Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo

Abstract

Introduction: Natural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary.

Methods: In this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence.

Results: We demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model.

Conclusion: Together with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals.

Keywords: B-cell malignances; CAR-NK cells; IL-15; IL-15 receptor; NK-92; adoptive cell therapy; allogeneic therapy.

Figure 1

Generation of NK-92-CAR.19-IL-15/IL15Rα cells. (A) Scheme of CAR constructs targeting CD19 (CAR.19) under the control of spleen focus-forming virus (SFFV) promoter. CAR consists of scFv, CD8 hinge and transmembrane region, 41BB costimulatory molecule and CD3ζ signaling molecule. Where indicated, the CAR sequences are followed by a self-cleaving peptide (T2A) and IL-15 or IL-15/IL-15Rα (IL-15 and IL-15Rα fused through a flexible linker). (B, C) NK-92 cells were transduced with the described CAR constructs followed by immunomagnetic enrichment in two steps. (B) Representative dot plots show flow cytometric analysis of CAR expression on freshly transduced and enriched NK-92-CAR.19 cells. (C) The graph shows the frequency of CAR-positive cells before selection, after first and second selection, and 30 days after second selection.

Figure 2

NK-92-CAR.19-IL-15/IL15Rα cells proliferate independently of exogenous IL-2. Proliferation of parental and genetically modified NK-92 cells: NK-92-CAR.19, NK-92-CAR.19-IL-15 and NK-92-CAR.19-IL-15/IL15Rα in X-Vivo 10 supplemented with 5% of human plasma. (A) Cells were cultured either in presence of IL-2 (500 UI/mL) or (B) in absence of exogenous cytokines. (C) Viability of cell culture with cytokines and (D) without cytokines. Cell culture was followed over 21 days. Pooled data of two independent experiments (each one performed in duplicates) are shown. (E) Quantification of soluble IL-15 in supernatant of NK cell culture by Luminex (n=2). Two-way ANOVA statistical test, Tukey's post-multiple comparison posttest. ****P<0.0001.

Figure 3

NK-92-CAR.19 cells coexpressing IL-15/Rα enhance CAR-mediated anti-tumor Ncell function. NK-92, NK-92-CAR.19, NK-92-CAR.19-IL-15 and NK-92-CAR.19-IL-15/IL-15Rα cells were co-cultivated with (A) Raji (B) NALM-6 and (C) K562 in 2:1 and 10:1 effector:target cells ratio, for 5 hours. Cytotoxicity was measured by flow cytometry cytotoxicity assay. One-way ANOVA statistical test, Tukey's multiple comparison posttest. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. Mean values + SD are shown. Data pooled from three independent experiments.

Figure 4

NK-92-CAR.19-IL-15/IL15Rα cells produce high amount of anti-tumor pro-inflammatory cytokines. NK-92, CAR.19, CAR.19-IL-15 and CAR.19-IL-15Rα NK-92 cells (2.5 × 10⁵) were incubated for 5 h with Raji lymphoma cells (A, C) or with Nalm-6 (B, D) at an E/T ratio of 10:1. Supernatants were collected, and the levels of IFN-γ (A, B) and TNF-α (C, D), granzyme A (E, F)), granzyme B (G, H) and perforin (I, J) were measured using the Luminex MAGPIX system. NK cells without target cells were included as controls. One-way ANOVA statistical test, Tukey's multiple comparison posttest. Mean values ± SD are shown; n=2. ***P < 0.001; **P < 0.01; *P < 0.05. Data pooled from three independent experiments.

Figure 5

Metascape functional analysis of transcriptome profiles of NK-92-CAR.19 cells after coculture with a target B cell line. (A) The Circos plot shows how genes from the different input gene lists after coculture setups overlap. On the outside, the arc represents the identity of each gene list. On the inside, the orange color represents the genes that appear in multiple lists, and the light orange color represents genes that are unique to that gene list. Purple lines link the same genes shared by multiple gene lists. Blue lines link the genes that fall into the same ontology term (the term has to be statistically significantly enriched). The greater the number of purple links and the longer the dark orange arcs, the greater is the overlap among the input gene lists. Blue links indicate the amount of functional overlap among the input gene lists. (B) Networks of Protein-Protein interaction (PPI) identified in the gene lists for comparing across NK92-CAR groups and NK92-WT, and illustrated using Cytoscape 3.9.1. (C) Summary of enrichment analysis done using the DEG_up from (A) and the Trancription_Factor_Targets as an ontology category (Metascape).

Figure 6

Clustering analysis of the most significantly upregulated and downregulated genes. (A) Heatmap of differentially expressed genes (DEGs) upregulated, with clustering of results was done to comparatively illustrate the expression across different samples (IL-15 and IL15/IL15Rα). (B) Enrichment analysis using the Enrichr bioinformatics tool following the input of the upregulated (DEG) gene list clusters derived from the DEGs in (A) (23).

Figure 7

Transgenic IL-15 or IL-15/Rα maintains low expression of immune checkpoint molecules in NK-92-CAR.19 cell variants. NK-92-CAR.19 cells were cultured unstimulated (-) or stimulated (+) with Raji cells at an E:T ratio of 1:2, followed by restimulation with fresh target cells after 24 and 48 hours. Three days after the beginning of the experiment, the surface expression of LAG-3 (A), PD-1 (B) and TIM-3 (C) were analyzed by flow cytometry. Three independent experiments were performed. One-way ANOVA statistical test, Tukey's post-hoc multiple comparison. Mean values + SD are shown. Gray: NK-92, Black: coculture of NK-92 and Raji cells, Orange: CAR.19, Brown: coculture of CAR.19 and Raji cells, Pink: CAR.19-IL-15, Red: cocultured of CAR.19-IL-15 and Raji cells, Light blue: CAR.19-IL-15RA, and Blue: coculture of CAR.19-IL-15 and Raji cells. (D) Representative flow cytometry histograms plots showing the mean fluorescence intensity of LAG-3, PD-1 and TIM-3 in NK-92 cell and, in the NK-92-CAR.19 cell variants. ***P < 0.001; **P < 0.01; *P < 0.05.

Figure 8

NK-92-CAR.19-IL15/IL-15Rα cells eliminate lymphoma B-cells and reduce tumor burden in vivo. (A) Scheme of an in vivo study demonstrating the anti-tumor activity of transduced NK-92-CAR.19 cells in a xenogeneic NSG mouse model of disseminated human B-cell hematologic malignancy. NSG mice were intravenously injected with 2 × 10⁴ Raji-Luc lymphoma B-cells. On days 4, 7, 10, 12, 15, and 19, animals were treated by intravenous injection of 7 × 10⁶ NK-92 cells (n=5). (B) Lymphoma development was monitored by in vivo bioluminescence imaging. Images were taken on days 8, 14, 21, 26, and 29. (C) Bioluminescence intensity was quantified and means + SEM are shown. (D) Bioluminescence of individual mice shown on day 29. Means + SEM are shown. Statistics were determined by the Mann-Whitney test. Each experimental group consisted of five mice. **P < 0.01; *P < 0.05.

Figure 9

Proposed model of action of engineered NK-92-CAR-19-IL-15/IL-15Rα cells. In the absence of exogenous IL-2: (A) NK-92-CAR.19 cells remain non-proliferative. (B) NK-92-CAR.19-IL-15 cells exhibit modest proliferation up to day 12, as they release soluble IL-15 that subsequently engages with IL-2/IL-15Rβ–γc receptors on their own membrane or neighboring NK cells. (C) NK-92-CAR.19-IL-15/IL-15Rα cells display robust proliferation, driven by the presence of IL-15/IL-15Rα complexes on their cell membrane. This activates the cells through cis-presentation or may stimulate adjacent NK cells expressing membrane-bound IL-2/IL-15Rβ–γc receptors via trans-presentation. The signaling from IL-15 prompts all three NK-92-CAR-19 cell variants to upregulate the PI3K/AKT and JAK/STAT pathways. However, this upregulation is notably more pronounced in NK-92-CAR.19-IL-15/IL-15Rα cells compared to the other variants. Consequently, this substantial enhancement in signaling leads to heightened proliferative capacity, increased secretion of proinflammatory cytokines, and exceptionally potent cytotoxic activity in these pioneering engineered NK-92 cells.