in large clinical studies. In the present study, we investigated variability and consistency in gene expression profiles between five of the most common post venipuncture methods of cell and RNA isolation: whole blood (PAXgene (PAX)), PBMCs, Epstein-Barr virus (EBV) transformed LCLs, CD19-specific B-cells subsets (B-cell CD19), CD20-specific B-cells subsets (B-cell CD20). Using samples from six individuals collected during two visits, we evaluated the differences and concordances of global gene expression profiles, the biological and technical variability seen in these approaches, cell-type specific gene expression signatures and their relevance to large-scale biobanking initiatives.

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

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