molecular cancer therapeutics - bispeciﬁc and trispeciﬁc killer … · 2012. 11. 29. · zhou...

Preclinical Development

Bispecific and Trispecific Killer Cell Engagers DirectlyActivate HumanNKCells throughCD16 Signaling and InduceCytotoxicity and Cytokine Production

Michelle K. Gleason1, Michael R. Verneris2, Deborah A. Todhunter3, Bin Zhang1, Valarie McCullar1, Sophia X.Zhou1, Angela Panoskaltsis-Mortari2, Louis M. Weiner4, Daniel A. Vallera3, and Jeffrey S. Miller1

AbstractThis study evaluates the mechanism by which bispecific and trispecific killer cell engagers (BiKEs and

TriKEs) act to trigger human natural killer (NK) cell effector function and investigates their ability to induce

NK cell cytokine and chemokine production against human B-cell leukemia. We examined the ability of BiKEs

and TriKEs to trigger NK cell activation through direct CD16 signaling, measuring intracellular Ca2þ mobili-

zation, secretion of lytic granules, induction of target cell apoptosis, and production of cytokine and chemokines

in response to the Raji cell line and primary leukemia targets. Resting NK cells triggered by the recombinant

reagents led to intracellular Ca2þ mobilization through direct CD16 signaling. Coculture of reagent-treated

resting NK cells with Raji targets resulted in significant increases in NK cell degranulation and target cell death.

BiKEs andTriKEseffectivelymediatedNKcytotoxicityofRaji targets athighand loweffector-to-target ratios and

maintained functional stability after 24 and 48 hours of culture in human serum. NK cell production of IFN-g ,TNF-a, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, macrophage inflam-

matory protein (MIP)-1a, and regulated and normal T cell expressed and secreted (RANTES) was differentially

induced in the presence of recombinant reagents and Raji targets. Moreover, significant increases in NK cell

degranulation and enhancement of IFN-g production against primary acute lymphoblastic leukemia and chronic

lymphocytic leukemia targetswere inducedwith reagent treatment of restingNK cells. In conclusion, BiKEs and

TriKEs directly trigger NK cell activation through CD16, significantly increasing NK cell cytolytic activity and

cytokine production against tumor targets, showing their therapeutic potential for enhancing NK cell immu-

notherapies for leukemias and lymphomas. Mol Cancer Ther; 11(12); 1–11. �2012 AACR.

IntroductionWith more than 70,000 cases anticipated in the United

States for 2012, non-Hodgkins lymphoma (NHL) is themost common adult hematologic malignancy, of which85% of cases are of B-cell origin (1, 2). Monoclonalantibodies (mAb), such as rituximab, have shown to betherapeutic for the treatment of NHL (3). Despite thissuccess, there are limitations that decrease the overallefficiency of mAb therapies (4). With the developmentof CD16-directed bispecific and trispecific single-chain

fragment variable (bscFv and tscFv) recombinant re-agents, most of these undesired limitations are avoidedwhile eliciting high effector function as they lack the Fcportion of whole antibodies and have a targeted specific-ity for CD16 (5–7). As a result, recombinant reagents areattractive for clinical use in enhancing natural killer (NK)cell immunotherapies.

The ability of NK cells to recognize and kill targets isregulated by a sophisticated repertoire of inhibitory andactivating cell surface receptors. NK cell cytotoxicity canoccur by natural cytotoxicity, mediated via the naturalcytotoxicity receptors (NCR), or by antibodies, such asrituximab, to trigger antibody-dependent cell-mediatedcytotoxicity (ADCC) through CD16, the activating low-affinity Fc-g receptor for immunoglobulin G (IgG) highlyexpressed by the CD56dim subset of NK cells (8–11).Natural cytotoxicity is triggered via NCRs by de novoexpression ofNK cell activating receptor ligands on targetcells. In the absence of cytokine stimulation, these recep-tors inefficiently elicit a cytotoxic or cytokine responseindependently, but together they are able to functionsynergistically to activate a resting NK cell and promoteeffector function (9, 12). In contrast, ADCC is mediatedwhen CD16 binds to opsonized targets through Fc

Authors' Affiliations: 1Adult and 2Pediatric Divisions of Hematology,Oncology, and Transplantation and 3Section on Molecular Cancer Ther-apeutics, Department of Therapeutic Radiology-Radiation Oncology, Uni-versity ofMinnesotaMasonic Cancer Center, Minneapolis, Minnesota; and4Department ofOncology, LombardiComprehensiveCancerCenter,Geor-getown University, Washington, DC

D.A. Vallera and J.S. Miller contributed equally to this work.

Corresponding Author: Jeffrey S. Miller, University of MinnesotaMasonic Cancer Center, MMC 806, Harvard Street at East River Road,Minneapolis, MN 55455. Phone: 612-625-7409; Fax: 612-626-3941;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0692

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org OF1

on July 21, 2021. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 17, 2012; DOI: 10.1158/1535-7163.MCT-12-0692

http://mct.aacrjournals.org/

engagement and signals through immunoreceptor tyro-sine-based activation motifs (ITAM) of the associatedFceRIg chain and CD3z chain subunits (13). The signaldelivered via CD16 is potent and induces both a cytotoxicand cytokine response in the absence of cytokine stimu-lation that can further be enhanced by coengagement ofother activating receptors (12).

In this study,we show the ability of a CD16/CD19BiKEand a CD16/CD19/CD22 TriKE to trigger NK cell acti-vation through direct signaling of CD16 and inducedirected secretion of lytic granules and target cell death.Furthermore, we show for the first time the ability of thesereagents to induceNKcell activation that leads to cytokineand chemokine production.

Materials and MethodsCell isolation and purification

Peripheral blood mononuclear cells (PBMC) were iso-lated from adult blood (Memorial Blood Center, Minnea-polis, MN) by centrifugation using a Histopaque gradient(Sigma-Aldrich), andNK cells were purified by removingT cells, B cells, stem cells, dendritic cells, monocytes,granulocytes, and erythroid cells via magnetic beadsaccording to the manufacturer’s protocol (Miltenyi Bio-tec). Primary acute myelogenous leukemia (AML), acutelymphoblastic leukemia (ALL), and chronic lymphobla-sitc leukemia (CLL) samples were obtained from theLeukemia MDS Tissue Bank (LMTB) at the University ofMinnesota (Minneapolis,MN).All sampleswere obtainedafter informed consent and in accordance with the Dec-laration of Helsinki, using guidelines approved by theCommittee on the Use of Human Subjects in Research atthe University of Minnesota.

Flow cytometryCell suspensions were stained with the following

mAbs: PE/Cy7-conjugated CD56 (HCD56; BioLegend),ECD-conjugated CD3 (UCHT1; Beckman Coulter), FITC-conjugated CD16 (3G8; BD Biosciences), PE-conjugatedCD19 (SJ25C1; BD Biosciences), APC-conjugated CD20(2H7; BioLegend), FITC-conjugated CD22 (H1B22; BioLe-gend), PerCP/Cy5.5-conjugated anti-human CD107a(LAMP-1; H4A3; BioLegend), Pacific Blue-conjugatedanti-human IFN-g (4S.B3; BioLegend), and AF488-conju-gated cleaved caspase-3 (9669; Cell Signaling Technolo-gy). Phenotypic acquisition of cells was carried out on theLSRII (BD Biosciences) and analyzed with FlowJo soft-ware (Tree Star Inc.).

Construction, expression, and purification of bscFvCD16/CD19 and tscFv CD16/CD19/CD22 reagents

Synthesis of hybrid genes encoding the bscFv andtscFv reagents were accomplished using DNA shufflingand DNA ligation techniques as previously described(14). The fully assembled gene (from 50 end to 30 end)consisted of an NcoI restriction site, an ATG initiationcodon, the VH and VL regions of anti-human CD16

(NM3E2; ref. 6), a 20-amino acid segment of humanmuscle aldolase, the VH and VL regions of humanizedanti-CD22 (14), humanized anti-CD19 (14), and a XhoIrestriction site. The resultant gene fragment was splicedinto the pET21d expression vector, and DNA sequencinganalysis (Biomedical Genomics Center, University ofMinnesota) was used to verify that the gene was correctin sequence and cloned in frame. Genes encoding theBiKEs and monospecific scFV controls were created inthe same manner. For bacterial protein expression andpurification by ion exchange and size-exclusion chro-matography, methods were used as previously des-cribed (14).

Proliferation assayThe Burkitt lymphoma Raji cell line (American Type

Culture Collection) was cultured at 37�C with 5% CO2 inRPMI-1640 medium (Gibco) supplemented with 10% FBS(Gibco). Then, 2� 104 Raji cells were treated with varyingconcentrations of CD16/CD19/CD22 or nonspecific con-trol CD16/EpCAMand 1 nmol/L of a targeted diphtheriatoxin-CD19/CD22 (DT-CD19/CD22) was added and a3H-thymidine proliferation was carried out as previouslydescribed (14).

Cytokine/chemokine production, CD107a, andcaspase-3 assay

PBMCor purifiedNK cells were incubated overnight at37oC, 5%CO2 in basalmedium (RPMI supplementedwith10% fetal calf serum). Cells were washed, treated withbscFv CD16/CD19, tscFv CD16/CD19/CD22, rituximab(Genentech), CD22 parental antibody (derived from theRFB4 hybridoma), CD19 parental antibody (derived fromthe HD37 hybridoma), scFv anti-CD16 or scFv CD19/CD22 (negative controls), and CD107a and intracellularIFN-g assays were conducted as previously described(15). For evaluation of caspase-3 activation, Raji targetcell cleaved caspase-3 expression was evaluated by fluo-rescence-activated cell sorting (FACS) analysis using alive forward/side scatter gate and a CD56�/CD20þ gateto determine cleaved caspase-3–positive Raji popula-tions relative to an isotype control (rabbit IgG; CellSignaling Technology). For Luminex analysis of cyto-kines/chemokines, NK cells, Raji cells, and NK cellscocultured with Raji cells were treated with the BiKE,TriKE, rituximab, negative controls, or no reagent for 6hours at 37oC in 5%CO2. Supernatant levels of IFN-g ,TNF-a, granulocyte macrophage colony-stimulating fac-tor (GM-CSF), interleukin (IL-8), macrophage inflamma-tory protein (MIP-1a), and RANTES were determined bymultiplex assay using the Luminex system (LuminexCo.) and human-specific bead sets (R&D systems; sen-sitivity 0.5–3.0 pg/mL) and the final pg/mL valuesadjusted for background for each condition were deter-mined using the following equation: (NK cells þ Rajicells pg/mL) � [(NK cells alone pg/mL) þ (Raji cellsalone pg/mL)].

Gleason et al.

Mol Cancer Ther; 11(12) December 2012 Molecular Cancer TherapeuticsOF2




51Chromium release cytotoxicity assayDirect cytotoxicity assays were conducted by standard

4-hour 51Chromium (51Cr) release assays using effectorcells pretreated with reagents and Raji cells as targets. Forexamination of reagent stability, reagents were preincu-bated for 24 and 48 hours at 37�C, 5%CO2 in 100% humanserum. The 51Cr released by specific target cell lysis wasmeasured by a gamma scintillation counter and the per-centage of specific cell lysis was calculated.

Ca2+ flux assayCa2þ flux was measured using the Fluo-4 NW Calcium

Assay Kit (Invitrogen) within effector NK cells accordingto the manufacturer’s protocol. Ca2þ mobilization wasevaluated by FACS analysis. After 30 seconds of acquisi-tion, 10 mg/mL of purified anti-CD16 mAb (3G8; BectonDickinsonBiosciences), biotinylated scFv anti-CD16, puri-

fiedmIgG (negative control; R&DSystems), or 1 mg/mLofionomycin (positive control; Sigma)was addedandeventswere acquired for 1 minute. After 1 minute, 10 mg/mL ofpurified goat anti-mouse (GAM; BioLegend) or purifiedstreptavidin (SA; Pierce Thermo Scientific) was added tocross-link receptors and events were acquired for anadditional 4 to 5 minutes. The median Fluo-4 relativefluorescencewasanalyzedas a functionof time in seconds.

ResultsGeneration of bscFv CD16/CD19 BiKE and tscFvCD16/CD19/CD22 TriKE

Recombinant bscFv and tscFv reagents were generatedtargeting the NK cell receptor CD16 and B-cell antigensCD19 and CD22, antigens that have been shown to betherapeutic for the treatment of diffuse large B-cell lym-phoma and ALL (refs. 16, 17; Fig. 1A). BiKEs and TriKEs

Figure 1. BiKEs and TriKEs specifically target both effector and target cells. A, construct diagrams of bscFv CD16/CD19 and tscFv CD16/CD19/CD22reagents. B, ion exchange and size-exclusion chromatography purification trace of the CD16/CD19/CD22 TriKE. C, Coomassie Blue staining of TriKE proteinisolates (molecular weight of �96 kDa). Lane 1, nonreduced recombinant protein; Lane 2, reduced recombinant protein; and Lane 3, molecular weightladder. D, proliferation assay of 3H-pulsed Raji cells treated with CD16/CD19/CD22 (top graph) or a nonspecific control (bottom). E, intracellular Ca2þ fluxassay in resting NK cells after CD16 receptor cross-linking. Plots display a representative donor (of 4 experiments) showing the median Fluo-4 relativefluorescence plotted as a function of time in seconds.

BiKEs and TriKEs Enhance NK Cell Effector Function

www.aacrjournals.org Mol Cancer Ther; 11(12) December 2012 OF3




were purified by ion exchange and size-exclusion chro-matography. The fractions collected during the final stepof purification of theCD16/CD19/CD22TriKE are shown(Fig. 1B). High (95%) purity was obtained as shown byCoomassie Blue staining (Fig. 1C). Similar results wereobtained for the CD16/CD19 BiKE (data not shown).

BiKE and TriKE antigen binding is cell specific anddirectly signals through CD16 to induce NK cellactivation

Raji cellswere coatedwithvarying concentrations of theTriKE, treated with a CD19/CD22-targeted diphtheriatoxin (DT-CD19/CD22), anda 3H-thymidineproliferationassay was conducted (Fig. 1D). All concentrations of theTriKE blocked the effects of the inhibitory toxin, whereasthe nonspecific control reagent (CD16/EpCAM) had noeffect, showing effective targeting of the B-cell antigens bythe CD19 and CD22 end of the reagents.

We next evaluated the ability of the BiKE and TriKE todirectly target and trigger NK cell activation. Ca2þ mobi-lization, a primary indicator of cell activation, was mea-sured after specifically cross-linking CD16 on the surfaceof NK cells. Resting NK cells were loaded with a Ca2þ

binding dye solution and a time-course analysis ofchanges in intracellular Ca2þ concentration was carriedout by flow cytometry (Fig. 1E). Without cross-linking,both the CD16 mAb and the biotinylated scFv CD16elevated baseline Ca2þ levels, which were furtherenhanced upon cross-linking with the secondary anti-body. The Ca2þ flux kinetics induced by the biotinylatedscFv CD16 were faster than the response elicited by theCD16mAb, which may be explained by the high-affinityinteraction of streptavidin and biotin molecules. Theseresults show that the CD16 end of the BiKE and TriKE iscapable of engaging the NK cell and inducing a potentsignal through CD16, which results in Ca2þ mobiliza-tion.

BiKEs and TriKEs induce directed secretion of NKcell lytic granules and lysis of B-cell targets

As Ca2þ flux is a prerequisite for function (18), weevaluated the ability of these recombinant reagents tomediate killing of Raji targets, a lymphoma cell line withhigh expression of CD19, CD20, and CD22 (Fig. 2A).Resting NK cells were cocultured with Raji targets withor without reagent treatment and CD107a expression, amarker of NK cell cytotoxicity (19, 20), was measured viaFACS analysis (Fig. 2B). NK cell CD107a expression sig-nificantly increased in the presence of the BiKE and TriKEcomparedwith untreatedNK cells or parental antibodies.This response was target cell restricted as no functionalresponsewas inducedwith treatedNKcells in the absenceof targets (data not shown). The level of induced degran-ulation was equal to the function induced by the mAbrituximab. To analyze the correlation of NK cell CD107aexpression induced by the recombinant reagents withdirect target cell lysis (19, 20), 51Cr-labeled Raji targetswere cocultured with resting NK cells treated with or

without reagents and target cell killing was determined(Fig. 2C). Both the BiKE [for effector-to-target (E:T) ratio¼2.2:1, 70.6%� 4.5%;P < 0.01] and TriKE (59.8%� 3.9%; P <0.01) mediated significant lysis of the target cells at all E:Tratios compared with the untreated (18.7% � 3.0%) andcontrol-treatedNK cells. Furthermore, analysis of caspaseactivation in Raji target cells after 2 or 4 hours of coculturewithNK cells led to increases in cleaved caspase-3 expres-sion in BiKE and TriKE coated-Raji targets remaining inthe live cell gate compared with the uncoated-Raji targets(Fig. 2D). These results show that BiKE and TriKEreagents specifically engage both targets and effectors tomediate target cell killing, activating NK cells to secretelytic granules and induce target cell death via a caspase-3apoptosis pathway.

BiKEs and TriKEs are stable and mediate target cellkilling at high and lowE:T ratios in a dose-dependentmanner

To determine the potency of the BiKE and TriKEreagents, resting PBMC were pretreated with or withoutreagents at varying concentrations, exposed to Raji targetcells, and NK cell function was measured by FACS anal-ysis (Fig. 3A). Both NK cell degranulation and IFN-gproduction increased in a dose-dependentmanner.Whilerituximab induced NK cell function equally well at lowand high concentrations, it peaked without reaching thefunctional levels seen for the higher concentrations of theBiKEandTriKE reagents. ThehumanFcgR family consistsof 6 known members, which are expressed by variouseffector immune cells (4). As PBMCs contain multipleFcgR receptor-expressing populations in addition to NKcells (21), these cells may compete with NK cells forengagement of the Fc portion of rituximab diluting theNK cell effect. As the effector-targeting end of BiKEs andTriKEs is specific for the low affinity FcgRIII (CD16; ref. 6),this further supports the notion of increased NK cellspecificity of the bscFv and tscFv recombinant reagents.

NK cells were next tested at varying E:T ratios (Fig. 3B).NK cells efficiently degranulated at both high and lowE:Tratios. The BiKE induced greater CD107a expression com-pared with the TriKE at the 1:1, 1:5, and 1:10 E:T ratios,suggesting that the BiKE may be more efficient at recruit-ing effector NK cells for target cell lysis. Furthermore,production of IFN-g byNK cells against Raji targets in thepresence of the BiKE and TriKE was also maintained atboth high and low E:T ratios (Fig. 3B). As the activationthreshold for NK cell cytokine production differs fromthat of NK cell cytotoxicity (12, 22, 23), these resultsindicate the recombinant reagents are capable of inducingboth activation requirements and function consistently atboth low and high E:T ratios.

The stability of antibody-derived proteins is a vitalproperty that greatly affects the therapeutic efficacy. Toevaluate the functional stability, BiKEs and TriKEs wereincubated in 100% human serum for 24 or 48 hours, afterwhich resting NK cells were treated with or withoutreagents, cocultured with 51Cr-labeled Raji target cells

Gleason et al.





and target cell lysiswasmeasured (Fig. 4A). As the resultsshow, the reagents remain stable and are capable ofmediating target cell lysis. In addition, after human serumincubation, the TriKE induced greater degranulation (Fig.4B), target cell lysis, and IFN-g production (Fig. 4C)compared with the BiKE, implying structural variationsin the recombinant reagents may impact their biologicproperties, which has been suggested (24). In addition,these data support the premise that degranulation andcytotoxicity data correlate well as readouts for CD16triggering.

BiKEs and TriKEs induce NK cell chemokine andcytokine production against B-cell targetsAnalysis of intracellular IFN-g production of resting

NK cells with or without reagent treatment against Rajitarget cells showed significant increases in cytokine pro-duction for both BiKE- and TriKE-treated NK cells com-pared with untreated NK cells (Fig. 5A). Further analysisof cytokine and chemokine productionwas carried out via

Luminex on supernatants harvested from restingNK cellscoculturedwith Raji targets. The BiKE induced significantincreases inNKcell production of IFN-g , GM-CSF, TNF-a,RANTES, MIP-1a, and IL-8 and treatment with the TriKEresulted in a different profile with significant increases inGM-CSF, TNF-a, RANTES, and MIP-1a compared withuntreated NK cells (Fig. 5B). Interestingly, the potencywith which rituximab induced NK cell IFN-g (rituximabvs. bscFv, P ¼ 0.003; vs. tscFv, P ¼ 0.001), GM-CSF (P ¼0.026; P¼ 0.003), TNF-a (P¼ 0.108; P¼ 0.0002), andMIP-1a (P¼ 0.207;P¼ 0.03) productionwasgreater than that ofthe recombinant reagents. Furthermore, there were sig-nificant differences observed between theBiKE andTriKEreagents for NK cell production of GM-CSF (P < 0.05) andRANTES (P < 0.01), showing that CD16 triggering by eachreagent generates a unique profile of significantly increas-ed chemokine and cytokine production. As differences insignaling thresholds for the induction of NK cell chemo-kine and cytokine responses have been shown (25), thedifferential effects inducedbyBiKEs, TriKEs, and themAb

Figure 2. BiKEs and TriKEs enhance NK cell killing of Raji tumor target cells. A, FACS plots of CD19, CD20, and CD22 (open histogram; isotype control,shaded histogram) receptor expression on Raji targets. B, CD107a expression of resting NK cells cocultured with Raji target cells (E:T ¼ 2:1) with orwithout (10 mg/mL) reagent treatment (�, P < 0.01 for test condition compared with no reagent, n ¼ 3; error bars represent SEM). C, 51Cr-labeled Raji targetcell lysis by resting treated (10 mg/mL) or untreated NK cells. Each experiment was carried out in triplicate and repeated with 3 donors, with aggregateresults shown (�, P < 0.01 for test condition compared with no reagent; error bars represent SEM). D, cleaved caspase-3 expression in Raji targets inducedby treated or untreated resting NK cells (E:T ¼ 2:1; experiment was conducted twice with 2 NK donors; error bars represent SEM).






rituximab suggest other receptor–ligand interactions maybe differentially engaged to ultimately determine effectorfunction.

NK cell function against primary ALL and CLLtumors is enhanced in the presence of BiKEs andTriKEs

Primary leukemia cells, pre-B ALL and B-CLL cells,were next tested with allogeneic normal NK cells. NK celldegranulation was significantly enhanced against bothALL and CLL primary targets in the presence of the BiKEand TriKE compared with untreated NK cells with noincreases observed for the primary AML targets, furthershowing B-cell target specificity (Fig. 6A). Moreover,CD107a expression induced by the TriKEwas significant-ly greater than the levels induced by rituximab. To eval-uate whether this increased function was due to thesurface expression levels of CD19, CD20, and CD22 onthe primary ALL, CLL, and AML leukemia targets,expression of these B-cell antigens was measured viaFACS analysis (Fig. 6B and C). Variations in surfaceexpression of these receptors between patient sampleswere observedwithin each type of primary leukemia. The

overall CD19, CD20, and CD22 expression profile of theprimary leukemia targets suggests that there may be anadvantage to targeting 2 tumor antigens rather than 1.Finally, evaluation of NK cell IFN-g production againstprimary leukemia targets was also carried out. NK cellstreated with BiKE and TriKE reagents displayed signifi-cant increases in IFN-g production against primary CLLtargets and enhancement (not statistically significant)against primary ALL targets compared with untreatedNK cells (Fig. 6D).

DiscussionIn this study, we examined the ability of CD16/CD19

BiKE and CD16/CD19/CD22 TriKE recombinantreagents to enhance NK cell function against B-cell tumortargets. While in vitro NK cell targeting of B-cell malig-nancies with bispecific recombinant reagents has beendescribed (5, 7, 24), the mechanism by which thesereagents directly function to enhance NK cell activity hasnot been addressed. Our results show engagement ofCD16 by the CD16 end of the recombinant reagentsactively induces Ca2þmobilization in the effector NK cell.

Figure3. BiKE andTriKE reagents display a dose-dependent functional effect. A, PBMC treatedwith varying dosesof reagentswere coculturedwithRaji targetcells (E:T¼10:1) andpercentageofNKcellsCD107a (left)- or IFN-g (right)-positivewere evaluated (n¼3; error bars represent SEM).B, purifiedNKcells treatedwith orwithout 10 mg/mLof reagent were coculturedwithRaji target cells at varying E:T ratios andNKcell CD107a expression (left) and IFN-g production (right)were evaluated (�, P < 0.05, n ¼ 2; error bars represent SEM).

Gleason et al.





As triggering of CD16 leads to phospholipase C-g activa-tion and Ca2þ mobilization via signaling through theassociated ITAM-bearing FceRIg chain and CD3z chain(13), our data definitively show that BiKE and TriKEreagents directly signal through CD16 to activate NKcells. Furthermore, it has been shown that NK cell cyto-toxicity results from a combination of distinct signals,which includes CD16 (26, 27). As NK cell engagement byBiKEs and TriKEs led to significant increases in NK celldegranulation and directed target cell lysis, this furthershows the ability of these reagents to actively propagatesignals for NK cell activation.Cytokine and chemokine production is another critical

component of NK cell function. We definitely show theability of BiKE and TriKE reagents to induce a proinflam-matory profile of cytokines and chemokines. When com-paring the effects of the BiKE to the TriKE, there is ageneral increase in overall cytokine and chemokine pro-duction induced by the BiKE. Furthermore, NK cell cyto-kine and chemokine production induced by the mAbrituximab was significantly enhanced compared with therecombinant reagents. It has been shown that CD16engagement is sufficient to induce significant TNF-asecretion, but requires the addition of coactivating recep-tor engagement to induce an equivalent IFN-g response(25). Therefore, the functional potency of the recombinantreagents is likely influenced by the presence of coactivat-ing receptor ligands present on the engaged target cell,whose expressionmaybe altered by signaling through the

target antigen, which has been shown for CD20 engage-ment by rituximab (28). CD19 is a B-cell coreceptorinvolved in the positive regulation of B-cell function andupregulation of B-cell costimulatory ligands (29–33). Asour data show enhancedNK cell effector function, both incytotoxicity and cytokine and chemokine production,after engagement of target cells via the recombinantreagents targeting CD19, it is likely that a CD19 triggeringevent in the target cell occurs upon cross-linking thatupregulates B-cell costimulatory molecules that serve tofurther drive NK cell activation through interactions withactivating coreceptors, such as 2B4, ICOS, OX40, 4-1BB,andCD28, all ofwhich are expressedon activatedNKcells(34–38). In contrast to CD19, CD22 is an ITIM-containingB-cell receptor involved in the negative regulation ofB-cell function (31, 39). Our data show that the BiKEinduced greater NK cell degranulation and cytokine pro-duction compared with the TriKE. As CD22 and CD19deliver opposing signals that regulate B-cell activation(39) and as antigen expression on Raji target cells isvirtually uniform for both (shown in Fig. 2A), this couldsuggest engagement of the inhibitory CD22 receptor bythe TriKE modulates the CD19 triggering event, whichmay lead to a lower surface density of costimulatoryligand expression and, thus, lower reagent-induced effec-tor function as observed for the TriKE.

The ability of rituximab to induce significantly greateramounts of proinflammatory cytokines suggests it eithermediates stronger cross-linking of CD16 or CD20 ligation

Figure 4. BiKE and TriKE reagentsare functionally stable after culture inhuman serum. Reagents werecultured in 100% human serum at37oC for 24 or 48 hours. Resting NKcells were treated with or without 10mg/mL of reagent and coculturedwith 51Cr-labeled Raji target cells (A)or unlabeled Raji target cells (B andC). Percentages of target cell killing(A), CD107a expression (B), andintracellular IFN-g production (C)were evaluated. (For the 51Cr releaseassay, each experiment was carriedout in triplicate and repeated with 3donors, with aggregate resultsshown. For the degranulation andIFN-g assay,n ¼ 3, error bars represent SEM.)






leads to target cell modifications capable of enhancingeffector function. As surface densities of CD19, CD20, andCD22were essentially the same on the Raji target cells and

the binding affinity of the scFv CD16 is higher (Kd ¼ 10�8

mol/L; ref. 6) than that of IgG (Kd ¼ 10�6 mol/L; ref. 40),this points to the latter. Activated B cells express CD40

Figure 5. NK cell chemokine andcytokine production induced byRaji target cells is enhanced in thepresence of BiKEs and TriKEs. A,FACS analysis of treated (10 mg/mL) or untreated NK cell IFN-gproduction against Raji targets.(�P < 0.01 for test conditioncompared with no reagent, n ¼ 3;error bars represent SEM). B,Luminex analysis of cytokine/chemokine levels onsupernatants harvested fromresting treated(10 mg/mL) or untreated NK cellscocultured with Raji targets. Finalpg/mL values were determinedfor each condition using thefollowing equation: (NK cellþRajicell pg/mL) � [(NK cell alone pg/mL)þ (Raji cell alone pg/mL)]. �, P< 0.05, n¼ 6; error bars representSEM.

Gleason et al.





and the CD40-CD40L ligand interaction has been shownto induce B-cell IL-12 production (41, 42). As activatedNKcells express CD40L, effector–target cell interaction couldpromote B-cell production of IL-12, a known stimulus forIFN-g production and a cytokine that enhances the effec-tiveness of mAb therapy (43). NK cell IFN-g productionhas a greater threshold of activation (i.e., requires theaddition of coactivating signals; ref. 25) and, as our resultsshow that rituximab induces a stronger cytokine andchemokine NK cell response compared with the BiKEdespite the lower affinity interaction with CD16, thissuggests an important role for target cell costimulatoryligand expression for which CD19 engagement may be aweaker stimulus.Donor-derived NK cells are among the first cells to

reconstitute the recipient’s immune cell repertoire afterHCT and are key mediators of graft versus leukemiareactions againstminimal residual disease providing pro-tection against relapse (44). Consequently, the use ofBiKEs and TriKEs to enhance NK cell target recognition

and function holds great therapeutic promise for thetreatment of cancer. We have shown that BiKEs andTriKEs successfully enhance NK cell function againstprimary ALL and CLL tumors. Notably, the TriKE wassignificantly superior to rituximab in targeting ofALLandCLL. Lower expression of CD20 compared with CD19 onthe ALL tumors could explain these differences. Howev-er, expression of CD20 on CLL was greater than that ofCD19, which suggests that targeting CD22 in addition toCD19 provided an advantage. This has been shown withthe combinatorial use of the epratuzumab, a humanizedanti-CD22 mAb, and rituximab, which producedenhanced antitumor activity compared with either mAbalone (16). In contrast, as B cell ALL has been shown toexpress the inhibitory receptor CD32 (FcgRIIA; ref. 45),it is likely this receptor competes with CD16 for Fcbinding of rituximab resulting in decreased effectorfunction. This notion may further be supported by thefact that B cell CLL has been shown to be CD32 negative(46) and our results show a greater ability of rituximab

Figure 6. NKcell function against primary leukemia targets is enhanced in the presenceof BiKEs and TriKEs. A andB, FACSanalysis of CD19,CD20, andCD22(open histograms; isotype control, shaded histogram) surface expression on ALL, CLL, and AML primary tumor cells. For each primary tumor, the histogramplots represent 1 of 6 donors (A), with the scatter-plot displaying aggregate data (B). C and D, resting NK cells were cocultured with primary ALL, CLL, or AMLtumor cells with or without 10 mg/mL of reagent andCD107a (C) or IFN-g (D) expressionwas evaluated (�,P < 0.01; ��,P < 0.001; and ��,P < 0.0001; 6 primaryleukemia samples of each tumor type were tested against 2 allogeneic normal NK donors; error bars represent SEM).






to induce NK cell effector function against CLL com-pared with ALL.

Altogether, ourdata show thatBiKEs andTriKEsdirect-ly activate NK cells through CD16, overcoming inhibitionby MHC class I molecules and inducing target-specificcytotoxicity and cytokine and chemokine production.Moreover, there are implications that the tumor antigenstargeted by the recombinant reagents influence effectorfunction. Therefore, careful evaluation of effector–targetcell interactions induced by BiKEs and TriKEswill furtherserve to optimize their clinical efficacy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M.K. Gleason, M.R Verneris, D.A. Vallera, J.S.MillerDevelopment ofmethodology:M.K. Gleason, D.A. Todhunter, V.McCul-lar, L.M. Weiner, J.S. MillerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.K. Gleason, M.R Verneris, V. McCullar, A.Panoskaltsis-Mortari, B. Zhang, J.S. MillerAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): M.K. Gleason, M.R Verneris, V. McCullar,A. Panoskaltsis-Mortari, L.M. Weiner, D.A. Vallera, J.S. Miller

Writing, review, and/or revision of the manuscript: M.K. Gleason, M.RVerneris, A. Panoskaltsis-Mortari, L.M. Weiner, J.S. MillerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D.A. Todhunter, S.X. ZhouStudy supervision: J.S. Miller

AcknowledgmentsThe authors thank Ellen S. Vitetta for generously providing the RFB4

and HD37 hybridoma cell lines used to generate the CD19 and CD22parent antibodies, sample procurement and cell-processing services fromthe Translational Therapy Core supported from the National CancerInstitute (P30-CA77598), and Minnesota Masonic Charities through theCancer Experimental Therapeutics Initiative (CETI).

Grant SupportThis work was supported, in part, by the grants P01 CA65493, P30

CA77598, the Minnesota Masonic Charities, the US Public Health ServiceGrants R01-CA36725, the Randy Shaver Foundation, the Lion’s Chil-dren’s Cancer Fund, and the William Lawrence and Blanche HughesFund.

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 July 5, 2012; revised September 12, 2012; accepted September28, 2012; published OnlineFirst October 17, 2012.

References1. American Cancer Society. Cancer facts & figures 2012. Atlanta, GA:

American Cancer Society; 2012.2. Armitage JO, Weisenburger DD. New approach to classifying non-

Hodgkin's lymphomas: clinical features of the major histologic sub-types. Non-Hodgkin's Lymphoma Classification Project. J Clin Oncol1998;16:2780–95.

3. Keating GM. Spotlight on rituximab in chronic lymphocytic leukemia,low-grade or follicular lymphoma, and diffuse large B-cell lymphoma.BioDrugs 2011;25:55–61.

4. Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic anti-bodies: successes, limitations andhopes for the future. Br JPharmacol2009;157:220–33.

5. Bruenke J, Barbin K, Kunert S, Lang P, Pfeiffer M, Stieglmaier K, et al.Effective lysis of lymphoma cells with a stabilised bispecific single-chain Fv antibody against CD19 and FcgammaRIII (CD16). Br JHaematol 2005;130:218–28.

6. McCall AM, Adams GP, Amoroso AR, Nielsen UB, Zhang L, Horak E,et al. Isolation and characterization of an anti-CD16 single-chain Fvfragment and construction of an anti-HER2/neu/anti-CD16 bispecificscFv that triggers CD16-dependent tumor cytolysis. Mol Immunol1999;36:433–45.

7. Kellner C, Bruenke J, Horner H, Schubert J, Schwenkert M, Mentz K,et al. Heterodimeric bispecific antibody-derivatives against CD19 andCD16 induce effective antibody-dependent cellular cytotoxicityagainst B-lymphoid tumor cells. Cancer Lett 2011;303:128–39.

8. Cooper MA, Fehniger TA, Caligiuri MA. The biology of human naturalkiller-cell subsets. Trends Immunol 2001;22:633–40.

9. Bryceson YT, March ME, Ljunggren HG, Long EO. Activation, coacti-vation, and costimulation of resting human natural killer cells. ImmunolRev 2006;214:73–91.

10. Lanier LL. NK cell recognition. Annu Rev Immunol 2005;23:225–74.11. Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC,

et al. Activating receptors and coreceptors involved in humannatural killer cell-mediated cytolysis. Annu Rev Immunol 2001;19:197–223.

12. Bryceson YT, March ME, Ljunggren HG, Long EO. Synergy amongreceptors on resting NK cells for the activation of natural cytotoxicityand cytokine secretion. Blood 2006;107:159–66.

13. Vivier E, Nunes JA, Vely F. Natural killer cell signaling pathways.Science 2004;306:1517–9.

14. Vallera DA, Todhunter DA, Kuroki DW, Shu Y, Sicheneder A, Chen H. Abispecific recombinant immunotoxin, DT2219, targeting human CD19and CD22 receptors in a mouse xenograft model of B-cell leukemia/lymphoma. Clin Cancer Res 2005;11:3879–88.

15. Gleason MK, Lenvik TR, McCullar V, Felices M, O'Brien MS, CooleySA, et al. Tim-3 is an inducible human natural killer cell receptor thatenhances interferon gamma production in response to galectin-9.Blood 2012;119:3064–72.

16. Micallef IN,MaurerMJ,WisemanGA,NikcevichDA, Kurtin PJ, CannonMW, et al. Epratuzumab with rituximab, cyclophosphamide, doxoru-bicin, vincristine, and prednisone chemotherapy in patients with pre-viously untreated diffuse large B-cell lymphoma. Blood 2011;118:4053–61.

17. Raetz EA, Cairo MS, Borowitz MJ, Blaney SM, KrailoMD, Leil TA, et al.Chemoimmunotherapy reinduction with epratuzumab in children withacute lymphoblastic leukemia in marrow relapse: a Children's Oncol-ogy Group Pilot Study. J Clin Oncol 2008;26:3756–62.

18. Trotta R, Puorro KA, Paroli M, Azzoni L, Abebe B, Eisenlohr LC, et al.Dependence of both spontaneous and antibody-dependent, granuleexocytosis-mediated NK cell cytotoxicity on extracellular signal-reg-ulated kinases. J Immunol 1998;161:6648–56.

19. Alter G,Malenfant JM, AltfeldM. CD107a as a functionalmarker for theidentification of natural killer cell activity. J Immunol Methods 2004;294:15–22.

20. Aktas E, Kucuksezer UC, Bilgic S, Erten G, Deniz G. Relationshipbetween CD107a expression and cytotoxic activity. Cell Immunol2009;254:149–54.

21. Nimmerjahn F, Ravetch JV. Antibodies, Fc receptors and cancer. CurrOpin Immunol 2007;19:239–45.

22. Huntington ND, Xu Y, Nutt SL, Tarlinton DM. A requirement for CD45distinguishes Ly49D-mediated cytokine and chemokine productionfrom killing in primary natural killer cells. J ExpMed 2005;201:1421–33.

23. Hesslein DG, Takaki R, Hermiston ML, Weiss A, Lanier LL. Dysregula-tion of signaling pathways in CD45-deficient NK cells leads to differ-entially regulated cytotoxicity and cytokine production. ProcNatl AcadSci U S A 2006;103:7012–7.

Gleason et al.





24. Kellner C, Bruenke J, Stieglmaier J, Schwemmlein M, Schwenkert M,Singer H, et al. A novelCD19-directed recombinant bispecific antibodyderivative with enhanced immune effector functions for human leu-kemic cells. J Immunother 2008;31:871–84.

25. Fauriat C, Long EO, Ljunggren HG, Bryceson YT. Regulation of humanNK-cell cytokine and chemokine production by target cell recognition.Blood 2010;115:2167–76.

26. Barber DF, FaureM, Long EO. LFA-1 contributes an early signal for NKcell cytotoxicity. J Immunol 2004;173:3653–9.

27. Bryceson YT,MarchME, Barber DF, LjunggrenHG, LongEO.Cytolyticgranule polarization and degranulation controlled by different recep-tors in resting NK cells. J Exp Med 2005;202:1001–12.

28. Jazirehi AR, Bonavida B. Cellular and molecular signal transductionpathways modulated by rituximab (rituxan, anti-CD20 mAb) in non-Hodgkin's lymphoma: implications in chemosensitization and thera-peutic intervention. Oncogene 2005;24:2121–43.

29. Kozono Y, Abe R, Kozono H, Kelly RG, Azuma T, Holers VM. Cross-linking CD21/CD35 or CD19 increases both B7-1 and B7-2 expressionon murine splenic B cells. J Immunol 1998;160:1565–72.

30. Fujimoto M, Fujimoto Y, Poe JC, Jansen PJ, Lowell CA, DeFranco AL,et al. CD19 regulates Src family protein tyrosine kinase activation in Blymphocytes through processive amplification. Immunity 2000;13:47–57.

31. FujimotoM, Poe JC,HasegawaM, Tedder TF. CD19 regulates intrinsicB lymphocyte signal transduction and activation through a novelmechanism of processive amplification. Immunol Res 2000;22:281–98.

32. Fujimoto M, Poe JC, Jansen PJ, Sato S, Tedder TF. CD19 amplifies Blymphocyte signal transduction by regulating Src-family protein tyro-sine kinase activation. J Immunol 1999;162:7088–94.

33. Roifman CM, Ke S. CD19 is a substrate of the antigen receptor-associated protein tyrosine kinase in human B cells. Biochem BiophysRes Commun 1993;194:222–5.

34. Assarsson E, Kambayashi T, Persson CM, Chambers BJ, LjunggrenHG. 2B4/CD48-mediated regulation of lymphocyte activation andfunction. J Immunol 2005;175:2045–9.

35. AzumaM, Cayabyab M, Buck D, Phillips JH, Lanier LL. Involvement ofCD28 in MHC-unrestricted cytotoxicity mediated by a human naturalkiller leukemia cell line. J Immunol 1992;149:1115–23.

36. Croft M. Control of immunity by the TNFR-related molecule OX40(CD134). Annu Rev Immunol 2010;28:57–78.

37. Ogasawara K, Yoshinaga SK, Lanier LL. Inducible costimulator costi-mulates cytotoxic activity and IFN-gamma production in activatedmurine NK cells. J Immunol 2002;169:3676–85.

38. Vinay DS, Kwon BS. 4-1BB signaling beyond T cells. Cell Mol Immunol2011;8:281–4.

39. Fujimoto M, Bradney AP, Poe JC, Steeber DA, Tedder TF. Modulationof B lymphocyte antigen receptor signal transduction by aCD19/CD22regulatory loop. Immunity 1999;11:191–200.

40. Ravetch JV, Bolland S. IgG Fc receptors. Annu Rev Immunol 2001;19:275–90.

41. Schultze JL, Michalak S, Lowne J, Wong A, Gilleece MH, Gribben JG,et al. Humannon-germinal center B cell interleukin (IL)-12 production isprimarily regulated by T cell signals CD40 ligand, interferon gamma,and IL-10: role of B cells in the maintenance of T cell responses. J ExpMed 1999;189:1–12.

42. Sartori A, Ma X, Gri G, Showe L, Benjamin D, Trinchieri G. Interleukin-12: an immunoregulatory cytokine produced by B cells and antigen-presenting cells. Methods 1997;11:116–27.

43. Parihar R, Dierksheide J, Hu Y, CarsonWE. IL-12 enhances the naturalkiller cell cytokine response to Ab-coated tumor cells. J Clin Invest2002;110:983–92.

44. Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A,et al. Effectiveness of donor natural killer cell alloreactivity in mis-matched hematopoietic transplants. Science 2002;295:2097–100.

45. Suzuki T, Coustan-Smith E, Mihara K, Campana D. Signals mediatedby FcgammaRIIA suppress the growth of B-lineage acute lympho-blastic leukemia cells. Leukemia 2002;16:1276–84.

46. DamleRN,Ghiotto F, Valetto A, AlbesianoE, Fais F, YanXJ, et al. B-cellchronic lymphocytic leukemia cells express a surface membranephenotype of activated, antigen-experienced B lymphocytes. Blood2002;99:4087–93.






Published OnlineFirst October 17, 2012.Mol Cancer Ther Michelle K. Gleason, Michael R. Verneris, Deborah A. Todhunter, et al. and Cytokine ProductionHuman NK Cells through CD16 Signaling and Induce Cytotoxicity Bispecific and Trispecific Killer Cell Engagers Directly Activate

Updated version

10.1158/1535-7163.MCT-12-0692doi:

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/early/2012/11/29/1535-7163.MCT-12-0692To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-12-0692

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/early/2012/11/29/1535-7163.MCT-12-0692


molecular cancer therapeutics - bispeciﬁc and trispeciﬁc killer … · 2012. 11. 29. · zhou...

Documents