SOT101 induces NK cell cytotoxicity and potentiates antibody-dependent cell cytotoxicity and anti-tumor activity

Abstract

SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8⁺ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56^brightCD16^- and CD56^dimCD16⁺, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.

Keywords: NK cells; RLI-15; antibody-dependent cytotoxicity; immunotherapy; interleukin-15; therapeutic antibodies.

Figure 1

SOT101 expands and activates human NK cell subtypes in vitro. Human PBMCs were incubated with the increasing concentrations of SOT101 at 0.001 – 10 nM for 7 days (NT – non-treated). NK cells (CD3^-CD56⁺) and their subpopulations (CD56^dimCD16⁺ and CD56^brightCD16^-) were analyzed by flow cytometry at day 3 and 7. (A) representative dotplots from day 7 are shown (B) SOT101-stimulated proliferation and the relative expansion human NK cells and (C) their subpopulations CD56^dimCD16⁺ and CD56^brightCD16^- in a concentration-dependent manner already at day 3 in vitro. Statistical significance from SOT101 at 0.01 nM. NK cell proliferation was detected as a percentage of Ki67⁺ cells. (D) SOT101 increased the relative number of NK cells expressing the activating receptors DNAM-1, NKp30, and at low concentrations also NKG2D, and increased their expression on NK cells (fold change of MFI with SOT101 at 1 nM) already at day 3. Statistical significance from SOT101 at 0.01 nM for NKp30, DNAM-1 and from SOT101 at 0.001 nM for NKG2D. SOT101 did not change the expression levels of the inhibitory receptors NKG2A, CD158a and CD158b on NK cells. Data represent mean ± SEM from 4-8 healthy donors. The results were considered statistically significant if * p < 0.05, ** p < 0.01, *** p < 0.001 or **** p < 0.0001.

Figure 2

SOT101 induces cytotoxic and tumor cell-killing activity of human NK cell subtypes in vitro.PBMCs were pre-stimulated with 0, 0.1, 1 and 10 nM SOT101 for 3 days, then added to the target tumor cells DiD⁺K562 at E:T ratio 1:5. The cytotoxic activity was determined by (A) LAMP-1 degranulation on NK cells and their subpopulations and (B) by an intracellular IFN-γ staining after 4h. (C) The percentage of killed DiD⁺DAPI⁺ K562 cells was determined after 6 h by flow cytometry. Representative dotplots of LAMP-1 and IFN-γ staining in NK cells are shown in (B) (NT – non-treated NK cells with K562; SOT101 + K562 – NK cells pre-stimulated with SOT101 at 1 nM with K562; SOT101 – NK cells pre-stimulated with SOT101 at 1 nM without K562). Data represent mean ± SEM from 4 healthy donors. The results were considered statistically significant if * p < 0.05, ** p < 0.01 or *** p < 0.001.

Figure 3

SOT101 enhances the tumor cell killing activity of therapeutic antibodies in vitro (A) Concomitant treatment: PBMCs were added to the target tumor cells together with SOT101 at 1 nM and the therapeutic antibodies at 0.1, 1 and 10 nM and incubated for 20 h E:T ratio was 25:1 for Daudi (Daratumumab, Obinutuzumab) and 150:1 for SCC25 (Cetuximab) cells, respectively. (B) Sequential treatment: PBMCs were first pre-stimulated with 0.1 nM SOT101 for 3 days (SOT101-PBMC) and then added to the target tumor cells together with therapeutic antibodies at 0.1, 1 and 10 nM and incubated for 24 h E:T ratio was 15:1 for Daudi and 100:1 for SCC25 cells, respectively. The percentage of cytotoxicity was determined by a LDH release assay. (C, E) PBMCs were pretreated with SOT101 (0.1 nM, 3 days) and then incubated with Daratumumab and Daudi cells (E:T 1:1) for 4 h (C) The percentage of dead DiD⁺DAPI⁺ tumor cells was determined by flow cytometry. (E) Representative dotplots for NK cell degranulation as determined by CD3^-CD56⁺LAMP-1⁺ staining Daratumumab was used at 0.1 nM here. (D) Isolated NK cells were pretreated with SOT101 (0.1 nM, 2 days) and then incubated with Daratumumab (0.1, 1 and 10 nM) and Daudi tumor cells (E:T 10:1) for 4 h. The percentage of cytotoxicity was determined by a LDH release assay. Data represent mean ± SEM from 3-6 healthy donors. The results were considered statistically significant if ** p < 0.0021, *** p < 0.0002 or **** p < 0.0001.

Figure 4

SOT101 activates NK cells and induces anti-tumor efficacy in dependance on dose, administration route and schedule in vivo. (A) SOT101 was administered IP or SC once daily at the indicated doses into C57BL/6 mice at day 1,2,3,4 in 2 mice/group. NK cell proliferation and the relative expansion was analyzed from splenocytes by flow cytometry at day 5. Lung weight was determined at day 5 to detect vascular leakage syndrome (VLS). (B) SOT101 was administered SC at 2.5 mg/kg in 2 mice per time point. Serum was collected at 2 min, 15 min, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h and 32 h after dose administration and analyzed by ELISA. Pharmacokinetic parameters were calculated. (C) SOT101 was administered SC at 1 mg/kg once daily at the indicated day. NK cell proliferation and the relative expansion was determined by flow cytometry at day 5. (D) SOT101 was administered SC at 0.5 or 1 mg/kg at day 1,2,3,4 in the Renca mouse tumor model. Lung metastases were evaluated as the lung weight at day 16. (E) Dosing schedule dependency of SOT101 at 1 mg/kg SC in Renca mouse model for prevention of lung metastases. NK cell activity from spleen was evaluated at day 5 and 12. (F) Dose and schedule dependency of SOT101 administered SC in solid multiple myeloma xenograft tumor model. Data represent mean ± SEM from 2-4 mice/group for PD and PK, and 8 mice/group for the anti-tumor efficacy experiments in vivo. The results were considered statistically significant if * p < 0.05, ** p < 0.01, *** p < 0.001 or **** p < 0.0001.

Figure 5

SOT101 potentiates the therapeutic activity of Daratumumab in a solid human multiple myeloma xenograft model in vivo in dependence on the combination schedule. SOT101 was administered SC at 1 mg/kg and Daratumumab was administered IP at 20 mg/kg at the indicated timepoints (A) sequential drug schedules in the early therapeutic setting where treatment started at randomization day 1 (~ 100 mm³). (B) sequential drug schedules in the late therapeutic setting where the treatment started 4 days post randomization (day 5) (C). Concomitant drug schedule in the late therapeutic setting. Data are presented as mean ± SEM from 8 mice and show a representative study of n = 2. The results were considered statistically significant if * p < 0.05, ** p < 0.01 or *** p < 0.001; ns, not significant.