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NK cell-based immunotherapies have been gaining traction in the clinic for treatment of cancer. IL-15 is currently being used in number of clinical trials to improve NK cell expansion and function. The objective of this study is to evaluate the effect of repetitive IL-15 exposure on NK cells. An in vitro model in which human NK cells are continuously (on on on) or intermittently (on off on) treated with IL-15 was used to explore this question. After treatment, cells were evaluated for proliferation, survival, cell cycle gene expression, function, and metabolic processes. Our data indicate that continuous treatment of NK cells with IL-15 resulted in decreased viability and a cell cycle arrest gene expression pattern. This was associated with diminished signaling, decreased function both in vitro and in vivo, and reduced tumor control. NK cells continuously treated with IL-15 also displayed a reduced mitochondrial respiration profile when compared with NK cells treated intermittently with IL-15. This profile was characterized by a decrease in the spare respiratory capacity that was dependent on fatty acid oxidation (FAO). Limiting the strength of IL-15 signaling via utilization of an mTOR inhibitor rescued NK cell functionality in the group continuously treated with IL-15. The findings presented here show that human NK cells continuously treated with IL-15 undergo a process consistent with exhaustion that is accompanied by a reduction in FAO. These findings should inform IL-15-dosing strategies in NK cell cancer immunotherapeutic settings.

Purpose The selective elimination of cancer stem cells (CSCs) in tumor patients is a crucial goal because CSCs cause drug refractory relapse. To improve the current conventional bispecific immune-engager platform, a 16133 bispecific natural killer (NK) cell engager (BiKE), consisting of scFvs binding FcγRIII (CD16) on NK cells and CD133 on carcinoma cells, was first synthesized and a modified interleukin (IL)-15 crosslinker capable of stimulating NK effector cells was introduced. Materials and methods DNA shuffling and ligation techniques were used to assemble and synthesize the 1615133 trispecific NK cell engager (TriKE). The construct was tested for its specificity using flow cytometry, cytotoxic determinations using chromium release assays, and lytic degranulation. IL-15-mediated expansion was measured using flow-based proliferation assays. The level of interferon (IFN)-γ release was measured because of its importance in the anti-cancer response. Results 1615133 TriKE induced NK cell-mediated cytotoxicity and NK expansion far greater than that achieved with BiKE devoid of IL-15. The drug binding and induction of cytotoxic degranulation was CD133+ specific and the anti-cancer activity was improved by integrating the IL-15 cross linker. The NK cell-related cytokine release measured by IFN-γ detection was higher than that of BiKE. NK cytokine release studies showed that although the IFN-γ levels were elevated, they did not approach the levels achieved with IL-12/IL-18, indicating that release was not at the supraphysiologic level. Conclusion 1615133 TriKE enhances the NK cell anti-cancer activity and provides a self-sustaining mechanism via IL-15 signaling. By improving the NK cell performance, the new TriKE represents a highly active drug against drug refractory relapse mediated by CSCs.

Purpose: The effectiveness of NK cell infusions to induce leukemic remission is limited by lack of both antigen specificity and in vivo expansion. To address the first issue, we previously generated a bispecific killer engager (BiKE) containing single-chain scFv against CD16 and CD33 to create an immunologic synapse between NK cells and CD33+ myeloid targets. We have now incorporated a novel modified human IL15 crosslinker, producing a 161533 trispecific killer engager (TriKE) to induce expansion, priming, and survival, which we hypothesize will enhance clinical efficacy. Experimental Design: Reagents were tested in proliferation and functional assays and in an in vivo xenograft model of AML. Results: When compared with the 1633 BiKE, the 161533 TriKE induced superior NK cell cytotoxicity, degranulation, and cytokine production against CD33+ HL-60 targets and increased NK survival and proliferation. Specificity was shown by the ability of a 1615EpCAM TriKE to kill CD33-EpCAM+ targets. Using NK cells from patients after allogeneic stem cell transplantation when NK cell function is defective, the 161533 TriKE restored potent NK function against primary AML targets and induced specific NK cell proliferation. These results were confirmed in an immunodeficient mouse HL-60-Luc tumor model where the 161533 TriKE exhibited superior antitumor activity and induced in vivo persistence and survival of human NK cells for at least 3 weeks. Conclusions: Off-the-shelf 161533 TriKE imparts antigen specificity and promotes in vivo persistence, activation, and survival of NK cells. These qualities are ideal for NK cell therapy of myeloid malignancies or targeting antigens of solid tumors. Clin Cancer Res; 22(14); 3440–50. ©2016 AACR. See related commentary by Talmadge, p. 3419

Natural Killer (NK) cells represent an exciting immunotherapeutic approach to treat cancer. We have shown that in vivo expansion and activation of donor NK cells supported by administration of IL-2 induces remission in patients with refractory AML. Recent clinical studies by our group have shown that IL-15 is superior to IL-2 to support NK cell persistence 14 days after adoptive transfer. However, only 36% of patients treated with 12 consecutive days of IL-15 had NK cell expansion to the level of ≥100 donor derived NK cells/µL blood compared to 10% in subjects treated with IL-2 (p=0.02). This leads us to conclude that we might not know the optimal route and interval to administer in vivo IL-15. We hypothesized that daily uninterrupted IL-15 dosing could lead to exhaustion or NK cellular stress. Therefore we designed an in vitro model system in which enriched NK cells are treated with three 3-day cycles of continuous IL-15 (IL-15cont) or were rested with a "gap" (skipping the middle cycle [IL-15gap]) before returning to the last cycle of IL-15. IL-15cont treatment yielded more proliferation and higher cell numbers compared to IL-15gap (4.8±0.44 vs. 1.9±0.26 million cells/ml, p < 0.0001) when cells were analyzed at the end of the three cycles (on day 9, where all in vitro measurements were taken). However, NK cell death, measured by flow cytometry, in the IL-15cont group was higher (18.9±2.2 vs 14.9±1.7 % cell death, p = 0.035) and this group also had an enrichment in genes involved in cell cycle checkpoint/ arrest, perhaps indicating more cellular stress in the IL-15cont. In an in vitro flow cytometric functional assay, the IL-15cont group had decreased activation when compared to the IL-15 gap group against K562 targets (43.6±2.1 vs 55.6±2.7 % CD107a [degranulation], p < 0.0001; 1.9±0.41 vs 7.1±0.93 % IFNg [inflammatory cytokine production], p = 0.0055). The decrease in NK cell activation correlated with a strong decrease in tumor target killing in an in vitro chromium release assay (Figure 1A) measuring killing of acute promyelocytic leukemia (HL-60) cell targets, in which the IL-15cont NK cells were potently outperformed by the IL-15gap cells (6.4±2.6 vs 51.5±4.8 % killing at 2.5:1 effector:target ratio, p < 0.0001). We used an in vivo xenogeneic model of AML, where conditioned NSG (NOD scid gamma) mice are engrafted with HL-60luc tumor targets 3 days prior to infusion with nothing, IL-15cont or IL-15gap human NK cells prepared within our 9 day culture system. Only the IL-15gap NK group mediated statistically significant tumor control when compared to tumor alone at two weeks following NK cell infusion (Figure 1B). To probe deeper into the functional defect we evaluated signaling after these treatments and noted decreased phosphorylation of several proteins in the IL-15cont group. These data led us to explore proteins involved in metabolism and we noted that CPT1A, a critical enzyme involved in fatty acid oxidation (FAO), was strongly increased in the IL-15gap treated NK cells (protein MFI of 15,759±2,603 [IL-15gap] vs 5,273±744 [IL-15cont], p = 0.009). Metabolic analysis using a Seahorse XFe24 analyzer showed an increased mitochondrial spare respiratory capacity (SRC) in the IL-15gap group, denoting better capability of the IL-15gap NK cells to respond to energetic demands (Figure 1C). In a separate experiment the groups were treated with etomoxir to inhibit CPT1A, and the SRC phenotype was reversed, with the IL-15gap group containing lower SRC than the IL-15cont group. To test these findings in a functional assay we repeated the IL-15cont treatment in combination with rapamycin, which can induce CPT1A through inhibition of mTORC1, and saw restoration of function to levels similar to IL-15gap (40.8±2.0 vs 49.3±2.9 % CD107a in the IL-15cont vs IL-15cont + rapamycin, p = 0.005; 2.4±0.47 vs 4.8±1.0 % IFNg in the IL-15cont vs IL-15cont + rapamycin, p = 0.03). These data indicate that NK cell functional exhaustion via continuous IL-15 signaling is mediated by a decrease in FAO. Intermittent IL-15 dosing or altering metabolism through other mechanisms may overcome this competition. These findings could impact ongoing clinical trials through simple alterations in dosing strategies in order to minimize NK cell exhaustion in the immunotherapeutic setting. Disclosures Cooley: Fate Therapeutics: Research Funding. Miller:Oxis Biotech: Consultancy, Other: SAB; Fate Therapeutics: Consultancy, Research Funding.

Although adoptive transfer of NK cells with IL-2 can induce complete remissions in 30-50% of patients with refractory AML, efficacy is limited by IL-2 mediated induction of Tregs and by lack of antigen specificity. Thus we generated a 161533 trispecific killer engager (TriKE) molecule containing an anti-CD16 scFv to engage NK cells, an anti-CD33 scFv to engage myeloid targets (including myelodysplastic syndrome [MDS]), and a modified IL-15 linker. We have previously shown that this molecule is superior to a 1633 bispecific killer engager (BiKE) in killing of AML targets and that it promotes enhanced survival and in vivo expansion of NK cells. We questioned the mechanism for the increased potency of the 161533 TriKE and if the TriKE could activate the dysfunctional and suppressed NK cells found in patients with MDS. Cryopreserved mononuclear cells (obtained from the NMDP Sample Repository) collected from 8 patients with advanced MDS were tested to investigate how the TriKE might enhance functionality in this setting. Previously we reported that MDS patients have significantly decreased frequencies of NK cells due to increased CD33+ myeloid derived suppressor cells. Our 161533 TriKE enhanced the function of NK cells derived from MDS patients against acute promyelocytic CD33+ leukemia HL-60 tumor targets (Figure 1A), when compared to 1633 BiKE, in a flow cytometry assay measuring NK cell degranulation (% CD107a: 41.8±3.8 vs. 30.3±3.2, p=0.004), and inflammatory cytokine production (% IFNg: 40.7±5.0 vs. 30.0±4.9, p=0.009; % TNFa: 36.9±5.5 vs. 28.4±4.8, p=0.009). State of the art microchip-based live cell imaging was then employed to evaluate NK cell function and contact-to-target dynamics (Figure 1B). Briefly, resting NK cells and HL-60 target cells were stained with distinct dyes and then co-incubated in the presence of BiKE or TriKE in microwells at 37°C and 5% CO2. Cells were then imaged for 12 hours using a Zeiss 880 microscope and analyzed by Matlab. In contrast to those incubated with BiKE, NK cells cultured in the presence of TriKE had augmented cytotoxicity (37%±6% vs. 59%±6%, p=0.02) and killed their targets remarkably faster (time to first target kill = 148±30 min vs. 75±26 min, p< 0.0001). In addition, NK cell serial killers were more common in the presence of TriKE compared to BiKE (number of killed targets ≥3: 18%±7% vs. 9%±1%, p=0.04). Having shown the robust killing dynamics of TriKE primed NK cells, we next designed an in vivo dose escalation study to evaluate HL-60 tumor control in our xenogeneic mouse model. NSG (NOD scid gamma) mice were conditioned with a sub-lethal dose of radiation (275 cGy) and engrafted with 750,000 HL-60luc cells, which allow for tracking of tumor growth using bioluminescent imaging (BLI). Mice were infused with 1 million fresh healthy donor human NK cells and treated daily with recombinant IL-15 (5 ug/injection), 20-200 ug of 161533 TriKE, or left untreated. Higher doses of TriKE provided better tumor control (6.3x109±2.9x109 [200 ug 1615133] vs. 1.6 x1010±2.7x109 [20 ug 161533] p/sec/cm2/sr) demonstrating dose responsiveness at days 14 and 21 This was not due to increased proliferation of effectors (NK cell numbers) induced by the IL-15 segment of the TriKE as the absolute PBNK cell counts in mice were not different across the TriKE concentrations. However, the day 14 and 21 absolute PBNK counts were higher in all of the TriKE groups when compared to the IL-15 treated mice, including in the 20 ug 161533 TriKE group which best matched the 5 ug IL-15 dose. These data indicated that the TriKE molecule mediates superior NK cell expansion or maintenance in vivo. Taken together the in vivo data suggests that 161533 TriKE not only mediates tumor control by inducing NK cell proliferation and survival in a methodology that exceeds signals provided by IL-15 alone, but also increases tumor killing through enhanced killing kinetics. In summary, we have shown that the 161533 TriKE can rescue dysfunctional NK cells suppressed in patients with MDS and can mediate potent in vivo tumor killing presumably through better NK cell maintenance and enhanced killing kinetics This is of translational importance and demonstrates that the cancer induced immune suppression is reversible by the activating signals induced by the TriKE molecule. The 161533 TriKE represents a promising modality to maximizing NK cell based immunotherapies against MDS and AML and will be in phase I clinical testing the first half of 2017. Disclosures Cooley: Fate Therapeutics: Research Funding. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.