il15 trispeciﬁc killer engagers (trike) make targets while ... · trike restored potent nk...

Cancer Therapy: Clinical

IL15 Trispecific Killer Engagers (TriKE) MakeNatural Killer Cells Specific to CD33þ TargetsWhile Also Inducing Persistence, In VivoExpansion, and Enhanced FunctionDaniel A. Vallera1, Martin Felices2, Ron McElmurry3, Valarie McCullar2,Xianzheng Zhou4, Joerg Uwe Schmohl1, Bin Zhang2, Alexander J. Lenvik2,Angela Panoskaltsis-Mortari3, Michael R. Verneris3, Jakub Tolar3, Sarah Cooley2,Daniel J.Weisdorf2, Bruce R. Blazar3, and Jeffrey S. Miller2

Abstract

Purpose: The effectiveness of NK cell infusions to induceleukemic remission is limited by lack of both antigen specificityand in vivo expansion. To address the first issue, we previouslygenerated a bispecific killer engager (BiKE) containing single-chain scFv against CD16 and CD33 to create an immunologicsynapse between NK cells and CD33þ myeloid targets. We havenow incorporated a novel modified human IL15 crosslinker,producing a 161533 trispecific killer engager (TriKE) to induceexpansion, priming, and survival, which we hypothesize willenhance clinical efficacy.

Experimental Design: Reagents were tested in proliferationand functional assays and in an in vivo xenograft model ofAML.

Results: When compared with the 1633 BiKE, the 161533TriKE induced superior NK cell cytotoxicity, degranulation,and cytokine production against CD33þ HL-60 targets and

increased NK survival and proliferation. Specificity was shownby the ability of a 1615EpCAM TriKE to kill CD33-EpCAMþ

targets. Using NK cells from patients after allogeneic stem celltransplantation when NK cell function is defective, the 161533TriKE restored potent NK function against primary AML targetsand induced specific NK cell proliferation. These results wereconfirmed in an immunodeficient mouse HL-60-Luc tumormodel where the 161533 TriKE exhibited superior antitumoractivity and induced in vivo persistence and survival of humanNK cells for at least 3 weeks.

Conclusions: Off-the-shelf 161533 TriKE imparts antigenspecificity and promotes in vivo persistence, activation, andsurvival of NK cells. These qualities are ideal for NK cell therapyof myeloid malignancies or targeting antigens of solid tumors.Clin Cancer Res; 22(14); 3440–50. �2016 AACR.

See related commentary by Talmadge, p. 3419

IntroductionNatural killer (NK) cells are cytotoxic lymphocytes of the innate

immune system capable of immune surveillance. Like cytotoxic Tcells, upon activation NK cells deliver a store of membranepenetrating and apoptosis-inducing molecules, including granu-lysin, granzyme, and perforin (1). Unlike T cells, NK cells do notrequire antigen priming and recognize targets by engaging acti-

vating receptors in the absence of self-MHC recognition byinhibitory receptors. We have shown that adoptive transfer ofhaploidentical NK cells after lymphodepleting chemotherapy caninduce complete remissions in 30% to 50% of patients withrefractory acute myeloid leukemia (AML) when given with IL2to stimulate in vivo donor NK cell expansion (2,3). However, thisapproach is limited by lack of antigen specificity and by IL2-mediated induction of regulatory T (Treg) cells that suppress NKcell proliferation and function (3,4). Thus, we have developed areagent that targets NK cells to specific tumor antigens and drivesNK expansion and persistence, while bypassing the negativeeffects of Tregs and the morbidity of IL2.

NK cells mediate antibody-dependent cellular cytotoxicity(ADCC) through the highly potent CD16 (FcgRIII) activatingreceptor. Signaling through CD16 induces a calcium flux andphosphorylation of ITAMs triggering the release of lytic granulesand cytokines such as interferon (IFNg) and tumor necrosis factor(TNFa; refs. 5–7). Tobetter targetNK cells tomalignant targets,wecreated and tested bispecific or trispecific killer engagers (BiKE andTriKE, respectively; refs. 8–11) each incorporating an antihumananti-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 highlyactivating CD16 receptor on NK cells and specific tumor antigens

1Masonic Cancer Center, Laboratory of Molecular Cancer Therapeu-tics, Department of Therapeutic Radiology-Radiation Oncology,University of Minnesota, Minneapolis, Minnesota. 2Division of Hema-tology, Oncology, and Transplantation, Department of Medicine,University of Minnesota, Minneapolis, Minnesota. 3Department ofPediatrics, University of Minnesota, Minneapolis, Minnesota. 4NewYork Medical College, New York, New York.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

D.A. Vallera and M. Felices contributed equally to this article.

Corresponding Author: Daniel A. Vallera, University of Minnesota MasonicCancer Center, MMC:494, 420 Delaware St. SE, Minneapolis, MN 55455. Phone:612-626-6664; Fax: 612-626-4842; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2710

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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andmarkedly enhance cytotoxic killing of various human cancers(8–11). Our 1633 BiKE enhances NK cell responses to CD33þ

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 shareseveral signaling components including the IL2/IL15Rb(CD122) and the common gamma chain (CD132). However,unlike IL2, IL15 does not stimulate CD25þ Tregs, allowing forNK cell activation without inducing concurrent Treg-mediatedimmune inhibition (14). IL15 also activates NK cells, and canrestore functional defects in engrafting NK cells after hemato-poietic stem cell transplantation (HSCT; ref. 15). IL15 alsostimulates CD8þ cytotoxic T cells, which further enhances itsimmunotherapeutic potential. Importantly, based on preclin-ical animal studies, the toxicity profile of low dose IL15 may bemore favorable than that of IL2 (14,16). This report describesthe generation of a 161533 TriKE that utilizes IL15 as anintramolecular linker between CD16 and CD33 scFvs to directNK cell–mediated killing of CD33þ tumors while simulta-neously generating an NK cell self-sustaining proliferation/survival signal through IL15 with a lesser effect on T cells.

Materials and MethodsCell isolation, patients, and samples

Peripheral blood mononuclear cells (PBMC) were isolatedfrom normal adult donor blood obtained from Memorial BloodCenter by centrifugation using a Histopaque gradient (Sigma-Aldrich) and cryopreserved. Where noted, NK cells were enrichedwith magnetic beads using Stemcell EasySep Human NK cellenrichment kit whereas CD3/CD19–depleted products were gen-erated withMiltenyi's CliniMACS beads. Post-HSCT PBMCs frompatients undergoing HLA-matched sibling transplants wereobtained from an immune reconstitution biobank protocol.Recipient blood was collected and cryopreserved early (day 20to 100; n ¼ 10) after HSCT. All samples were obtained afterinformed consent, using guidelines approved by the Committeeon the Use of Human Subjects in Research at the University ofMinnesota (Minneapolis, MN) in accordance with the Declara-tion of Helsinki.

Cell linesTwo cell lines were used and authenticated in these studies. HL-

60, a CD33þ human acute promyelocytic leukemia cell line, wasobtained in 2013 from ATCC, and authenticated by flow cyto-metry (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 wascultured in Iscove's medium (Invitrogen) supplementedwith 20% FBS (Gibco, Invitrogen) and 100 U/mL penicillin and100 U/mL streptomycin (Invitrogen) at 37�C and 5% CO2. Thecontrol, murine colorectal carcinoma cell line HT-29 was alsoobtained fromATCC in 2013 and authenticated against a panel ofBV7 anti-Integrin b1 antibody,MOC-31 epithelial glycoprotein 2,and anti-EpCAM as positive markers and anti-CD19, anti-CD22,and anti-CD45 as negativemarkers. The line was cultured at 37�Cwith 5% CO2 in high glucose DMEM (Invitrogen) supplementedwith 10% FBS, and 100 U/mL penicillin and 100 U/mL strepto-mycin and last tested in 2015.

Construction, expression, and purification of BiKEs and TriKEsThe hybrid gene encoding 161533 was synthesized using DNA

shuffling and DNA ligation techniques (10,17). The VL and VHgenes of each sFv were linked by a fragment encoding G4S linker.In its final configuration, the 161533 NcoI/XhoI gene fragmentencoded a start codon followed first by antihuman CD16 scFv(12), a 20 amino acidflanking sequence PSGQAGAAASESLFVSN-HAY, human IL15, and then a seven amino acidflanking sequenceEASGGPE, and then the anti-CD33 scFv. The final target gene wasspliced into the pET21d expression vector and inclusion bodiesexpressed. DNA-sequencing analysis (Biomedical Genomics Cen-ter, University ofMinnesota,Minneapolis,MN)was used to verifythat 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 Brothsupplemented with 100 mg/mL carbenicillin in a 2-liter flask at37�Cwith shaking. Expression of the hybrid gene was induced bythe addition of isopropyl-b-D-thiogalactopyranoside (IPTG; Fish-erBiotech). Two hours after induction, the bacteria were harvestedby centrifugation. The cell pellets were suspended and homoge-nized using a polytron homogenizer. After sonication and cen-trifugation, the pellets were extracted with 0.3% sodium deox-ycholate, 5% Triton X-100, 10% glycerin, 50 mmol/L Tris, 50mmol/L NaCl, 5 mmol/L EDTA, pH 8.0, and inclusion bodiesextensively washed to remove endotoxin.

The proteins were refolded using a sodium N-lauroyl-sarcosine(SLS) air oxidation method modified from a previously reportedprocedure for isolating scFv (18). Refolded 161533was purifiedbyFPLC ion exchange chromatography (Q Sepharose Fast Flow)using a stepwise gradient from 0.2 to 0.5 mol/L NaCl in20 mmol/L Tris-HCl, pH 9.0 over four column volumes.

Flow cytometryCells were immunophenotypedwith the followingfluorescent-

labeled mAbs against: PE-Cy7-conjugated CD56 (HCD56; Bio-Legend), ECD/PE-CF594-conjugated CD3 (UCHT1; BeckmanCoulter), APC-Cy7-conjugated CD16 (3G8; BioLegend), PacificBlue-conjugatedCD45(HI30;BioLegend),PerCP-Cy5.5/FITC-con-jugated anti-human CD107a (LAMP-1; H4A3; BioLegend), PacificBlue/BV421-conjugated antihuman IFN-g (4S.B3; BioLegend),FITC/Alexa Fluor 647-conjugated TNFa (MAb11; BioLegend),

Translational Relevance

IL15 is a signal for NK cell development, proliferation,survival, and activation. A major advantage of IL15 therapyover IL2 therapy is that the IL15 does not engage Treg sup-pressor cells and may minimize toxicities associated with IL2therapy. In this article, we report for the first time that we havestably integrated IL15 into a bispecific antibody platformrecognizing CD16 on NK cells and CD33 on myeloid cancercells. This trispecific 16 � 15 � 33 recombinant moleculesuccessfully boosted antibody-dependent cell-mediated tox-icity (ADCC) in vitro and in vivo compared with the bispecificantibody without IL15. Importantly, the new molecule hadthe ability to expand and sustain human NK cells in vivo in axenograft model. This off-the-shelf drug is cost effective andeasy to use, desirable qualities for enhancing the selectivity ofNK cell therapy of myeloid malignancies or targeting antigensof solid tumors.

Improvement of Bispecific Antibody by Insertion of IL15

www.aacrjournals.org Clin Cancer Res; 22(14) July 15, 2016 3441




FITC/PE-conjugated CD33 (P67.6; BD Biosciences), and APC-con-jugated CD45 (HI30; BioLegend), FITC-conjugated EpCAM; (Bio-Legend). Phenotypic acquisition of cells was performed on theLSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar Inc.).

CD107a and IFNg/TNFa functional flow assayPost-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 TriKEwas addedto the cultures. The next morning, cells were washed and anotherround of 50 nmol/L 1633 BiKE or 161533 TriKE was added. HL-60 targets or primary AML blasts were added immediately after togenerate a 5:1 effector to target ratio. PBMCs, HL-60 targets orprimary AML blasts, and BiKE or TriKEmolecules were coculturedfor 4 hours and CD107a expression and intracellular IFNg andTNFa production were evaluated as described previously (10).

Proliferation assayPBMCs from posttransplant patients (day 22 to 44 or day 100)

were labeled with CellTrace Violet Cell Proliferation Dye (Invitro-gen), per manufacturer's protocol, placed in culturemediumwithHL-60 target cells at 5:1 (E:T) ratio, and treated with 50 nmol/L1633 BiKE or 161533 TriKE. Cells were then harvested 7 dayslater and analyzed for viability, through Live/Dead staining,and proliferation, through dilution of CellTrace, in the NK cell(CD56þCD3�) population.

51Chromium release cytotoxicity assayCytotoxicity was evaluated by 4-hour 51Cr-release assays. Brief-

ly, resting PBMC from normal donors treated with 161533 TriKEor 1633 BiKE (10 mg/mL), scFvCD16 control reagent (10 mg/mL),or no reagentwere cocultured for 4 hourswith 51Cr-labeledHL-60targets at varying E:T ratios. For posttransplant study, PBMCs cellswere cocultured with HL-60 targets at a 20:1 (E:T) ratio in thepresence 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).

In vivo mouse study and imagingHL-60-luc cells were incorporated into a previously described

NK cell xenogeneic mouse model system (19). NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, n ¼ 5/group) were conditioned with275 cGy and injected intravenously with 7.5 � 105 HL-60-luc S4subcultured for tumor invasiveness. The control group did notreceive NK cells whereas the 1633 BiKE and the 161533 TriKEgroups received 1 � 106 NK cells, calculated from a CD3/CD19magnetically depleted product, on day 3. BiKE and TriKE treat-ment was also initiated after injection of the NK cells on day 3. Asingle course of treatment consisted of an intraperitoneal injec-tion of 20 mg of drug given 5 days/week (MTWThF) andmiceweretreated for 2weeks. TheHL-60-luc cells are a subline ofHL-60 andhave been transfected with a luciferase reporter gene, allowing forimaging of themice each week to determine their bioluminescentactivity and to monitor leukemia progression as described previ-ously (20). Briefly, mice were injected with 100 mL of 30 mg/mLluciferin substrate 10 minutes prior to imaging and then anes-thetized via inhalation of isoflurane gas. The mice were thenimaged using the Xenogen IVIS 100 imaging system and analyzedwith Living Image 2.5 software (Xenogen Corporation). On

day 20, all the animals were bled and 2-minute exposures weremade and units for the regions of interest (ROI) were expressed ascounts. The bloodwas analyzed by flow cytometry for presence ofhuman CD45þCD56þCD3� NK cells. A second experiment wasperformed to verify reproducibility of data.

Cytokine measurement by luminexFor analysis of cytokines/chemokines, 500,000 NK cells cocul-

tured with 250,000 HL-60 cells were treated with the BiKE, TriKE,or no reagent for 24 hours at 37�C, 5% CO2. Supernatant levelswere determined using a human-specific panel for IFNg , TNFa,GM-CSF, or MIP-1a (EMD Millipore), and the Luminex system(Luminex). Final pg/mL values were interpolated from standardcurves of the corresponding recombinant human proteins withBioplex software v.5.1 (Bio-Rad).

Statistical analysisGrouped data were expressed as mean � SEM. Differences

between two groups were analyzed by Student t test. Multiplecomparisons were analyzed by paired one-way ANOVA withTukey correction. Analysis was carried out in GraphPad Prismsoftware. On figures statistical significance is indicated as: �, P �0.05; ��, P < 0.01; ��, P < 0.001.

ResultsSynthesis and purity of a 161533 TriKE

We introduced a human modified IL15 cross-linker into our1633 BiKE to create a 161533 TriKE designed to mediateantigen specificity and to deliver a proliferation and survivalsignal to NK cells (Fig. 1A). The bacterial expression system,which requires refolding, delivered high yield 1633 BiKE andthe 161533 TriKE products as shown by fast protein liquidchromatography (FPLC) profiles (Fig. 1B). The addition of IL15to the molecule reduced its isoelectric point by two pH units,creating more favorable conditions for purification, resulting innearly double the yield compared with 1633 BiKE after pur-ifications that began with identical amounts of inclusion bod-ies. Products were >95% pure by SDS-PAGE gel analysis andCoomasie Blue Staining (Fig. 1C). To verify that binding andspecificity remained intact in the new TriKE molecule, selec-tivity was measured by direct binding and competition flowcytometry assays against CD33þ HL-60 cells and CD33� HT-29cells (Supplementary Tables S1 and S2). The IL15 flankingsequences were critical for optimal TriKE activity.

161533 TriKE increases NK cell functionTo test whether the 161533 TriKE retained the ability to

mediate ADCC, as demonstratedwith the 1633 BiKE (8,9), killingof CD33þ HL-60 targets by normal donor PBMCs was measuredwith both agents in a 4-hour 51Cr release assay (Fig. 2A). The161533 TriKE induced higher NK cell–mediated killing than the1633BiKE, particularly at the 20:1 ratio (58.3%� 2.3%vs. 33%�4%,P¼0.02). Control conditionswith anti-CD16 andanti-CD33scFv alone did not increase killing compared with untreatedPBMC controls, demonstrating that these components alone haveminimal activity. To address the input of the IL15 moiety on NKcell cytotoxicity, we compared the 161533 TriKE to equivalentmolar concentrations (30 nmol/L) of IL15 or IL15 plus 1633 BiKE(Supplementary Fig. S1A). Results indicate that combining 1633BiKE and IL15 yields similar NK cell–mediated cytotoxicity

Vallera et al.

Clin Cancer Res; 22(14) July 15, 2016 Clinical Cancer Research3442




when compared with utilization of the 161533 TriKE.Although a trend of enhanced cytotoxicity can be seen withaddition of IL15 alone, likely because of enhanced priming ofnatural cytotoxicity, the 1633 specificity elicited by the scFvs in theTriKE or BiKE þ IL15 clearly enhance killing of targets indicatingthat combination of thesemolecules are paramount to enhancingNK cell function. The specificity of 161533 TriKE was furtherdemonstrated by its inability to mediate killing of CD33� HT-29

target cells after coincubation with PBMCs (Fig. 2B). A novel IL15TriKE made with an anti-EpCAM scFv in place of anti-CD33 wasused as a positive control to confirm that the HT-29 target cellswere not inherently resistant to NK-mediated killing. The robustkilling of CD33�EpCAMþ HT-29 cells mediated by the anti-EpCAM TriKE highlights the versatility of the IL15 linker TriKEplatform to induce antigen specificity against both hematologicand solid tumor malignancies.

Figure 1.161533 TriKE elicits superiorpurification properties over 1633 BiKE.A, schematic of gene placement of theBiKE (left) and TriKE (right) moieties inthe pET expression vector. B,absorbance tracings for 1633 BiKE(left) and 161533 TriKE (right) elutedfrom the ion exchange column as thefirst phase in drug purification using athree-step elution protocol. For bothagents, a similar quantity of inclusionbodies was refolded and purified. Thefirst peak eluted from the columnrepresents the product. C, SDS-PAGEgel and Coomasie Blue staining ofthe BiKE and TriKE agents after asecond-step purification over a sizeexclusion column. MWS, MolecularWeight Standards.

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Figure 2.161533 TriKE elicits superior NK cell function against CD33þ targets. A, freshly isolated PBMCs were cultured with chromium loaded HL-60 cells for 4 hoursat E:T ratios of 20:1, 6.6:1, and 2:1. The listed reagents were added at the beginning of coculture at a 20 nmol/L concentration. Data are displayed as percent NKcell cytolytic activity. Statistical significance only shown for 161533 TriKE and 1633 BiKE comparison (n ¼ 3). B, to evaluate the specificity of 161533 TriKE, achromium release assaywas performed against CD33�EpCAMþHT29 targets. EpCAM1533 TriKEwas used as a positive control (n¼ 2). Data are displayed as percentNK cell cytolytic activity. C, NK cells were enriched from normal donor PBMCs utilizing magnetic beads and placed in culture with HL-60 targets at an E:Tratio of 2:1 alone, in the presence of 1633BiKE or in the presence of 161533 TriKE for 24 hours. Supernatants collected and frozen at the endof the incubationwere laterassessed for levels of secreted IFNg , TNFa, GM-CSF, and MIP1a using a Luminex-based multiplex assay (n ¼ 5). Points and bars represent mean � SEM.






To directly investigate the role of the 1633 BiKE and 161533TriKE on NK cell activation, PBMCs were incubated alone, withHL-60 cells, or with HL-60 cells plus 1633 BiKE or 161533 TriKEfor 24 hours and activation markers were evaluated on the NKcells (Supplementary Fig. S1B). A clear hierarchy of activation canbe noted through induction of CD69 expression, which is higheston NK cells incubated with 161533 and targets, followed by NKcells incubated with 1633 and targets, NK cells incubated withtargets alone, and NK cells that were left untreated. CD25 expres-sion was also highest on the NK cells incubated with 161533 andHL-60s, but the magnitude of the increase is likely also contrib-uted by the effect of IL15 signaling onCD25 induction. A trend foran increase in HLA-DR expression, which has slower inductiondynamics, can also be noted in the NK cells treated with 1633 and161533 and targets. Taken together these data indicate potent NKcell activation through the 161533 TriKE.

161533 TriKE affects cytokine productionIn addition to cytotoxicity, another key anticancer function of

NK cells is the production of cytokines and chemokines upontarget cell recognition. Cytokine production was measured byincubating enriched NK cells and HL-60 targets alone, with 1633BiKE, or with 161533 TriKE. Inflammatory cytokines and chemo-kines were measured in supernatants collected after 24 hours ofincubation (Fig. 2C). 161533 TriKE induced significantly moreIFNg and TNFa secretion compared with 1633 BiKE (left). Nosignificant differences were noted between levels of IL6, IL10, orIL8 (data not shown). GM-CSF and MIP-1a (right) were alsoinduced by the 161533 TriKE, whereas RANTES remainedunchanged from baseline (data not shown). These data indicatethat incorporation of the IL15 molecule into the TriKE enhancesthe production of proinflammatory cytokines and chemokines byNK cells, augmenting their antitumor activity.

161533 TriKE induces survival and proliferation ofposttransplant NK cells

The ability of the IL15 linker in our 161533 TriKE to deliverhomeostatic and expansion signals to NK cells was tested in aphysiologically relevant context using early post-HSCT recipientsamples. This time point is of particular interest because we haveshown major defects in NK cell–mediated target cell inducedcytokine production at this time, when engrafting NK cells areneeded 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; n¼ 8) was measured afterlabeling with CellTrace dye and 7 days of incubation with HL-60targets. Cell survival at 7 days, measured with Live/Dead dye, wassignificantly greater in the 161533 TriKE condition comparedwith the 1633 BiKE condition (Fig. 3A and B; 96.9% � 0.5% vs.21% � 5.4%; P < 0.0001). The proliferation induced by the IL15moiety in the 161533 TriKE was measured by CellTrace dyedilution in the viable NK and T-cell populations. Unexpectedly,the 161533 TriKE induced robust and specific NK cell prolifera-tion, with minimal T-cell proliferation in the same samples(Fig. 3C and D, top; cell division in 79.1% � 2.5% of NK cellsvs. 2.3%� 1.1%of T cells, P < 0.0001). Comparedwith T cells, theexpansion index or total fold expansion of the NK cells wassignificantly higher in the posttransplant setting (Fig. 3D, bottom:7.2% � 0.8% vs. 1.1% � 0.1%, P < 0.0001). However, in normaldonors, the 161533 TriKE was capable of expanding T cells

(Supplementary Fig. S2A), albeit to a much lower extent thanNK cells, indicating that the reason there is no expansion of T cellsin the posttransplant setting might be that at early time pointsafter HSCT T cells capable of responding to the IL15 moiety arediminished or their activity is blunted by concomitant pharma-cologic agents given for GVHD prophylaxis.

We compared the biologic activity of the IL15 moiety in the161533 TriKE molecule to that of the clinical grade recombi-nant human IL15 produced by the NCI. Enriched NK cells fromnormal donors were labeled with CellTrace and incubated for 7days with no reagent, NCI IL15, 1633 BiKE, or 161533 TriKE.NK cell proliferation required IL15, and was maintained byeither NCI IL15 or the 161533 TriKE (Supplementary Fig. S2Band S2C). In marked contrast, the NK cells in the no reagent or1633 BiKE groups failed to proliferate. Together, these dataindicate that the IL15 linker incorporated into the 161533TriKE is functionally active and capable of delivering survivaland proliferation signals to normal donor or post-HSCT recip-ient NK cells, with minimal effect on T cells.

161533 TriKE potently restores function in defective post-HSCTrecipient NK cells

Given the potential clinical development of 1633 BiKE and161533 TriKE as post-HSCT immunotherapy, the ability of eachmolecule to restore defective NK cell function was tested usingpost-HSCT recipient PBMCs (Fig. 4). Thawed and rested PBMCwere incubated with 1633 BiKE or 161533 TriKE overnight,washed, and reincubated with the same agent immediately priorto addition of targets. Responses after a 4-hour incubation withHL-60 targets were measured by chromium release and flowcytometry. Although the 1633 BiKE promoted double the cyto-toxicity against HL-60 targets compared with PBMCs alone(18.9% � 2.1% vs. 9 � 1.5%, P < 0.0001), incubation with the161533 TriKE further enhancedNK cell–mediated cytotoxic func-tion (52.1 � 3.5%; Fig. 4A). The increased cytotoxicity mediatedby 161533 TriKE correlated with increased NK cell degranulationmeasured by CD107a expression (Fig. 4B and C, left). Defectivetarget cell-induced production of IFNg by NK cells was notrestored by 1633 BiKE, whereas 161533 TriKE promoted a potentresponse (2.6% � 0.5% vs. 21.7% � 4.4%, respectively; P ¼0.001; Fig. 4B and C, middle). A similar rescue of TNFa produc-tion by 161533 TriKE, but not 1633 BiKE, was observed (29.9%�3.8%vs. 6.1%�1%, respectively;P<0.0001; Fig. 4B andC, right).The magnitude of post-HSCT recipient NK cell responses toCD33þ targets mediated by 161533 TriKE clearly demonstratethe immunotherapeutic potential of the this agent.

161533 TriKE increases NK cell function against primary AMLblasts

We compared the function of post-HSCT recipient NK cellsincubated with 1633 BiKE or 161533 TriKE against primary AMLblasts from two different patients (AML1 and AML2). Althoughthe overall production of CD107a, IFNg , and TNFa by post-HSCTNK cells exposed to primary blasts was reduced when comparedwithHL-60 targets, the 161533 TriKE againmediated significantlymore degranulation and cytokine production compared with the1633 BiKE (Fig. 5A and B). Reduced expression of CD33 on theprimary AML blasts compared with HL-60 (Supplementary Fig.S2D) could explain the decreased NK cell response to primaryAML blasts. Importantly, the post-HSCT recipient NK cells

Vallera et al.





exhibited similar 161533 TriKE-mediated responses to activationby AML1 and AML2. Taken together, these in vitro data indicatethat the 161533 TriKE induces antigen-specific NK cell cytotoxicand cytokine-producing responses against primary CD33þ tumorcells, and that the costimulation provided by the IL15 linkerincreases NK responses compared with those seen with the1633 BiKE agent.

161533 TriKE induces enhanced in vivo survival of NK cells withantitumor function

Wedeveloped amurine xenograft model incorporating humanNK cells and CD33þ tumor cells to compare the activity of the1633 BiKE and the 161533 TriKE in vivo. NSG mice were condi-tioned and injected intravenously with HL-60 cells containing a

luciferase reporter gene (7.5� 105 cells/mouse), followed 3 dayslater by intravenous infusion of 1� 106 normal humandonorNKcells activated overnightwith IL15.Mice thenwere treatedwith nodrug (andnoNKcells in the control group), 1633BiKE, or 161533TriKE (20 mg) administered intraperitoneally MTWThF throughthefirst 2weeks of the study (10doses total).Micewere imagedonday 14 and day 21 to measure tumor burden (Fig. 6A and B). Atday 14, the 1633 BiKE and 161533 TriKE groups showed similarcontrol of tumor burden, both of which were significantly betterthan the control group.However, on day 21, bywhich time2micefrom the no-drug control group had died, only the 161533 TriKEgroup showed significantly reduced tumor burden, indicatingsuperior sustained control of the HL-60 tumor burden with161533 TriKE therapy.

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Figure 3.The 161533 TriKE mediates NK cellsurvival and proliferation. Post-HSCTrecipient PBMCs were loaded withCellTrace proliferation dye andcocultured with HL-60 Targets at a 5:1(E:T) ratio for 7 days in the presence of50 nmol/L 1633 BiKE or 161533 TriKE.At the end of the incubation CD56þ

CD3�, NK cells were assessed forviability through Live/Dead Near IRstaining. A, an individual histogram; B,pooled analyses of viability in the NKcell population treated with the 1633BiKE (gray) or 161533 TriKE (black) areshown. Individual dots representseparate post-HSCT samples treatedwith noted reagents (n ¼ 8). C, after7 days, significantly fewer live cellsremained in the 1633 BiKE conditionprecluding proliferation analysis.Proliferation in the 161533 TriKEcondition was assessed by CellTracedilution on live CD56þCD3� NK cells(black) and CD56�CD3þ T cells (gray).D, pooled analyses depict the percentof dividing cells (top) and expansionindex (bottom). The expansion index iscalculated based on fold expansionwithin the population for a givenamount of CellTrace dilution.Individual dots represent separatepost-HSCT samples for notedpopulations (n ¼ 8).






To evaluate the effect of the 1633 BiKE and the 161533 TriKEon in vivo NK cell maintenance, accounting for NK cell survivaland proliferation, we bled the mice (20 mL) at day 20 and

measured the number of human NK cell (CD45þCD56þCD3�)events in 60 seconds of acquisition by flow cytometry. Essen-tially, no circulating human NK cells were detected in the no-

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Figure 4.161533 TriKE potently restores function in defective post-HSCT recipient NK cells. The NK cell function induced by 161533 TriKE was compared with 1633 BiKEusing post-HSCT recipient PBMCs that were thawed and rested overnight with no cytokines. The next night they were incubated with no drug, 1633 BiKE(50 nmol/L), or 161533 TriKE (50 nmol/L). The next morning they were washed and reincubated with the specified agent immediately prior to addition of targets.A, cytotoxicity against chromium loaded HL-60 targets was measured after 4 hours and the percent cytolytic activity was calculated. Dots denote mean �SEM (n¼ 9). B, target cell-induced function is shown in representative histograms; C, pooled data of CD56þCD3�NK cell expression of CD107a (left), IFNg (center),and TNFa (right) after 4-hour incubation with HL-60 targets. Dots denote individual patient samples treated with noted reagents (n ¼ 10).

Vallera et al.





drug control or the 1633 BiKE-treated animals (17.3� 14.3 and11.6 � 2.3 events, respectively; Fig. 6C and D). In markedcontrast, all of the 161533 TriKE-treated animals had a sub-stantial number circulating human NK cells (4261 � 410.6events) during the same acquisition period. Thus, mice treatedwith 161533 TriKE had over 350-fold more human NK cellscirculating in the peripheral blood than did BiKE-treated mice,indicating that the IL15 linker within the TriKE moleculedelivers robust proliferation and survival signals.

A second experiment was performed using this same murinexenograftmodel (n¼4 to5/group) to reproduce this data.Only inthe second experiment, a higher number (106) of highly HL-60cells were administered. Fifty percent of the TriKE-treated micewere robust responders to therapy. Again, the BiKE-treated miceshowed only minimal effects on day 21 (data not shown). Also, aseparate group of animals were given high dose IL15 alonewithout BiKE or TriKE (5 mg MWF). These showed a reducedresponse indicating that IL15 itself is not sufficient to potentlymediate an NK cell–mediated antitumor effect.

Discussion

The original contribution of this work is a self-sustaining161533 TriKE using anti-CD16 and anti-CD33 scFvs with amodified IL15 linker capable of directing antigen-specific ADCCwhile inducing NK cell proliferation and survival. We previouslyreported that our 1633 BiKEmolecule successfully drives antigen-specific ADCC against primary AML and MDS (8,9), and we havesubstantially improved this platform by introducing an IL15signal that costimulates the NK cell response to CD33þ targets,including primary AML blasts, with significantly enhanced cyto-toxic and cytokine production compared to the 1633 BiKE. The161533 TriKE induces potent responses by NK cells from normaldonor samples as well as frompost-HSCT recipient NK cells.Mostimportantly, using our novel xenogeneic tumor model we dem-onstrate that the 161533 TriKE is capable of driving human NKcells to proliferate and survive in vivowhile mediating an antigen-specific sustained anticancer response. Neither 1633 BiKE or IL15alone had all these properties.

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Figure 5.161533 TriKE induces enhanced NK cell function against primary AML blasts. Posttransplant patient PBMCs were thawed and rested overnight. The next nightthey were incubated with 1633 BiKE (50 nmol/L) or 161533 TriKE (50 nmol/L). The next morning they were washed and reincubated with the specified agentimmediately prior to addition of targets. Primary AML blasts from apheresis products of two separate patients were thawed and rested overnight. Treatedposttransplant patient PBMCs (n¼ 6) were incubated with the two different primary AML blasts (n¼ 12 total) for 4 hours and NK cell function was assessed by flowcytometry. A, representative histograms denoting CD107a (left), IFNg (center), and TNFa (right) B, expression on posttransplant patient NK cells treated with1633 BiKE (gray) or 161533 TriKE (black) after 4-hour incubationwith primary AMLblasts. B, pooled data for CD107a (left), IFNg (center), and TNFa (right) expressionon posttransplant patient NK cells treated with 1633 BiKE and 161533 TriKE and incubated with primary AML blasts. Each box represents a separateposttransplant patient sample incubated against two separate patient AML blast targets, denoted by filled and open boxes (n ¼ 12 total).






Combining cytokines with antibodies is not a new concept andearly efforts focused on IL2. IL2 cytobodies were generated in theearly 2000s (21–24), but unfortunately clinical responses weremarginal and IL2 toxicities were dose limiting. The effectiveness ofIL2 as a cancer immunotherapy is also limited by its potentinduction of CD25þ Tregs. IL15 plays a critical role in NK celldevelopment, homeostasis, proliferation, activation and survival(13,25–30). IL15 also may be less toxic than IL2, as has beendemonstrated by Munger and colleagues who used a vascular leakmouse model that measures the extravasation of 125I-labeledalbumin. They showed that high dose IL2 caused vascular leaksyndrome (VLS) inmice, but high dose IL15 did not (16). Thus wechose to incorporate a modified IL15 molecule into our 161533TriKE to simultaneously function as an intramolecular linker and as

a stimulator of NK function. Although IL15 is considered less toxicthan IL2 based on its reduced propensity to induce VLS, it doestrigger a cytokine cascade including TNa, IL1, IL6, IL8,MCP-1,GM-CSF, and pro-inflammatory chemokines (31,32). The most com-mon severe side effects are fever, fatigue, nausea, vomiting, weightloss, severeneutropenia, anda generalized skin rash associatedwithcytokine inflammatory syndrome. In mice, weight loss precedesdeath, so weight loss can be used as a measure of toxicity. We didnot observeweight loss inNSGmice that receivedNK cells andNCIrH IL15or IL15 in the formof the 161533TriKE (10 injectionsof 20mg; 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 whetherIL15-activatedNKcells can induce toxicity indirectly,ourpreclinical

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Figure 6.161533 TriKE limits HL-60 tumor growth in vivo better than 1633 BiKE through enhanced maintenance of NK cells. An in vivo tumor model was established byconditioning NSG mice (275 cGy) and then injecting HL-60-luc cells intravenously (7.5 � 105 cells/mouse). Three days later 1 � 106 normal human donorNK cells (calculated from a magnetically depleted CD3/CD19 product) activated overnight with IL15 were infused. 1633 BiKE and 161533 TriKE (50 mg) wasadministered MTWThF through the next 2 weeks of the study (10 doses total), and a control group only received HL-60-luc cells. A, individual mousephotoluminescence after 2-minute exposures on day 14 (left) and day 21 (right) after NK infusion are shown (n ¼ 5 per treatment group). B, quantification ofluminescence inmice from the three treatment groups at day 14 (top) andday21 (bottom). Eachdot represents adifferentmouse andbars denotemean�SEM (n¼ 5per treatment group, representative of two separate experiments). C, blood was collected on day 20 from the mice in each of the experiential treatmentgroups. Circulating CD45þCD56þCD3� humanNK cellswere quantified by flow cytometry. Eventswere collected over 60 seconds and the number of humanNK cellevents was calculated. Representative dot plots are shown denoting the number of NK (CD56þCD3�) cell events within the CD45þ gate. D, aggregate datademonstrating the number of human NK cell events in each treatment group at day 20. Individual dots represent different mice and bars denote mean � SEM(n ¼ 3 for HL-60-luc group [two mice died], n ¼ 5 for the 1633 BiKE and 161533 TriKE groups).

Vallera et al.





data indicate that IL15 activation of NK cells by the 161533 TriKEagent may result in decreased toxicity by more localized delivery.

Other potential toxicities associated with exogenous IL15administration should be considered. In certain settings, IL15has been found to participate in the development of leukemiasand 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), metas-tasis, and tumor angiogenesis (36). Therefore, we believe thatselectively delivering IL15 signals to NK cells in the context of theTriKE molecule will facilitate more direct interactions with theimmune synapse formed between NK effector cells and tumortargets and will potentially decrease systemic effects.

We have demonstrated that unlike the 1633 BiKE, the 161533TriKE is capable of inducing proliferation and increasing survivalin normal donor and post-HSCT recipient NK cells. As shown inour xenogeneic tumor model, the increased number of live NKcells will likely translate to increased antitumor activity. Impor-tantly, our in vitro assays showed that the TriKE-induced prolif-eration of NK but not T cells in post-HSCT samples, which isimportant in the post-HSCT setting where T cells mediate graftversus disease. We have previously described functional defects inrecipient NK cells early post-HSCT (15,44), which may accountfor early relapses (45).Comparedwith the1633BiKE, our 161533TriKE restored robust NK cell function in post-HSCT recipient NKcells. Thus, there may be great benefit of this agent in the HSCTarena.

We and others have previously reported BiKEs (8–11,45,46).Our preclinical data show that 161533 TriKE holdsmore promisefor clinical testing to treat AML and MDS. Allogeneic HSCT isstandard therapy for MDS and AML, but older patients trans-planted with reduced intensity conditioning have an unaccept-ably high relapse rate of 30%. Thus, new therapeutic strategies areurgently needed to reduce relapse of MDS and AML after HSCTand to treat patients who are not transplant candidates. Our161533 TriKE adds a critical component to the BiKE scaffold byinducing maintenance and expansion of the effector NK cellsmediating tumor killing. This is particularly important becauseresting NK cell function is diminished in MDS and AML patientsfrom the disease itself or its therapy. A unique advantage of usingtheCD33 scFv in our TriKE is that themoleculemay also direct NKcells to eliminate CD33þ myeloid-derived suppressor cells(MDSC). Thus, the agent may directly target tumor as well asreducing tumor-induced immune suppression.

The 161533 TriKE molecules could also be used to enhanceendogenousNKcell function againstNK sensitivemalignancies inthe posttransplant setting, with minimal effects on T cells asdemonstrated by our in vitro studies, particularly in refractorydisease or in patients undergoing reduced intensity conditioning.Although studies shown here target the CD33 antigen in myeloidtumors, the IL15 TriKE platform can easily be adapted to target

other tumors by switching the anti-CD33 scFv portion for an scFvtargeting a different tumor antigen such as EpCAM present onsolid tumor targets, making this a versatile platform for self-sustained NK cell–based immunotherapies.

Disclosure of Potential Conflicts of InterestD.A. Vallera has ownership interest (including patents) in and is a consul-

tant/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 arelisted as co-inventors on a provisional patent application on IL15 TriKEs that isowned byUniversity ofMinnesota and is not yet licensed. No potential conflictsof interest were disclosed by the other authors.

Authors' ContributionsConception and design: D.A. Vallera, M. Felices, B. Zhang, M.R. Verneris,J. Tolar, B.R. Blazar, J.S. MillerDevelopment of methodology: M. Felices, B. Zhang, J. Tolar, B.R. Blazar,J.S. MillerAcquisition 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. BlazarAnalysis 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. ValleraWriting, 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. MillerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.A. Vallera, V. McCullar, B. ZhangStudy supervision: D.A. Vallera, M. Felices, B. Zhang, B.R. Blazar, J.S. MillerOther (providing critical reagents): X. Zhou

AcknowledgmentsThe authors acknowledge cell-processing services from the Translational

Therapy and FACS Cores of the Masonic Cancer Center (P30 CA77598). Theauthors also acknowledge the excellent technical assistance of Elizabeth Taras,Deborah Todhunter, Andy Sicheneder, Sami Chu, and Seunguk Oh.

Grant SupportThis work was supported in part by theU.S. Public Health Service Grant R01-

CA36725, R01-CA72669, P01-CA65493, P01-CA111412, and R35 CA197292awarded by the NCI and the NIAID, DHHS. It was also supported by an NIHResearch Evaluation and Commercialization Hub (REACH) Award (U01), theMayo Partnership Award, the Lion Fund, William Lawrence and BlancheHughes Fund, the Randy Shaver Foundation, the Atwater Cancer Drug Devel-opment Award, the Deutsche Krebshilfe (DS), and a CETI translational awardfrom the University of Minnesota Masonic Cancer Center.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 25, 2015; revised January 21, 2016; accepted January 23,2016; published OnlineFirst February 4, 2016.

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Vallera et al.




2016;22:3440-3450. Published OnlineFirst February 4, 2016.Clin Cancer Res Daniel A. Vallera, Martin Felices, Ron McElmurry, et al. Expansion, and Enhanced Function

In Vivo Targets While Also Inducing Persistence, +Specific to CD33IL15 Trispecific Killer Engagers (TriKE) Make Natural Killer Cells

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