Harnessing features of adaptive NK cells to generate iPSC-derived NK cells for enhanced immunotherapy

Abstract

Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD⁺. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer.

Keywords: NK cell; acute myeloid leukemia; adaptive; iPSC; immunotherapy; induced pluripotent stem cell; multiple myeloma; natural killer cell; off-the-shelf.

Figure 1.. CMV-induced adaptive NK cells downregulate CD38 and are more resistant to oxidative stress-induced death.

Peripheral blood mononuclear cells were isolated from healthy donors for examination of CD38 surface levels on major immune subsets. (A) Flow cytometry gating strategy used to identify immune subsets. (B) Representative histogram plots of CD38 surface expression (left) and cumulative data from 4 donors (right). (C) Heat map of CD38 mRNA transcript fold expression values assessed by RNA-seq in the indicated NK cell subsets sorted from the peripheral blood of 4 healthy CMV seropositive donors. Values are normalized to the CD56^dimCD57⁻NKG2C⁻ NK cell population. (D) Representative FACS plots of CD57 and NKG2C on gated CD3^𢈒CD56^dim NK cells from CMV seronegative and seropositive donors and histograms of surface CD38 expression on the indicated NK cell subsets (left). Cumulative data of CD38 mean fluorescence intensity (MFI) on the indicated NK cell subsets from 4 CMV seronegative and 4 CMV seropositive donors (right). Statistical significance was determined by one-way ANOVA with multiple comparisons. *p < 0.05 (E) NKG2C^𢈒 and NKG2C⁺ NK cells were isolated by magnetic selection from 3 CMV seropositive donors and assayed for intracellular concentrations of NAD⁺. Statistical significance was determined by paired Student’s t-test. *p < 0.05 (F) Peripheral blood NK cells from 4 CMV seropositive were isolated by negative selection and cultured in the presence or absence of 50 μM H₂O₂ for overnight followed by analysis of apoptosis and cell death by flow cytometry. Show are representative FACS plots of dead cell dye and Annexin V staining for the indicated NK cell subsets (left) and cumulative data plotting the percentages of viable cells after H₂O₂ treatment (right). Statistical significance was determined by one-way ANOVA with multiple comparisons, and results are from 2 independent experiments. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01

Figure 2.. hnCD16/CD38KO iNK cells exhibit enhanced metabolic fitness and avoid daratumumab-mediate fratricide.

CRISPR-Cas9 was used to knock out CD38 in iPSCs with transgenic expression of hnCD16. Knockout efficiency was assessed using (A) a T7E1 nuclease assay, (B) Western blot, and (C) flow cytometry. (D) The concentrations of NAD⁺ and NADH were determined in hnCD16 iNK cells and hnCD16/CD38KO iNK cells using a calorimetric cyclase assay, and ATP concentrations were determined by bioluminescence. Statistical significance was determined by paired Student’s t-test, and results are from 2 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (E) Real time metabolic profiling of hnCD16 iNK cells and hnCD16/CD38KO iNK cells was performed by Seahorse analysis. Shown are the oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) for 1 of 4 experiments performed. (F) hnCD16 iNK cells and hnCD16/CD38KO iNK cells were cultured in media alone or the indicated concentrations of H₂O₂ for 1 hour. Levels of mitochondrial superoxide were assessed by MitoSox dye fluorescence. Shown are FACS plots for a representative experiment (left) and cumulative data from 3 independent experiments. Statistical significance was determined by paired Student’s t-test. *p < 0.05 (G) To evaluate daratumumab-induced fratricide, iNK cells and peripheral blood NK cells were cultured with increasing concentrations of daratumumab for 3 hours, and viability was assessed by flow cytometry with staining for 7-AAD and a fixable viability dye. Data is graphed as specific cytotoxicity (% specific death - % spontaneous death) / (1 - % spontaneous death) x 100. Results were compiled from 2 independent experiments. (H) In 3D tumor killing assays, RPMI-8826 cells were cultured in low binding plates for 2 days to form tumor spheroids. The indicated iNK cells were then added at a 2:1 ratio with or without daratumumab. After 5 days, cultures were disrupted, and the remaining viable tumor and iNK cell numbers were quantified by flow cytometry. Results are representative of 2 independent experiments. Data are represented as mean ± SEM.

Figure 3.. hnCD16/CD38KO iNK cells exhibit elevated concentrations of glycolytic and antioxidant metabolites.

Expanded primary peripheral blood NK cells (n = 7), hnCD16 iNK cells (n = 3) and hnCD16/CD38KO iNK cells (n = 3) were analyzed by mass spectrometry to assess concentrations of key metabolites. Select data are represented in heat map (left) and column form (right). (B) NK cells isolated from peripheral blood by negative selection and (C) non-transduced iNK cells were cultured overnight in media alone or in media containing 50 μM L-CySSG. Cells were then cultured with or without 50 μM H₂O₂ for 1 hour. Shown are FACS plots of MitoSox dye fluorescence from a representative experiment (left) and cumulative data from 2 independent experiments (right). Statistical significance was determined by paired Student’s t-tests. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01

Figure 4.. hnCD16/CD38KO/IL-15RF iNK cells and CMV-induced adaptive NK cells share a set of highly upregulated transcripts.

(A) Violin plots of genes that were differentially expressed with statistical significance between hnCD16/CD38KO/IL-15RF iNK cells, hnCD16/CD38KO iNK cells, and non-transduced iNK cells in scRNA-seq analyses. (B) Analysis of the same genes in sorted canonical (CD3⁻CD56^dimNKG2C⁻) and adaptive (CD3⁻CD56^dimNKG2C⁺) NK cells from 4 donors analyzed by RNA-seq. Statistical significance was determined by paired Student’s t-test. *p < 0.05, **p < 0.01

Figure 5.. iADAPT NK cells sustain natural cytotoxicity and ADCC after multiple rounds of killing.

Expanded peripheral blood NK cells from 3 donors and iADAPT NK cells were thawed and co-cultured with MM.1R myeloma cells transduced with NucLight Red at the indicated E:T ratios in the presence or absence of daratumumab. (A) Target cell killing was assessed over 48 hours by live cell imaging. The remaining effector cells in all wells were collected and transferred to wells containing fresh MM.1R cells. Cytotoxicity was assessed for another 48 hours (round 2). The remaining effector cells in all wells were harvested for a second time and transferred to wells containing fresh MM.1R cells (round 3). Cytotoxicity was assessed by live imaging for another 48 hours. (B) Calculated cytotoxicity index values for each assay condition in IncuCyte assays. All data are normalized to MM.1R myeloma cells alone. Results are representative of 2 independent experiments.

Figure 6.. iADAPT NK cells mediate ADCC and produce IFN-γ when challenged with primary MM and AML cells.

(A) CD138⁺ myeloma cells were isolated from a bone marrow aspiration of a patient with relapsed MM and labeled with CellTrace dye and co-cultured overnight at a 2:1 E:T ratio with expanded peripheral blood NK cells or iADAPT NK cells that were thawed from cryopreservation. The percentages of live MM cells, defined as CellTrace⁺ and CD138⁺ were assessed by flow cytometry. (B) Primary AML cells from two patients with > 90% blasts were thawed, labeled with CellTrace dye, and cultured overnight with or without ATRA. FACS histogram plots showing surface CD38 expression on HL-60 AML cells and primary, patient-derived AML cells after overnight culture with or without ATRA are shown. Tumor cells were co-cultured at a 2:1 E:T ratio overnight with expanded peripheral blood NK cells or iADAPT NK cells thawed from cryopreservation. The percentages of live (C) HL-60 AML cells and (D) primary, patient-derived AML cells remaining at the end of the co-culture period was determined by flow cytometry and gating on the viable, CellTrace⁺ population. Intracellular IFN-γ frequencies in expanded NK cells and iADAPT NK cells were also assessed by flow cytometry. Cumulative data for % specific killing and IFN-γ production is shown from primary AML experiments performed with 2 peripheral blood NK cell products and 2 iADAPT iNK cell products in 2 independent experiments. Statistical significance was determined by one-way ANOVA with multiple comparisons. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01

Figure 7.. iADAPT NK cells mediate robust antitumor function in vivo.

(A) To assess persistence, NSG mice were injected with 1.2x10⁷ expanded peripheral blood NK cells or iADAPT NK cells thawed from cryopreservation on days 1, 8, and 15. Mice were bled on days 8 (before second NK injection), 15 (before 3rd NK injection), 16, 22, 29, 36, and 43. (B) peripheral blood NK cells and iADAPT NK cells were identified by flow cytometry as hCD45⁺hCD56⁺hCD16⁺mCD45⁻ cells. To assess antitumor function, NSG mice were engrafted with 1.5x10⁶ HL-60 cells transduced with the firefly luciferase gene. After 4 days, groups of mice (5 mice per group) received no treatment (HL-60 alone), 3 infusions of thawed expanded peripheral blood NK cells, hnCD16/CD38KO iNK cells, hnCD16/IL-15RF iNK cells, iADAPT NK cells. Each dose consisted of 1x10⁷ cells, and mice were treated weekly for 3 weeks. Bioluminescent imaging (BLI) was performed weekly to track tumor burden. Shown are (C) raw BLIs and (D) quantification of BLI data through day 28. (E) NSG mice were engrafted with 5x10⁵ luciferase-expressing MM.1S cells. After 3 days, groups of mice (4 mice per group) received no treatment (MM.1S alone), 3 infusions of daratumumab alone, 3 infusions of iADAPT NK cells alone, or 3 infusions of iADAPT NK cells in combination with daratumumab. Each dose consisted of 1x10⁷ cells, and mice were treated weekly for 3 weeks. Bioluminescent imaging (BLI) was performed weekly to track tumor burden. (F) Quantification of BLI data through day 35. (G) Area under the curve (AUC) data quantified from BLI imaging. AUC values were statistically significant for the daratumumab alone and hnCD16/CD38KO/IL-15R fusion iNK cells + daratumumab groups relative to the MM.1S alone group. Data is representative of 3 independent experiments. P values were determined by one-way ANOVA. Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001