IL15 Trispecific Killer Engagers (TriKE) Make Natural Killer Cells Specific to CD33⁺ Targets While Also Inducing Persistence, In Vivo Expansion, and Enhanced Function

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Like cytotoxic T cells, upon activation NK cells deliver a store of membrane penetrating and apoptosis-inducing molecules, including granulysin, granzyme, and perforin (1). Unlike T cells, NK cells do not require antigen priming and recognize targets by engaging activating receptors in the absence of self-MHC recognition by inhibitory receptors. We have shown that adoptive transfer of haploidentical NK cells after lymphodepleting chemotherapy can induce complete remissions in 30% to 50% of patients with refractory acute myeloid leukemia (AML) when given with IL2 to stimulate in vivo donor NK cell expansion (2,3). However, this approach is limited by lack of antigen specificity and by IL2-mediated induction of regulatory T (Treg) cells that suppress NK cell proliferation and function (3,4). Thus, we have developed a reagent that targets NK cells to specific tumor antigens and drives NK expansion and persistence, while bypassing the negative effects of Tregs and the morbidity of IL2.

NK cells mediate antibody-dependent cellular cytotoxicity (ADCC) through the highly potent CD16 (FcγRIII) activating receptor. Signaling through CD16 induces a calcium flux and phosphorylation of ITAMs triggering the release of lytic granules and cytokines such as interferon (IFNγ) and tumor necrosis factor (TNFα; refs. 5–7). To better target NK cells to malignant targets, we created and tested bispecific or trispecific killer engagers (BiKE and TriKE, respectively; refs. 8–11) each incorporating an antihuman anti-CD16 scFv derived from a human phage display library (12) with other scFvs directed against epitopes on malignant cells. These agents form an immunologic synapse between the highly activating CD16 receptor on NK cells and specific tumor antigens and markedly enhance cytotoxic killing of various human cancers (8–11). Our 1633 BiKE enhances NK cell responses to CD33⁺ AML (9) and myelodyplastic syndrome (MDS) targets (8).

IL15 plays a key role in NK cell development, homeostasis, proliferation, survival, and activation (13). IL15 and IL2 share several signaling components including the IL2/IL15Rβ (CD122) and the common gamma chain (CD132). However, unlike IL2, IL15 does not stimulate CD25⁺ Tregs, allowing for NK cell activation without inducing concurrent Treg-mediated immune inhibition (14). IL15 also activates NK cells, and can restore functional defects in engrafting NK cells after hematopoietic stem cell transplantation (HSCT; ref. 15). IL15 also stimulates CD8⁺ cytotoxic T cells, which further enhances its immunotherapeutic potential. Importantly, based on preclinical animal studies, the toxicity profile of low dose IL15 may be more favorable than that of IL2 (14,16). This report describes the generation of a 161533 TriKE that utilizes IL15 as an intramolecular linker between CD16 and CD33 scFvs to direct NK cell–mediated killing of CD33⁺ tumors while simultaneously generating an NK cell self-sustaining proliferation/survival signal through IL15 with a lesser effect on T cells.

Peripheral blood mononuclear cells (PBMC) were isolated from normal adult donor blood obtained from Memorial Blood Center by centrifugation using a Histopaque gradient (Sigma-Aldrich) and cryopreserved. Where noted, NK cells were enriched with magnetic beads using Stemcell EasySep Human NK cell enrichment kit whereas CD3/CD19–depleted products were generated with Miltenyi's CliniMACS beads. Post-HSCT PBMCs from patients undergoing HLA-matched sibling transplants were obtained from an immune reconstitution biobank protocol. Recipient blood was collected and cryopreserved early (day 20 to 100; n = 10) after HSCT. All samples were obtained after informed consent, using guidelines approved by the Committee on the Use of Human Subjects in Research at the University of Minnesota (Minneapolis, MN) in accordance with the Declaration of Helsinki.

Two cell lines were used and authenticated in these studies. HL-60, a CD33⁺ human acute promyelocytic leukemia cell line, was obtained in 2013 from ATCC, and authenticated by flow cytometry (FC) against a panel of FITC-labeled anti-CD33 and anti-CD45 as positive markers and anti-CD19, anti-CD22, anti-CD3, and anti-EpCAM as negative markers (BioLegend). The line was cultured in Iscove's medium (Invitrogen) supplemented with 20% FBS (Gibco, Invitrogen) and 100 U/mL penicillin and 100 U/mL streptomycin (Invitrogen) at 37°C and 5% CO₂. The control, murine colorectal carcinoma cell line HT-29 was also obtained from ATCC in 2013 and authenticated against a panel of BV7 anti-Integrin β1 antibody, MOC-31 epithelial glycoprotein 2, and anti-EpCAM as positive markers and anti-CD19, anti-CD22, and anti-CD45 as negative markers. The line was cultured at 37°C with 5% CO₂ in high glucose DMEM (Invitrogen) supplemented with 10% FBS, and 100 U/mL penicillin and 100 U/mL streptomycin and last tested in 2015.

The hybrid gene encoding 161533 was synthesized using DNA shuffling and DNA ligation techniques (10,17). The VL and VH genes of each sFv were linked by a fragment encoding G4S linker. In its final configuration, the 161533 NcoI/XhoI gene fragment encoded a start codon followed first by antihuman CD16 scFv (12), a 20 amino acid flanking sequence PSGQAGAAASESLFVSNHAY, human IL15, and then a seven amino acid flanking sequence EASGGPE, and then the anti-CD33 scFv. The final target gene was spliced into the pET21d expression vector and inclusion bodies expressed. DNA-sequencing analysis (Biomedical Genomics Center, University of Minnesota, Minneapolis, MN) was used to verify that the gene was correct in sequence and cloned in frame.

Plasmid was transformed into the Escherichia coli strain BL21(DE3) (EMD). Bacteria were grown in 600 mL Luria Broth supplemented with 100 μg/mL carbenicillin in a 2-liter flask at 37°C with shaking. Expression of the hybrid gene was induced by the addition of isopropyl-b-d-thiogalactopyranoside (IPTG; FisherBiotech). Two hours after induction, the bacteria were harvested by centrifugation. The cell pellets were suspended and homogenized using a polytron homogenizer. After sonication and centrifugation, the pellets were extracted with 0.3% sodium deoxycholate, 5% Triton X-100, 10% glycerin, 50 mmol/L Tris, 50 mmol/L NaCl, 5 mmol/L EDTA, pH 8.0, and inclusion bodies extensively washed to remove endotoxin.

The proteins were refolded using a sodium N-lauroyl-sarcosine (SLS) air oxidation method modified from a previously reported procedure for isolating scFv (18). Refolded 161533 was purified by FPLC ion exchange chromatography (Q Sepharose Fast Flow) using a stepwise gradient from 0.2 to 0.5 mol/L NaCl in 20 mmol/L Tris-HCl, pH 9.0 over four column volumes.

Cells were immunophenotyped with the following fluorescent-labeled mAbs against: PE-Cy7-conjugated CD56 (HCD56; BioLegend), ECD/PE-CF594-conjugated CD3 (UCHT1; Beckman Coulter), APC-Cy7-conjugated CD16 (3G8; BioLegend), Pacific Blue-conjugated CD45 (HI30; BioLegend), PerCP-Cy5.5/FITC-conjugated anti-human CD107a (LAMP-1; H4A3; BioLegend), Pacific Blue/BV421-conjugated antihuman IFN-γ (4S.B3; BioLegend), FITC/Alexa Fluor 647-conjugated TNFα (MAb11; BioLegend), FITC/PE-conjugated CD33 (P67.6; BD Biosciences), and APC-conjugated CD45 (HI30; BioLegend), FITC-conjugated EpCAM; (BioLegend). Phenotypic acquisition of cells was performed on the LSRII (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc.).

Post-HSCT recipient PBMCs and primary AML blasts were thawed and placed in RPMI10 overnight. After 16 to 24 hours, the PBMCs and 50 nmol/L 1633 BiKE or 161533 TriKE was added to the cultures. The next morning, cells were washed and another round of 50 nmol/L 1633 BiKE or 161533 TriKE was added. HL-60 targets or primary AML blasts were added immediately after to generate a 5:1 effector to target ratio. PBMCs, HL-60 targets or primary AML blasts, and BiKE or TriKE molecules were cocultured for 4 hours and CD107a expression and intracellular IFNγ and TNFα production were evaluated as described previously (10).

PBMCs from posttransplant patients (day 22 to 44 or day 100) were labeled with CellTrace Violet Cell Proliferation Dye (Invitrogen), per manufacturer's protocol, placed in culture medium with HL-60 target cells at 5:1 (E:T) ratio, and treated with 50 nmol/L 1633 BiKE or 161533 TriKE. Cells were then harvested 7 days later and analyzed for viability, through Live/Dead staining, and proliferation, through dilution of CellTrace, in the NK cell (CD56⁺CD3⁻) population.

Cytotoxicity was evaluated by 4-hour ⁵¹Cr-release assays. Briefly, resting PBMC from normal donors treated with 161533 TriKE or 1633 BiKE (10 μg/mL), scFvCD16 control reagent (10 μg/mL), or no reagent were cocultured for 4 hours with ⁵¹Cr-labeled HL-60 targets at varying E:T ratios. For posttransplant study, PBMCs cells were cocultured with HL-60 targets at a 20:1 (E:T) ratio in the presence of 50 nmol/L 1633 BiKE or 50 nmol/L 161533 TriKE. ⁵¹Cr release was measured by a gamma scintillation counter (Perkin Elmer) and specific target lysis was determined (10).

HL-60-luc cells were incorporated into a previously described NK cell xenogeneic mouse model system (19). NSG mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ, n = 5/group) were conditioned with 275 cGy and injected intravenously with 7.5 × 10⁵ HL-60-luc S4 subcultured for tumor invasiveness. The control group did not receive NK cells whereas the 1633 BiKE and the 161533 TriKE groups received 1 × 10⁶ NK cells, calculated from a CD3/CD19 magnetically depleted product, on day 3. BiKE and TriKE treatment was also initiated after injection of the NK cells on day 3. A single course of treatment consisted of an intraperitoneal injection of 20 μg of drug given 5 days/week (MTWThF) and mice were treated for 2 weeks. The HL-60-luc cells are a subline of HL-60 and have been transfected with a luciferase reporter gene, allowing for imaging of the mice each week to determine their bioluminescent activity and to monitor leukemia progression as described previously (20). Briefly, mice were injected with 100 μL of 30 mg/mL luciferin substrate 10 minutes prior to imaging and then anesthetized via inhalation of isoflurane gas. The mice were then imaged using the Xenogen IVIS 100 imaging system and analyzed with Living Image 2.5 software (Xenogen Corporation). On day 20, all the animals were bled and 2-minute exposures were made and units for the regions of interest (ROI) were expressed as counts. The blood was analyzed by flow cytometry for presence of human CD45⁺CD56⁺CD3⁻ NK cells. A second experiment was performed to verify reproducibility of data.

For analysis of cytokines/chemokines, 500,000 NK cells cocultured with 250,000 HL-60 cells were treated with the BiKE, TriKE, or no reagent for 24 hours at 37°C, 5% CO₂. Supernatant levels were determined using a human-specific panel for IFNγ, TNFα, GM-CSF, or MIP-1α (EMD Millipore), and the Luminex system (Luminex). Final pg/mL values were interpolated from standard curves of the corresponding recombinant human proteins with Bioplex software v.5.1 (Bio-Rad).

Grouped data were expressed as mean ± SEM. Differences between two groups were analyzed by Student t test. Multiple comparisons were analyzed by paired one-way ANOVA with Tukey correction. Analysis was carried out in GraphPad Prism software. On figures statistical significance is indicated as: *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001.

We introduced a human modified IL15 cross-linker into our 1633 BiKE to create a 161533 TriKE designed to mediate antigen specificity and to deliver a proliferation and survival signal to NK cells (Fig. 1A). The bacterial expression system, which requires refolding, delivered high yield 1633 BiKE and the 161533 TriKE products as shown by fast protein liquid chromatography (FPLC) profiles (Fig. 1B). The addition of IL15 to the molecule reduced its isoelectric point by two pH units, creating more favorable conditions for purification, resulting in nearly double the yield compared with 1633 BiKE after purifications that began with identical amounts of inclusion bodies. Products were >95% pure by SDS-PAGE gel analysis and Coomasie Blue Staining (Fig. 1C). To verify that binding and specificity remained intact in the new TriKE molecule, selectivity was measured by direct binding and competition flow cytometry assays against CD33⁺ HL-60 cells and CD33⁻ HT-29 cells (Supplementary Tables S1 and S2). The IL15 flanking sequences were critical for optimal TriKE activity.

To test whether the 161533 TriKE retained the ability to mediate ADCC, as demonstrated with the 1633 BiKE (8,9), killing of CD33⁺ HL-60 targets by normal donor PBMCs was measured with both agents in a 4-hour ⁵¹Cr release assay (Fig. 2A). The 161533 TriKE induced higher NK cell–mediated killing than the 1633 BiKE, particularly at the 20:1 ratio (58.3% ± 2.3% vs. 33% ± 4%, P = 0.02). Control conditions with anti-CD16 and anti-CD33 scFv alone did not increase killing compared with untreated PBMC controls, demonstrating that these components alone have minimal activity. To address the input of the IL15 moiety on NK cell cytotoxicity, we compared the 161533 TriKE to equivalent molar concentrations (30 nmol/L) of IL15 or IL15 plus 1633 BiKE (Supplementary Fig. S1A). Results indicate that combining 1633 BiKE and IL15 yields similar NK cell–mediated cytotoxicity when compared with utilization of the 161533 TriKE. Although a trend of enhanced cytotoxicity can be seen with addition of IL15 alone, likely because of enhanced priming of natural cytotoxicity, the 1633 specificity elicited by the scFvs in the TriKE or BiKE + IL15 clearly enhance killing of targets indicating that combination of these molecules are paramount to enhancing NK cell function. The specificity of 161533 TriKE was further demonstrated by its inability to mediate killing of CD33⁻ HT-29 target cells after coincubation with PBMCs (Fig. 2B). A novel IL15 TriKE made with an anti-EpCAM scFv in place of anti-CD33 was used as a positive control to confirm that the HT-29 target cells were not inherently resistant to NK-mediated killing. The robust killing of CD33⁻EpCAM⁺ HT-29 cells mediated by the anti-EpCAM TriKE highlights the versatility of the IL15 linker TriKE platform to induce antigen specificity against both hematologic and solid tumor malignancies.

To directly investigate the role of the 1633 BiKE and 161533 TriKE on NK cell activation, PBMCs were incubated alone, with HL-60 cells, or with HL-60 cells plus 1633 BiKE or 161533 TriKE for 24 hours and activation markers were evaluated on the NK cells (Supplementary Fig. S1B). A clear hierarchy of activation can be noted through induction of CD69 expression, which is highest on NK cells incubated with 161533 and targets, followed by NK cells incubated with 1633 and targets, NK cells incubated with targets alone, and NK cells that were left untreated. CD25 expression was also highest on the NK cells incubated with 161533 and HL-60s, but the magnitude of the increase is likely also contributed by the effect of IL15 signaling on CD25 induction. A trend for an increase in HLA-DR expression, which has slower induction dynamics, can also be noted in the NK cells treated with 1633 and 161533 and targets. Taken together these data indicate potent NK cell activation through the 161533 TriKE.

In addition to cytotoxicity, another key anticancer function of NK cells is the production of cytokines and chemokines upon target cell recognition. Cytokine production was measured by incubating enriched NK cells and HL-60 targets alone, with 1633 BiKE, or with 161533 TriKE. Inflammatory cytokines and chemokines were measured in supernatants collected after 24 hours of incubation (Fig. 2C). 161533 TriKE induced significantly more IFNγ and TNFα secretion compared with 1633 BiKE (left). No significant differences were noted between levels of IL6, IL10, or IL8 (data not shown). GM-CSF and MIP-1α (right) were also induced by the 161533 TriKE, whereas RANTES remained unchanged from baseline (data not shown). These data indicate that incorporation of the IL15 molecule into the TriKE enhances the production of proinflammatory cytokines and chemokines by NK cells, augmenting their antitumor activity.

The ability of the IL15 linker in our 161533 TriKE to deliver homeostatic and expansion signals to NK cells was tested in a physiologically relevant context using early post-HSCT recipient samples. This time point is of particular interest because we have shown major defects in NK cell–mediated target cell induced cytokine production at this time, when engrafting NK cells are needed to mediate graft versus leukemia (GvL) responses (15). The proliferation induced by 1633 BiKE or 161533 TriKE in post-HSCT recipient PBMCs (day 20 to 100; n = 8) was measured after labeling with CellTrace dye and 7 days of incubation with HL-60 targets. Cell survival at 7 days, measured with Live/Dead dye, was significantly greater in the 161533 TriKE condition compared with the 1633 BiKE condition (Fig. 3A and B; 96.9% ± 0.5% vs. 21% ± 5.4%; P < 0.0001). The proliferation induced by the IL15 moiety in the 161533 TriKE was measured by CellTrace dye dilution in the viable NK and T-cell populations. Unexpectedly, the 161533 TriKE induced robust and specific NK cell proliferation, with minimal T-cell proliferation in the same samples (Fig. 3C and D, top; cell division in 79.1% ± 2.5% of NK cells vs. 2.3% ± 1.1% of T cells, P < 0.0001). Compared with T cells, the expansion index or total fold expansion of the NK cells was significantly higher in the posttransplant setting (Fig. 3D, bottom: 7.2% ± 0.8% vs. 1.1% ± 0.1%, P < 0.0001). However, in normal donors, the 161533 TriKE was capable of expanding T cells (Supplementary Fig. S2A), albeit to a much lower extent than NK cells, indicating that the reason there is no expansion of T cells in the posttransplant setting might be that at early time points after HSCT T cells capable of responding to the IL15 moiety are diminished or their activity is blunted by concomitant pharmacologic agents given for GVHD prophylaxis.

We compared the biologic activity of the IL15 moiety in the 161533 TriKE molecule to that of the clinical grade recombinant human IL15 produced by the NCI. Enriched NK cells from normal donors were labeled with CellTrace and incubated for 7 days with no reagent, NCI IL15, 1633 BiKE, or 161533 TriKE. NK cell proliferation required IL15, and was maintained by either NCI IL15 or the 161533 TriKE (Supplementary Fig. S2B and S2C). In marked contrast, the NK cells in the no reagent or 1633 BiKE groups failed to proliferate. Together, these data indicate that the IL15 linker incorporated into the 161533 TriKE is functionally active and capable of delivering survival and proliferation signals to normal donor or post-HSCT recipient NK cells, with minimal effect on T cells.

Given the potential clinical development of 1633 BiKE and 161533 TriKE as post-HSCT immunotherapy, the ability of each molecule to restore defective NK cell function was tested using post-HSCT recipient PBMCs (Fig. 4). Thawed and rested PBMC were incubated with 1633 BiKE or 161533 TriKE overnight, washed, and reincubated with the same agent immediately prior to addition of targets. Responses after a 4-hour incubation with HL-60 targets were measured by chromium release and flow cytometry. Although the 1633 BiKE promoted double the cytotoxicity against HL-60 targets compared with PBMCs alone (18.9% ± 2.1% vs. 9 ± 1.5%, P < 0.0001), incubation with the 161533 TriKE further enhanced NK cell–mediated cytotoxic function (52.1 ± 3.5%; Fig. 4A). The increased cytotoxicity mediated by 161533 TriKE correlated with increased NK cell degranulation measured by CD107a expression (Fig. 4B and C, left). Defective target cell-induced production of IFNγ by NK cells was not restored by 1633 BiKE, whereas 161533 TriKE promoted a potent response (2.6% ± 0.5% vs. 21.7% ± 4.4%, respectively; P = 0.001; Fig. 4B and C, middle). A similar rescue of TNFα production by 161533 TriKE, but not 1633 BiKE, was observed (29.9% ± 3.8% vs. 6.1% ± 1%, respectively; P < 0.0001; Fig. 4B and C, right). The magnitude of post-HSCT recipient NK cell responses to CD33⁺ targets mediated by 161533 TriKE clearly demonstrate the immunotherapeutic potential of the this agent.

We compared the function of post-HSCT recipient NK cells incubated with 1633 BiKE or 161533 TriKE against primary AML blasts from two different patients (AML1 and AML2). Although the overall production of CD107a, IFNγ, and TNFα by post-HSCT NK cells exposed to primary blasts was reduced when compared with HL-60 targets, the 161533 TriKE again mediated significantly more degranulation and cytokine production compared with the 1633 BiKE (Fig. 5A and B). Reduced expression of CD33 on the primary AML blasts compared with HL-60 (Supplementary Fig. S2D) could explain the decreased NK cell response to primary AML blasts. Importantly, the post-HSCT recipient NK cells exhibited similar 161533 TriKE-mediated responses to activation by AML1 and AML2. Taken together, these in vitro data indicate that the 161533 TriKE induces antigen-specific NK cell cytotoxic and cytokine-producing responses against primary CD33⁺ tumor cells, and that the costimulation provided by the IL15 linker increases NK responses compared with those seen with the 1633 BiKE agent.

We developed a murine xenograft model incorporating human NK cells and CD33⁺ tumor cells to compare the activity of the 1633 BiKE and the 161533 TriKE in vivo. NSG mice were conditioned and injected intravenously with HL-60 cells containing a luciferase reporter gene (7.5 × 10⁵ cells/mouse), followed 3 days later by intravenous infusion of 1 × 10⁶ normal human donor NK cells activated overnight with IL15. Mice then were treated with no drug (and no NK cells in the control group), 1633 BiKE, or 161533 TriKE (20 μg) administered intraperitoneally MTWThF through the first 2 weeks of the study (10 doses total). Mice were imaged on day 14 and day 21 to measure tumor burden (Fig. 6A and B). At day 14, the 1633 BiKE and 161533 TriKE groups showed similar control of tumor burden, both of which were significantly better than the control group. However, on day 21, by which time 2 mice from the no-drug control group had died, only the 161533 TriKE group showed significantly reduced tumor burden, indicating superior sustained control of the HL-60 tumor burden with 161533 TriKE therapy.

To evaluate the effect of the 1633 BiKE and the 161533 TriKE on in vivo NK cell maintenance, accounting for NK cell survival and proliferation, we bled the mice (20 μL) at day 20 and measured the number of human NK cell (CD45⁺CD56⁺CD3⁻) events in 60 seconds of acquisition by flow cytometry. Essentially, no circulating human NK cells were detected in the no-drug control or the 1633 BiKE-treated animals (17.3 ± 14.3 and 11.6 ± 2.3 events, respectively; Fig. 6C and D). In marked contrast, all of the 161533 TriKE-treated animals had a substantial number circulating human NK cells (4261 ± 410.6 events) during the same acquisition period. Thus, mice treated with 161533 TriKE had over 350-fold more human NK cells circulating in the peripheral blood than did BiKE-treated mice, indicating that the IL15 linker within the TriKE molecule delivers robust proliferation and survival signals.

A second experiment was performed using this same murine xenograft model (n = 4 to 5/group) to reproduce this data. Only in the second experiment, a higher number (10⁶) of highly HL-60 cells were administered. Fifty percent of the TriKE-treated mice were robust responders to therapy. Again, the BiKE-treated mice showed only minimal effects on day 21 (data not shown). Also, a separate group of animals were given high dose IL15 alone without BiKE or TriKE (5 μg MWF). These showed a reduced response indicating that IL15 itself is not sufficient to potently mediate an NK cell–mediated antitumor effect.

The original contribution of this work is a self-sustaining 161533 TriKE using anti-CD16 and anti-CD33 scFvs with a modified IL15 linker capable of directing antigen-specific ADCC while inducing NK cell proliferation and survival. We previously reported that our 1633 BiKE molecule successfully drives antigen-specific ADCC against primary AML and MDS (8,9), and we have substantially improved this platform by introducing an IL15 signal that costimulates the NK cell response to CD33⁺ targets, including primary AML blasts, with significantly enhanced cytotoxic and cytokine production compared to the 1633 BiKE. The 161533 TriKE induces potent responses by NK cells from normal donor samples as well as from post-HSCT recipient NK cells. Most importantly, using our novel xenogeneic tumor model we demonstrate that the 161533 TriKE is capable of driving human NK cells to proliferate and survive in vivo while mediating an antigen-specific sustained anticancer response. Neither 1633 BiKE or IL15 alone had all these properties.

Combining cytokines with antibodies is not a new concept and early efforts focused on IL2. IL2 cytobodies were generated in the early 2000s (21–24), but unfortunately clinical responses were marginal and IL2 toxicities were dose limiting. The effectiveness of IL2 as a cancer immunotherapy is also limited by its potent induction of CD25⁺ Tregs. IL15 plays a critical role in NK cell development, homeostasis, proliferation, activation and survival (13,25–30). IL15 also may be less toxic than IL2, as has been demonstrated by Munger and colleagues who used a vascular leak mouse model that measures the extravasation of ¹²⁵I-labeled albumin. They showed that high dose IL2 caused vascular leak syndrome (VLS) in mice, but high dose IL15 did not (16). Thus we chose to incorporate a modified IL15 molecule into our 161533 TriKE to simultaneously function as an intramolecular linker and as a stimulator of NK function. Although IL15 is considered less toxic than IL2 based on its reduced propensity to induce VLS, it does trigger a cytokine cascade including TNα, IL1, IL6, IL8, MCP-1, GM-CSF, and pro-inflammatory chemokines (31,32). The most common severe side effects are fever, fatigue, nausea, vomiting, weight loss, severe neutropenia, and a generalized skin rash associated with cytokine inflammatory syndrome. In mice, weight loss precedes death, so weight loss can be used as a measure of toxicity. We did not observe weight loss in NSG mice that received NK cells and NCI rH IL15 or IL15 in the form of the 161533 TriKE (10 injections of 20 μg; data not shown). These studies do not demonstrate toxicity, either from the 161533 TriKE or from the activated NK cells. Although further investigation is warranted to assess whether IL15-activated NK cells can induce toxicity indirectly, our preclinical data indicate that IL15 activation of NK cells by the 161533 TriKE agent may result in decreased toxicity by more localized delivery.

Other potential toxicities associated with exogenous IL15 administration should be considered. In certain settings, IL15 has been found to participate in the development of leukemias and solid tumors (33–40), to inhibit apoptosis of tumor cells (41), and to promote their proliferation (38,42), migration (43), epithelial-to-mesenchymal transition (35), invasion (37), metastasis, and tumor angiogenesis (36). Therefore, we believe that selectively delivering IL15 signals to NK cells in the context of the TriKE molecule will facilitate more direct interactions with the immune synapse formed between NK effector cells and tumor targets and will potentially decrease systemic effects.

We have demonstrated that unlike the 1633 BiKE, the 161533 TriKE is capable of inducing proliferation and increasing survival in normal donor and post-HSCT recipient NK cells. As shown in our xenogeneic tumor model, the increased number of live NK cells will likely translate to increased antitumor activity. Importantly, our in vitro assays showed that the TriKE-induced proliferation of NK but not T cells in post-HSCT samples, which is important in the post-HSCT setting where T cells mediate graft versus disease. We have previously described functional defects in recipient NK cells early post-HSCT (15,44), which may account for early relapses (45). Compared with the 1633 BiKE, our 161533 TriKE restored robust NK cell function in post-HSCT recipient NK cells. Thus, there may be great benefit of this agent in the HSCT arena.

We and others have previously reported BiKEs (8–11,45,46). Our preclinical data show that 161533 TriKE holds more promise for clinical testing to treat AML and MDS. Allogeneic HSCT is standard therapy for MDS and AML, but older patients transplanted with reduced intensity conditioning have an unacceptably high relapse rate of 30%. Thus, new therapeutic strategies are urgently needed to reduce relapse of MDS and AML after HSCT and to treat patients who are not transplant candidates. Our 161533 TriKE adds a critical component to the BiKE scaffold by inducing maintenance and expansion of the effector NK cells mediating tumor killing. This is particularly important because resting NK cell function is diminished in MDS and AML patients from the disease itself or its therapy. A unique advantage of using the CD33 scFv in our TriKE is that the molecule may also direct NK cells to eliminate CD33⁺ myeloid-derived suppressor cells (MDSC). Thus, the agent may directly target tumor as well as reducing tumor-induced immune suppression.

The 161533 TriKE molecules could also be used to enhance endogenous NK cell function against NK sensitive malignancies in the posttransplant setting, with minimal effects on T cells as demonstrated by our in vitro studies, particularly in refractory disease or in patients undergoing reduced intensity conditioning. Although studies shown here target the CD33 antigen in myeloid tumors, the IL15 TriKE platform can easily be adapted to target other tumors by switching the anti-CD33 scFv portion for an scFv targeting a different tumor antigen such as EpCAM present on solid tumor targets, making this a versatile platform for self-sustained NK cell–based immunotherapies.

D.A. Vallera has ownership interest (including patents) in and is a consultant/advisory board member for Oxis Biotech. J.S. Miller is a consultant/advisory board member for Oxis International. D.A. Vallera and J.S. Miller are listed as co-inventors on a provisional patent application on IL15 TriKEs that is owned by University of Minnesota and is not yet licensed. No potential conflicts of interest were disclosed by the other authors.

Conception and design: D.A. Vallera, M. Felices, B. Zhang, M.R. Verneris, J. Tolar, B.R. Blazar, J.S. Miller

Development of methodology: M. Felices, B. Zhang, J. Tolar, B.R. Blazar, J.S. Miller

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.A. Vallera, M. Felices, R. McElmurry, V. McCullar, J.U. Schmohl, B. Zhang, A.J.L. Lenvik, A. Panoskaltsis-Mortari, J. Tolar, B.R. Blazar

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Felices, R. McElmurry, V. McCullar, B. Zhang, A.J.L. Lenvik, A. Panoskaltsis-Mortari, J. Tolar, D.J. Weisdorf, B.R. Blazar, J.S. Miller, D.A. Vallera

Writing, review, and/or revision of the manuscript: D.A. Vallera, M. Felices, V. McCullar, B. Zhang, A. Panoskaltsis-Mortari, M.R. Verneris, S. Cooley, D.J. Weisdorf, B.R. Blazar, J.S. Miller

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.A. Vallera, V. McCullar, B. Zhang

Study supervision: D.A. Vallera, M. Felices, B. Zhang, B.R. Blazar, J.S. Miller

Other (providing critical reagents): X. Zhou

The authors acknowledge cell-processing services from the Translational Therapy and FACS Cores of the Masonic Cancer Center (P30 CA77598). The authors also acknowledge the excellent technical assistance of Elizabeth Taras, Deborah Todhunter, Andy Sicheneder, Sami Chu, and Seunguk Oh.

This work was supported in part by the U.S. Public Health Service Grant R01-CA36725, R01-CA72669, P01-CA65493, P01-CA111412, and R35 CA197292 awarded by the NCI and the NIAID, DHHS. It was also supported by an NIH Research Evaluation and Commercialization Hub (REACH) Award (U01), the Mayo Partnership Award, the Lion Fund, William Lawrence and Blanche Hughes Fund, the Randy Shaver Foundation, the Atwater Cancer Drug Development Award, the Deutsche Krebshilfe (DS), and a CETI translational award from the University of Minnesota Masonic Cancer Center.

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