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BiKE and TriKE selectively bind antigen expressing targets

Supplemental Figure Legends

Supplementary Figure 2. Studies of effector cells from normal donors indicate that NK cells proliferate moreso than T cells in response to TriKE, but T cells can expand.

Blocking studies show specificity of BiKEs and TriKEs.

Supplementary Figure 1. To address the input of the IL-15 moiety on NK cell cytotoxicity, we compared the 161533 TriKE to equivalent molar concentrations of IL-15 or IL-15 plus 1633 BiKE. Activation markers were studied.

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

Background B7H3 is a tumor associated antigen (TAA), found on numerous malignancies including prostate, lung, and breast cancers. High levels of B7H3 expression are correlated with late stage disease and poor prognosis. Furthermore, B7H3 is minimally expressed on normal tissue, making it an ideal TAA for broad cancer treatment strategy. We developed a tri-specific killer engager (TriKETM) consisting of a nanobody anti-CD16, IL-15, and nanobody anti-B7H3 joined by flexible linkers (camB7H3 TriKE) (figure 1A). The combination of B7H3 TriKE with an off-the-shelf NK cell therapy presents an appealing therapeutic strategy for the treatment of solid tumors with decreased risk of toxicity in allogeneic settings compared to T-cell derived products. Methods An anti-B7H3 nanobody was developed via biopanning and cloned into a TriKE vector. TriKE was produced in Expi293 cells and affinity purified using poly-His tag. NK cells were co-incubated with cell lines exhibiting a range of B7H3 expression and with 3nM of camelid B7H3 TriKE or control. We have previously derived NK cells expressing high affinity non-cleavable hnCD16, CD38 KO, and IL-15/IL-15R fusion from clonal master engineered iPSC lines. Engineered iNK cells were tested in conjunction with the TriKE. A repeated measures ANOVA was used for statistical comparisons as noted in figure legends Results Engineered iNK cells co-incubated with camB7H3 TriKE and C4-2 prostate cells significantly increased degranulation (CD107a) and cytokine production (IFN-gamma) compared to controls (figure 1B/C, P Conclusions camB7H3 TriKE dramatically increases function and activation on endogenous NK cells as well as engineered iNK cell, which can be adoptively transferred to patients with a broad range of cancers, including prostate cancer. TriKE activity was potent across a broad concentration spectrum and corresponded directly with B7H3 target expression. These studies represent the proof-of-concept of a novel pairing of off-the-shelf, engineered iNK cells with B7H3-directed pan-cancer engager molecules (TriKEs and CARs) to enhance specificity, persistence and anti-tumor function.

Objective Natural killer (NK) cells represent a powerful immunotherapeutic target as they lyse tumors directly, do not require differentiation, and can elicit potent inflammatory responses. The objective of these studies was to use an IL-15 super-agonist complex, ALT-803 (Altor BioScience Corporation), to enhance the function of both normal and ovarian cancer patient derived NK cells by increasing cytotoxicity and cytokine production. Methods NK cell function from normal donor peripheral blood mononuclear cells (PBMCs) and ovarian cancer patient ascites was assessed using flow cytometry and chromium release assays ±ALT-803 stimulation. To evaluate the ability of ALT-803 to enhance NK cell function in vivo against ovarian cancer, we used a MA148-luc ovarian cancer NOD scid gamma (NSG) xenogeneic mouse model with transferred human NK cells. Results ALT-803 potently enhanced functionality of NK cells against all ovarian cancer cell lines with significant increases seen in CD107a, IFNγ and TNFα expression depending on target cell line. Function was also rescued in NK cells derived from ovarian cancer patient ascites. Finally, only animals treated with intraperitoneal ALT-803 displayed an NK dependent significant decrease in tumor. Conclusions ALT-803 enhances NK cell cytotoxicity against ovarian cancer in vitro and in vivo and is able to rescue functionality of NK cells derived from ovarian cancer patient ascites. These findings suggest that ALT-803 has the potential to enhance NK cell-based immunotherapeutic approaches for the treatment of ovarian cancer.

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.

Natural Killer (NK) cells are components of innate immune response that are involved in tumor surveillance and clearance. Due to their ability to lyse tumor targets without prior priming they have gained some notoriety in the tumor immunotherapy setting. Although NK cells sense malignant transformation of cells through a number of stochastic receptors they posses no clonotypic receptors to drive specific recognition of tumor antigens, somewhat limiting their therapeutic applications. In order to address the antigen specificity issue we generated molecules, termed Tri-Specific Killer Engagers (TriKEs), that contain three arms: one arm contains a single chain variable fragment (scFv) that binds a potent NK cell activating receptor (CD16); one arm contains an scFv that binds a tumor antigen; and one arm contains the cytokine IL-15 to drive NK cell priming, expansion and survival. Initial pre-clinical studies with these first generation molecules showed that we could use them to target hematological malignancies (via a CD33 binding arm) both in vitro and in vivo or solid malignancies (via an EpCAM binding arm) in vitro. However, to improve on this platform we altered the molecule by swapping out the anti-CD16 scFv arm with a humanized camelid single domain antibody against CD16 (termed cam16 henceforth). This second generation cam16 TriKE platform potently enhanced NK cell activation against tumor targets and induced far greater NK cell expansion when compared to the first generation TriKE. Using our bacterial expression system we generated and isolated cam16 TriKEs against a number of different tumor antigens (CD33, EpCAM, Mesothelin and ROR1) to display the ability of the second generation TriKE platform to target a variety of cancers. To test these molecules we used flow cytometry based NK cell activation assays, where NK cells co-cultured with tumor targets +/- cam16 TriKEs are evaluated for NK cell degranulation (by expression of surface CD107a) and NK cell inflammatory cytokine production (by expression of intracellular IFNγ). We also used a real-time imaging platform, the IncuCyte Zoom, to evaluate the ability of NK cells to kill tumor targets in the presence or absence of cam16 TriKEs over a 2-3 day period. Our data indicates that cam16 TriKEs drive NK cell specific degranulation, inflammation, and killing of AML (CD33), ovarian cancer (EpCAM and Mesothelin), lung cancer (Mesothelin and EpCAM) and sarcoma (ROR1) tumor targets in vitro. Furthermore cam16 TriKE in vivo efficacy could be seen in humanized mouse models using both luciferase-labeled cell lines and patient derived xenografts. Taken together, these findings clearly highlight the translational potential of cam16 TriKEs in the tumor immunotherapy setting. Citation Format: Martin Felices, Behiye Kodal, Alexander J. Lenvik, Brianna Ettestad, Laura Bendzick, Caitlin Ryan, Dawn K. Schirm, Ron McElmurry, Craig E. Eckfeldt, Melissa A. Geller, Todd R. Lenvik, Daniel A. Vallera, Jeffrey S. Miller. Driving natural killer cell antigen-specific targeting of cancer via next generation trivalent molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4939.

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

Myelodysplastic syndromes (MDS) are stem cell disorders that can progress to acute myeloid leukemia. Although hematopoietic cell transplantation can be curative, additional therapies are needed for a disease that disproportionally afflicts the elderly. We tested the ability of a CD16xCD33 BiKE to induce natural killer (NK) cell function in 67 MDS patients. Compared with age-matched normal controls, CD7(+) lymphocytes, NK cells, and CD16 expression were markedly decreased in MDS patients. Despite this, reverse antibody-dependent cell-mediated cytotoxicity assays showed potent degranulation and cytokine production when resting MDS-NK cells were triggered with an agonistic CD16 monoclonal antibody. Blood and marrow MDS-NK cells treated with bispecific killer cell engager (BiKE) significantly enhanced degranulation and tumor necrosis factor-α and interferon-γ production against HL-60 and endogenous CD33(+) MDS targets. MDS patients had a significantly increased proportion of immunosuppressive CD33(+) myeloid-derived suppressor cells (MDSCs) that negatively correlated with MDS lymphocyte populations and CD16 loss on NK cells. Treatment with the CD16xCD33 BiKE successfully reversed MDSC immunosuppression of NK cells and induced MDSC target cell lysis. Lastly, the BiKE induced optimal MDS-NK cell function irrespective of disease stage. Our data suggest that the CD16xCD33 BiKE functions against both CD33(+) MDS and MDSC targets and may be therapeutically beneficial for MDS patients.

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.