Quadruple gene-engineered natural killer cells enable multi-antigen targeting for durable antitumor activity against multiple myeloma

Abstract

Allogeneic natural killer (NK) cell adoptive transfer is a promising treatment for several cancers but is less effective for the treatment of multiple myeloma. In this study, we report on quadruple gene-engineered induced pluripotent stem cell (iPSC)-derived NK cells designed for mass production from a renewable source and for dual targeting against multiple myeloma through the introduction of an NK cell-optimized chimeric antigen receptor (CAR) specific for B cell maturation antigen (BCMA) and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity when combined with therapeutic anti-CD38 antibodies. Additionally, these cells express a membrane-bound interleukin-15 fusion molecule to enhance function and persistence along with knock out of CD38 to prevent antibody-mediated fratricide and enhance NK cell metabolic fitness. In various preclinical models, including xenogeneic adoptive transfer models, quadruple gene-engineered NK cells consistently demonstrate durable antitumor activity independent of exogenous cytokine support. Results presented here support clinical translation of this off-the-shelf strategy for effective treatment of multiple myeloma.

Fig. 1. Phenotype of iDuo-MM CAR-NK cells.

a Flow cytometry analysis of the indicated cell-surface proteins on PBMCs and PBNK cells from three healthy donors, unmodified iNK cells (n = 3 batches), and iDuo-MM CAR-NK cells (n = 4 batches). Backbone iNK cells (n = 3 batches) were also stained for comparison of anti-BCMA CAR levels. Data are presented as mean values ± SD. b Flow cytometry analysis of CD56 and cell-surface anti-BCMA CAR levels on iNK cells differentiated from an iPSC clone with backbone edits only, an iPSC clone with backbone edits, and the anti-BCMA-CAR4 construct (iDuo-MM CAR-NK cells).

Fig. 2. iDuo-MM CAR-NK cells with the anti-BCMA CAR4 construct mediate sustained cytotoxic activity against MM.1S cells in a serial restimulation assay.

A serial restimulation cytotoxicity assay was performed with MM.1S cells transduced with NucLight Red as targets in the presence or absence of daratumumab, using backbone iNK cells or iDuo-MM CAR-NK cells as effectors. MM.1S cells were plated on the day of assay initiation followed by the addition of iNK cells at a 1:1 E:T ratio. After 48 hours of co-culture (round 1), non-adherent effector cells were transferred to new plates containing freshly plated MM.1S target cells (round 2) without recalibration of the E:T ratio. This procedure was repeated for a third round (round 3) of stimulation. MM.1S target cell survival was assessed in real-time using IncuCyte imaging. Data are representative of two independent experiments. Source data are provided as a Source Data file.

Fig. 3. iDuo-MM CAR-NK cells exhibit robust specific cytotoxicity and enhanced inflammatory cytokine production when combined with daratumumab.

a Two independent batches of iDuo-MM CAR-NK cells were assessed alongside iNK cells differentiated from backbone clones 1 and 2 for BCMA-specific cytotoxicity. Nalm6 cells overexpressing BCMA were mixed at a 1:1 ratio with WT Nalm6 lacking BCMA and used as targets in a 4-hour cytotoxicity assay at E:T ratios ranging from 0.03:1 to 31:1. Specific cytotoxicity was calculated independently for BCMA-negative WT Nalm6 cells (left) and BCMA-engineered Nalm6 cells (right) as the frequencies of target cells with active caspase-3/7 activity by flow cytometry. Data are from two independent experiments. b iDuo-MM CAR-NK cells from four independent differentiations and overnight (O/N) primed (10 ng/ml IL-15) PBNK cells isolated from three healthy donors were co-cultured with a mixture of normal donor PBMCs and MM.1S myeloma cells. Specific cytotoxicity against each cell type was assessed using a fluorescent caspase-3/7 reporter following a 4-hour incubation period. Statistical significance was determined by two-way ANOVA. Data are presented as mean values±SD. c The same batches of iDuo-MM CAR-NK cells (n = 4) and overnight primed PBNK cells (n = 3) were co-cultured with RPMI-8226 myeloma cells in the presence or absence of daratumumab for 6 hours. Control conditions included effector cells cultured alone and in the presence of daratumumab only. Supernatants were collected at the end of the co-culture period, and the levels of TNF and IFN-γ were measured using the MesoScale Diagnostics (MSD) platform. Statistical significance was determined with two-sided paired Student’s t tests. d Bone marrow biopsy samples were collected from two patients with relapsed MM. CD138⁺ MM cells were enriched by magnetic selection and co-cultured with iDuo-MM CAR-NK cells with and without daratumumab (dara) for 4 hours. No daratumumab conditions were included for functional assays with MM cells from patient 1 because of limited cell numbers. The percentages of remaining MM cells in each condition were assessed by flow cytometry. Source data are provided as a Source Data file.

Fig. 4. iDuo-MM CAR-NK cells display more durable cytotoxicity and persistence relative to expanded PBNK cells in serial restimulation assays.

a Serial restimulation assays were performed using NucLight Red-transduced MM.1 S cells as targets with iDuo-MM CAR-NK cells (n = 4 independent batches) and expanded PBNK cells (n = 3 healthy donors) as effectors at 3:1 and 10:1 E:T ratios in the presence and absence of daratumumab. Cytotoxicity was determined using IncuCyte imaging in three consecutive rounds of co-culture, and target cell frequencies were normalized at each time point to the target cell alone condition. At the end of the assays, cells were collected and analyzed by flow cytometry to determine the numbers of MM.1 S cells remaining in the b 3:1 E:T and c 10:1 E:T co-cultures and the numbers of NK cells remaining in the d 3:1 E:T and e 10:1 E:T co-cultures. f Serial restimulation assays were also performed with NucLight Red-transduced MM.1R cells as targets backbone iNK cells (n = 3 independent batches) and iDuo-MM CAR-NK cells (n = 3 independent batches) as effectors at 3:1 E:T ratios. g Flow cytometry was used to determine the numbers of MM.1R cells remaining in each co-culture condition. Statistical significance was determined using multiple unpaired t tests using the False Discovery Rate approach. All data are presented mean values ± SD. Source data are provided as a Source Data file.

Fig. 5. iDuo-MM CAR-NK cells mediate superior in vivo tumor control and persist at high levels in the peripheral blood.

NSG mice (n = 28) were engrafted with 2 × 10⁵ luciferase-transduced MM.1 S cells. After 2 days, groups of mice (n = 4/group) received no treatment, daratumumab alone, 1 i.v. injection of 1 × 10⁷ backbone iNK cells or iDuo-MM CAR-NK cells, 1 injection of 1 × 10⁷ backbone iNK cells or iDuo-MM CAR-NK cells with daratumumab, three injections of 1 × 10⁷ backbone iNK cells, iDuo-MM CAR-NK cells, and expanded PBNK cells thawed from cryopreservation. Second and third effector cell injections were given at days 9 and 16 post-tumor engraftment. a Bioluminescence imaging of mice at days 2, 17, and 31. b Graphical representation of the bioluminescence data for the single effector dose arm of the study. c Peripheral blood was drawn from mice at days 22, 28, and 35 for flow cytometry analysis to determine human NK cell counts for the single effector dose arm of the study. d Representative flow cytometry plots for analysis of mouse peripheral blood in the single effector dose experiments. e Graphical representation of the bioluminescence data for the multiple effector dose arm of the study. f Analysis of human NK cell counts in the peripheral blood for the multiple effector dose arm of the study. g Representative flow cytometry plots for analysis of mouse peripheral blood in the multiple effector dose experiments. Statistical significance was determined by two-way ANOVA. All graphed data are presented mean values ± SD. Source data are provided as a Source Data file.

Fig. 6. iDuo-MM CAR-NK cells maintain in vivo antitumor function in the absence of exogenous cytokine support.

NSG mice (n = 26) were engrafted with 2 × 10⁵ luciferase-transduced MM.1S cells. Groups of mice (n = 4/group) received either no treatment or i.v. injections of 1 × 10⁷ iDuo-MM CAR-NK cells thawed from cryopreservation on days 2, 9, and 16 post-tumor engraftment. Additional groups of mice were given i.v. injections of 2 × 10⁶ primary anti-BMCA-CAR-T cells on day 2. Two independent batches of iDuo-MM CAR-NK cells were used, and all three treated groups were split and either given no exogenous cytokine support or supplemented with twice-weekly injections of IL-2 and IL-15. a Bioluminescence imaging of mice at days 2, 9, 16, and 23 post-tumor engraftments. b Graphical representation of bioluminescence data comparing mice treated with both batches of iDuo-MM CAR-NK cells to mice with tumor alone. c Graphical representation of bioluminescence data comparing mice treated with anti-BCMA-CAR-T cells to mice with tumor alone. d Graphical representation of bioluminescence data from all treatment and control groups. Statistical significance was determined by two-way ANOVA. Data are presented mean values ± SD. Source data are provided as a Source Data file.

Fig. 7. Dual targeting in combination with daratumumab and BCMA stabilization via γ-secretase inhibition enhance iDuo-MM CAR-NK cell in vivo antitumor activity.

NSG mice engrafted with 2 × 10⁵ luciferase-transduced MM.1S cells (n = 18) were orally dosed with LY3039478 (1 mg/kg) or left untreated. Groups of mice (n = 3/group) were sacrificed after 8, 24, and 48 hours. Bone marrow was collected for analysis of BCMA surface density on MM.1S cells. a Representative flow cytometry plots showing BCMA levels on MM.1S cells from mice with and without GSI treatment. b Graphical representation of the cumulative data. Data are presented mean values ± SD. c NSG mice (n = 38) were engrafted with 2 × 10⁵ luciferase-transduced MM.1S cells. Groups of mice (n = 3–5/group) received either no treatment or i.v. injections of 5 × 10⁶ iDuo-MM CAR-NK cells thawed from cryopreservation on days 3, 10, and 17. Separate groups of mice received i.v. injections of 2 × 10⁶ primary anti-BCMA CAR-T cells on day 3. Some groups of mice were orally dosed with GSI twice weekly for 3 weeks beginning 1 day before MM.1S engraftment. Some groups of mice were administered daratumumab (dara) along with each iDuo-MM CAR-NK cell injection. Shown is bioluminescence imaging of mice at days 3, 8, 15, and 22. d Graphical representation of bioluminescence data comparing each treatment group to the MM.1S tumor alone group. e Graphical representation of all treatment groups along with the MM.1S tumor alone group. f Mouse peripheral blood was collected on days 6, 12, and 18 for assessment of human NK and T-cell numbers by flow cytometry. Shown are cumulative data with three mice analyzed per treatment group at each time point. Statistical significance was determined by two-way ANOVA. Data are presented mean values ± SD. Source data are provided as a Source Data file.