decitabine enhances anti-cd33 monoclonal antibody bi … · 2017-04-13 · regular article myeloid...

Regular Article

MYELOID NEOPLASIA

Decitabine enhances anti-CD33 monoclonal antibody BI836858–mediated natural killer ADCC against AML blastsSumithira Vasu,1,* Shun He,1,* Carolyn Cheney,1 Bhavani Gopalakrishnan,1 Rajeswaran Mani,1 Gerard Lozanski,1

Xiaokui Mo,2 Veronica Groh,3 Susan P. Whitman,1 Renate Konopitzky,4 Christian Kossl,4 Donna Bucci,1 David M. Lucas,1

Jianhua Yu,1 Michael A. Caligiuri,1 William Blum,1 Paul J. Adam,4 Eric Borges,4 Bjoern Rueter,5 Karl-Heinz Heider,4,†

Guido Marcucci,6,† and Natarajan Muthusamy1,†

1Division of Hematology, Department of Internal Medicine, The Ohio State University Comprehensive Cancer Center, and 2Center for Biostatistics,

Department of Biomedical Informatics, The Ohio State University, Columbus, OH; 3Clinical Research Division, Fred Hutchinson Cancer Research Center,

University of Washington, Seattle, WA; 4Research Oncology, Boehringer Ingelheim RCV, Vienna, Austria; 5Medicine Oncology, Boehringer Ingelheim

Pharma GmbH, Biberach/Riss, Germany; and 6Hematologic Malignancies and Stem Cell Transplantation Institute, City of Hope, Duarte, CA

Key Points

• BI 836858, an Fc-engineeredanti-CD33 antibody, mediatesautologous and allogeneicNK cell–mediated ADCC.

• Decitabine increases ligandsfor activating NK receptorspotentiating BI 836858activity, providing a rationalefor combination therapy.

Acutemyeloid leukemia (AML) is themostcommontypeof acute leukemia, affectingolder

individuals at a median age of 67 years. Resistance to intensive induction chemotherapy

is themajor cause of death in elderly AML; hence, novel treatment strategies are warranted.

CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to

improve overall survival, validating CD33 as a target for antibody-based therapy of AML.

Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33

antibodyusingAMLcell lines andprimaryAMLblasts as targets. BI 836858–opsonizedAML

cells significantly induced both autologous and allogeneic natural killer (NK)-cell de-

granulation andNK-cell–mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro

treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents

that show efficacy in older patients, did not compromise BI 836858–induced NK-cell–

mediatedADCC. Evaluation of BI 836858–mediatedADCC in serialmarrowAMLaspirates

in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC)

revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to

activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples

comparedwithpre-DACsamples;whenNKG2DLreceptorwasblockedusingantibodies,BI 836858–mediatedADCCwassignificantly

decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a

rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

(Blood. 2016;127(23):2879-2889)

Introduction

Acute myeloid leukemia (AML) is the most common acute leukemiain adults, causing .10 000 deaths per year in the United States.1-3

Antibody-based therapeutics in AML have targeted CD33 (sialicacid–binding immunoglobulin-like lectin 3)which is expressed in over80% of leukemic cells.4-7 Gemtuzumab ozogamicin (GO), an anti-CD33 immunoconjugate, is composed of a humanized immunoglob-ulin G4 (IgG4) antibody conjugated to the powerful antimitoticcalicheamicin which mediates cell death following rapid internal-ization of the antibody-antigen complex formation.5 However, GO(marketed as Mylotarg) was voluntarily withdrawn from the marketin June 2010 after a phase 3 trial in newly diagnosed AML showed atrend toward increased mortality in the GO arm.8 Since that time,data from phase 3 trials and a meta-analysis have shown an advantage

in overall survival in patients treatedwithGO combinedwith standardinduction chemotherapy in older AML patients.9,10 An unconjugatedhumanized anti-CD33 antibody, lintuzumab (HuM195), has alsoresulted in complete remissions in elderly patients,11 althoughrandomized studies have not shown improvement in overallsurvival.12

Therapeuticmonoclonal antibodies (mAbs) elicit responses throughdirect killing (ie, apoptosis induction) or via antibody-dependentcellular cytotoxicity (ADCC) or antibody-dependent cellular phago-cytosis mechanisms. Targeted Fc engineering either by glycosylationor by mutagenesis increases molecular affinity toward CD16 (Fcgreceptor IIIa [FcgRIIIa]) on natural killer (NK) cells and has beenshown to potentiate NK-mediated ADCC.13 Also, coengagement of

Submitted November 17, 2015; accepted March 9, 2016. Prepublished online

as Blood First Edition paper, March 24, 2016; DOI 10.1182/blood-2015-11-

680546.

*S.V. and S.H. contributed equally to this work.

†K.-H.H., G.M., and N.M. are cosenior authors.

The online version of this article contains a data supplement.

There is an Inside Blood Commentary on this article in this issue.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked “advertisement” in accordance with 18 USC section 1734.

© 2016 by The American Society of Hematology

BLOOD, 9 JUNE 2016 x VOLUME 127, NUMBER 23 2879

For personal use only.on April 13, 2017. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/content/127/23/2787

http://www.bloodjournal.org/

http://www.bloodjournal.org/site/subscriptions/ToS.xhtml

AML target cells viaCD33, andNKcells viaCD16, has been shown toresult in increased cytotoxicity of the target cells.14 In addition toCD16engagement, we evaluated whether receptor-ligand interactions be-tween blasts and effectors can potentiate NK-mediated cytotoxicityagainst AML blasts. Leukemic cells downregulate ligands for theNK-cell–activating receptor NKG2D as a mechanism for evadingNK-mediated ADCC.15,16 However, treatment of blasts with histonedeacetylase inhibitors and hypomethylating agents has been shown toupregulate NKG2D ligand (NKG2DL).15 In the setting of hypome-thylating agents, upregulation of NKG2DL was attributed to promoterDNAdemethylation andDNAdamage and correlates with improvedNK cytotoxicity.17,18 Whether agents that upregulate NKG2DL onAML blasts could also enhance the efficacy of Fc-engineered anti-bodies is unknown. Here, we sought to evaluate whether hypomethy-lating agents such as decitabine (DAC) or azacytidine modulatesusceptibility of AML blasts to Fc-engineered mAb directed againstCD33.

BI 836858 is a fully human anti-CD33 antibody, which is Fcengineered for increased binding to FcgRIIIa. It binds with lownanomolar affinity to human CD33 and displays deceleratedinternalization kinetics comparedwith previously developedCD33mAbs, thus making it suitable for exploitation of NK-mediatedADCC. We report here potent single-agent NK-cell–mediatedADCC activity of BI 836858 against primary CD331 AML blasts.Given that hypomethylating agents are commonly used in olderpatients, we wanted to evaluate whether DAC modulates BI 836858–mediated cytotoxicity. In vitro addition of DAC or azacytidine did notinhibit BI 836858–mediated ADCC. We then analyzed serial bonemarrow samples from patients who received a 10-day regimen ofDAC. BI 836858 mediated higher ADCC in post-DAC (day 28)samples than pre-DAC treatment. We show that day 28 samplesshow increased expression of ligands for NKG2D (an activatingreceptor for NK cells); blocking of NKG2DL diminished BI836858–mediated ADCC. Our studies highlight that DAC increasessusceptibility of AML blasts to an Fc-engineered anti-CD33antibody, providing a rationale for combination therapy of BI836858 with azanucleosides in the treatment of older AMLpatients.

Materials and methods

Primary AML samples

Under an Ohio State University Institutional Review Board (IRB)–approvedprotocol, newly diagnosed AML patients presenting with high leukocyte countswere consented to undergo leukapheresis to collect primary cells. The Ficoll-enriched mononuclear cells were cryopreserved and subsequently thawed andcultured in RPMI 1640 with 10% fetal bovine serum (FBS). The clinicalcharacteristics of the patients are presented in supplemental Table 2 (availableon the Blood Web site). Some patients were treated with DAC as part of IRB-approved clinical trials (OSU-0336, OSU-07017). Patients received 20 mg/m2

DAC IV daily on days 1 to 10 and this cycle was repeated every 28 days. Bonemarrow samples were collected prior to treatment with DAC, and on days 4, 11,and 28 post-DAC.

NK-cell isolation

Human NK cells were enriched from the peripheral blood of healthy donors(American Red Cross) with the Rosette-Sep NK-cell Enrichment Cocktail(StemCell Technologies) and Ficoll-Paque Plus centrifugation (Amersham/GEHealthcare Bio-Sciences). For autologous assays, frozen apheresed AMLsamples were thawed and stained with anti-human CD56, CD3, and CD45

antibodies, and NK cells were sorted as SSClowCD451CD32CD561 on Aria IIfluorescence-activated cell sorter, with Sytox used in the process for dead cellexclusion.

mAbs

BI 836858 was derived by immunizing human antibody producingtransgenic mice.19 BI 836858 is Fc engineered through the introduction of2 amino acid substitutions in the Fc CH2 domain of the wild-type IgG1 viaquick change mutagenesis.20 BI 836847 is an isotype-matched, Fc-engineered control antibody which does not bind human cells. Lintuzumab(HuM195), an IgG1-type humanized mAb directed against human CD33,was constructed from a published sequence.21 Antibodies were expressedin dihydrofolate reductase–deficient Chinese hamster ovary (CHO) DG44suspension cells under serum-free conditions and purified via MabSelectProtein A affinity chromatography (GE Healthcare). Additional detailsabout use of HDXMS and the internalization assay are described in thesupplemental Methods.

CD107a assay

CD107a is a lysosome-related protein that traffics to the cell membranewhen theNKcells are activated anddegranulates by releasing cytolytic granules into targetcells.22 Therefore, CD107a expression serves as a surrogate for the cytotoxicpotential of NK cells. HL60 or primary AML patient cells were incubated withBI 836858 or BI 836847 (nonbinding Fc-engineered isotype control; BI47)mAbs at the concentration of 5 mg/mL for 30 minutes on ice, and then washedand cocultured at an effector-to-target (E:T) ratio of 2:1 with 0.5 million allo-geneic NK cells enriched from leukopacks of healthy donors or autologous NKcells isolated from AML samples. CD107a antibody (BD Biosciences, clone:H4A3) was added and cocultured for 1 hour, then monensin (eBioscience) ata final concentration of 2 mM was added and incubated with cells for an addi-tional 3 hours. Cells were then harvested, stained for NK cells (CD32CD561),and analyzed by flow cytometry. The percentage of CD107a1 cells gated onCD32CD561NK cells is presented.

ADCC assay

ADCC was determined by standard 4-hour 51Cr-release assay. 51Cr-labeled(0.1mCi 51Cr permillion cells) target cells (53 103 primaryAML samples orHL60 cells) per well were placed in 96-well plates after incubating themwithmAbs (10 mg/mL, unless indicated otherwise) for 30 minutes. Effector cells(NK cells from healthy donors or autologous NK cells) were then added tothewells at E:T ratios of 3:1 (unless otherwise indicated). After 4-hour incubation,supernatantswere removed and counted in ag counter. The percentage of specificcell lysiswas calculatedby: percent lysis51003 (ER2SR)/ (MR2SR),whereER, SR, and MR represent experimental release (ER), spontaneous release (SR),and maximum release (MR), respectively.

NKG2DL receptor-blocking studies

The 51Cr-labeled target cells (53 103 cells per well) were incubated at 37°C for30 minutes with 10 mg/mL antibodies. For blocking with mAb, labeled targetcells were preincubated for 30minutes with either or in combination ofMICA/B(Biolegend), ULBP-1 phycoerythrin (PE; R&D Systems), ULBP-2 PE (R&DSystems), ULBP-3 PE (R&D Systems), and its isotype controls. The unboundantibodies were washed off and 53 103 cells were added in 96-well plates. Theeffector NK cells at different E:T ratios were then added to the plates. ADCCactivity was determined by standard 4-hour 51Cr-release assay (PerkinElmer).The supernatantwas removed after the incubation and counted on a PerkinElmerWizard g counter. The percentage of specific cell lysis was determined asdescribed previously.

Cell culture and treatment

Cell lines were cultured in RPMI 1640 media (Invitrogen) supplemented with10% heat-inactivated FBS (Sigma-Aldrich), 2 mM L-glutamine (Invitrogen),and 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen) at 37°C,5% CO2. Similarly, the AML blasts were cultured in the above-mentionedmedia in supplement with 10 ng/mL each of interleukin-3 (R&D Systems),

2880 VASU et al BLOOD, 9 JUNE 2016 x VOLUME 127, NUMBER 23




granulocyte-macrophage colony-stimulating factor (R&D Systems), and stemcell factor (R&DSystems). HumanNK cells or thawedAMLblasts were treatedwithDACat the concentrationof 500 nMor 5-azacytidine at the concentrationof 2 mM for 48 hours. Due to the instability of the drugs in solution, thesedrugs were reconstituted in vehicles and added to the culture medium daily.

NKG2DL expression analysis

RNA was isolated using TRIzol reagent (Gibco BRL) according to themanufacturer’s instructions, and complementary DNAwas generated usingSuperScript (Life Technologies). The expression of NKG2D ligandsULBP-1, ULBP-2, ULBP-3, MICA, MICB, and the DNAM ligands(DNAML) PVR and Nectin-2 were assessed by real-time quantitativereverse-transcription polymerase chain reaction (RT-PCR) using a ViiA 7Real-Time PCR System (Applied Biosystems) with TaqMan detection.The following primer/probe sets, all from Applied Biosystems wereused: ULBP-1 (Hs00360941_m1), ULBP-2 (Hs00607609_mH), ULBP-3(Hs00225909_m1), MICA (Hs00741286_m1), MICB (Hs00792952_m1),Nectin-2 (Hs01071562_m1), and PVR (Hs00197846_m1). CIR-neo and itstransfectants (generously gifted by V.G., Fred Hutchinson Cancer ResearchCenter, Seattle, WA) were used as positive control for the ligands. The messengerRNA transcript analysis for NKG2DL was performed using primers mentioned

in a published protocol.23 The NKG2DL and DNAML transcript levels werenormalized to glyceraldehyde-3-phosphate dehydrogenase and the 2DDCt (cyclethreshold [Ct]) method was used for determination of fold change.

Flow cytometry

Primary AML cells were suspended in flow buffer (phosphate-buffered salineplus 1% FBS) and incubated with specific fluorochrome-labeled antibodies at4°C for 30 minutes. CD33-BV421 (Biolegend) and CD34-BV421 (Biolegend)antibodies were used. The cells were thenwashedwith flow buffer. The sampleswere run on a Gallios flow cytometer (Beckman Coulter) and analyzed usingKaluza 1.2 software (Beckman Coulter).

Statistical analysis

For the in vitro cell line experiments, data were analyzed by analysis ofvariance. For experiments comparing intrapatient variability in varioustreatment conditions, data were analyzed by mixed effect models, incor-porating observational dependencies for the same subject.24 The Holmmethod was used to adjust the multiplicities to control the type I family-wise error rate at 0.05.25 NKG2DL expression by RT-PCR was normalizedto internal control glyceraldehyde-3-phosphate dehydrogenase. Normalized

A B

0 5 10 15 20 25

20

40

60

80

100

% s

urfa

ce e

xpre

ssio

n BI 836858HuM195

C

% c

ytol

ysis

Log [mAb] (ng/ml)

BI 836858HuM195

–3–20

20

40

60

80

0

–2 –1 0 1 2 3 4

D

0

HL60

GF-D8

KG-1

Molm

-13

MV4-

11TF-1

THP-1

HEL92.

1.7

20

40

60

Time (h)

% c

ytot

oxic

ityBI 836858

HuM195

MDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHENLYFQGSHHHHHH

Figure 1. Comparison of Fc-engineered anti-CD33 antibody BI 836858 and nonengineered HuM195. (A) Hydrogen-deuterium exchange mass spectrometry (HDXMS)

analysis of BI 836858 and HuM195 complexed with CD33. Protection from D2O exchange when bound to CD33 mapped onto the structure of CD33. Blue region indicates

protection of CD33 by BI 836858; red region indicates protection by HuM195. Resolution of the method is determined by the peptides produced by digestion with pepsin. The

antibodies bind to nonoverlapping amino acid residues in the N-terminal region of CD33. A structural model was calculated based on Siglec-526; the predicted epitopes for BI

836858 (blue) and Hum195 (red) are depicted. (B) Effect of BI 836858 and HuM195 on CD33 surface retention on HL60 cells. Cell surface–bound IgG was determined at

indicated time points with a secondary fluorescence-labeled anti-IgG antibody and data were normalized against time point 0. The means of 6 independent experiments are

shown. Incubation of HL60 cell lines with both antibodies results in decreased CD33 surface exposure over time, indicative of internalization of antibody/CD33 complexes. BI

836858 shows significantly higher CD33 levels over time compared with HuM195. (C) ADCC activity on HL60 cells at an E:T ratio of 20:1; peripheral blood mononuclear cells

from healthy individuals were used as effectors. (D) Summary of ADCC on leukemia cell lines and primary AML blasts. BI 836858 displays superior efficacy of cytolysis

compared with HuM195 under identical experimental conditions in both AML cell lines and 6 primary AML samples. Healthy donor NK cells were used as effectors and the51Cr-labeled ADCC assay was done to measure cytotoxicity.

BLOOD, 9 JUNE 2016 x VOLUME 127, NUMBER 23 DAC ENHANCES ENGINEERED ANTI-CD33 ACTIVITY IN AML 2881




data were analyzed by using the mixed effect model, incorporating repeatedmeasures for each subject. The primary test results (difference in geneexpression between pre-DAC and day 28 post-DAC) are summarized by foldchange in table format. Statistical analyses were conducted by using SAS 9.4(SAS, Inc).

Results

CD33-directed Fc-engineered BI 836858 antibody has

decelerated internalization, and binds a different CD33 epitope

than nonengineered HuM195

BI 836858 is a fully human anti-CD33 antibody which was derivedby Fc engineering of BI 836854, a standard IgG1-type antibodyderived from human antibody-producing transgenic mice. To assessthe effect of antibody incubation on the level of cell surface CD33,we initially compared BI 836858 to BI 836854 and later on toHuM195 (lintuzumab), an independent CD33-directed humanized

IgG1 mAb. Incubation of HL60 cells with all 3 mAbs results indecreased CD33 molecules on the cell surface in a time-dependentmanner, indicative of internalization of mAb/CD33 complexes.Interestingly, BI 836858 showed a higher degree of retained cellsurface CD33 compared with HuM195 (Figure 1B), whereas thenon–Fc-engineered version BI 836854 showed similar levels as BI836858 (not shown); in addition, the non–FC-engineered antibody BI836854 also had higher surface expression of CD33 in comparison withlintuzumab (supplemental Figure 1A). From these findings, we concludethat the difference in cell surface–retained CD33 observed between BI836858andHuM195 isnotdue toFcengineering.To investigatewhetherbinding to different regions of CD33 may cause the difference inCD33 surface retention, epitope mapping was performed by directcompetition experiments and HDXMS. A structural model wascalculated based on Siglec-3. Predicted epitopes for BI 836858(blue) and HuM195 (red) are depicted in Figure 1A.26 The resultsdemonstrate that BI 836858 and HuM195 bind to nonoverlappingamino acid residues in the N-terminal region of CD33.

Cyto

toxi

city

fold

cha

nge

0

5

10

15

0.01 0.1 1 10

Summary of 8 blasts

***

**

ns

Antibody concentration (ug/mL)–10

0

10

20

30

40

50

60 Representative blast

% c

ytot

oxic

ity

Antibody concentration (ug/mL)0 0.01 0.1 1 10

ABI836858

BI836847

0

5

10

15

CD10

7a f

old

chan

ge

Summary of 8 blasts

***

BI836858BI836847

DonorNK3

DonorNK4

Representative blastB

102

103

104

105

102

103

104

105

50 100 150 200 250(x 1,000) (x 1,000) (x 1,000)

50 100 150 200 250 50 100 150 200 250

102

103

104

105

102

103

104

105

102

103

104

105

50 100 150 200 250(x 1,000) (x 1,000) (x 1,000)

50 100 150 200 250 50 100 150 200 250

102

103

104

105CD

107a

Figure 2. BI 836858 elicits potent NK-cell–mediated ADCC effect as well as NK-cell activation against primary AML blasts. (A-B) AML primary blasts were precoated

with different concentrations of BI 836858, and then cocultured with NK cells from healthy donors at an E:T of 3:1 for the ADCC assays. The Fc-engineered, nonbinding BI 47

antibody was used as negative control antibody at the same E:T for comparison. Representative cytotoxicity for 1 blast donor (A) and summary fold changes for 8 different donors

(B) data are shown. (C-D) AML primary blasts were precoated with 5 mg/mL BI 836858 or BI 836847, and then cocultured with NK cells at an E:T of 2:1 for the CD107a assays.

Representative CD107a expression for 2 donors (C) and summary fold changes for 8 different donors (D) are shown. *P , .0001, **P , .01, ***P , .001. ns, not significant.





BI 836858 shows high-affinity binding to leukemic cell lines and

primary AML cells, and displays superior ADCC compared

with HuM195

BI 836858 binds CD33 on cell lines and primary blasts with lownanomolar affinity as determined by fluorescence-activated cellsorter Scatchard analysis (supplemental Table 1). Of note, the averageCD33 antigen density on AML cell lines was clearly higher than onprimary AML cells. Direct comparison of the cytolytic activity of theengineered antibody BI 836858 to BI 836854, the non–Fc-engineeredparental mAb, in the presence of peripheral blood mononuclear celleffector cells clearly indicated an increase in ADCC due to Fc engi-neering (supplemental Figure 1B). This is in line with previouslyreported data for a CD37-specific antibody.19 In a next step, theADCC of BI 836858 was tested on a panel of AML cell lines andprimary AML blasts and compared with the reference antibody,HuM195 (Figure 1C-D). BI 836858 displayed higher cytolyticactivity than HuM195 on all tested primary AML samples and celllines. It is noteworthy that there were 4 samples (cell lines MV4-11

and TF-1; primary samples E and F) where BI 836858 still displayedsignificant ADCC whereas HuM195 was devoid of ADCC. The50% inhibitory concentration of BI 836858 on HL60 cells is about5 ng/mL (0.2 nM). To further assess the effect of varying E:T ratioson cytolytic activity, different E:T ratios from 3:1 to 25:1 wereevaluated (supplemental Figure 2). Although the highest activitywas observed at a ratio of 25:1, significant activitywas still observed atthe lowest tested ratio of 3:1, suggesting potent activity of BI 836858even at lowE:T ratios. The low3:1 E:T ratiowas used in subsequentADCC assays. As AML cells have been shown to be negative forHER-2 expression,27 the therapeutic IgG1 antibody Herceptintargeting HER-2 was used as a negative control.

To further characterize the NK-cell–mediated ADCC activity byBI 836858 against primary AML blasts, we compared BI 836858 withFc-engineered isotype control. As shown in Figure 2, BI 836858 in-duced concentration-dependent ADCC effects while the isotypecontrol antibody BI 836847 showed only minimal effects that werenot concentration dependent.With n58 primary samples,ADCCwas10-fold higher at 10mg/mL (95% confidence interval [CI], 4.2-23.7;

0

20

40

60

80

100

Untreated Decitabine 5-azacytidine

% c

ytot

oxic

ity

NK treatment:

ns nsns

****

***

AML blast 1

0

20

40

60

80

Untreated Decitabine 5-azacytidine

% c

ytot

oxic

ity

ns

ns

ns

******

***

AML blast 2No Ab

BI836847

BI 836858

050

100150

200250300350

102

103

104

105

0102030405060708090

102

103

104

105

0

50

100

150

200

102

103

104

105

0

50

100

150

200

102

103

104

105

0

50

100

150

102

103

104

105

0

50

100

150

200

102

103

104

105

0

50

100

150

200

102

103

104

105

0

25

50

75

100

125

102

103

104

105

0

50

100

150

200

250

102

103

104

105

BI836847 BI836858 BI836847

0

50

100

150

200

250

102

103

104

105

BI836847 BI836858

0

25

50

75

100

102

103

104

105

0

25

50

75

100

102

103

104

105

BI836858

NK treatment

Untreated Decitabine

CD107a

Donor NK 2

Donor NK 1

5-azacytidine

0.9%

1.6% 40.5% 1.3% 40.1% 1.1% 40.3%

40.4% 0.9% 38.3% 0.7% 39.4%

A

B

Figure 3. Pretreatment of NK effector cells with DAC or 5-azacytidine did not compromise BI 836858–mediated ADCC or NK-cell activation against AML blasts. NK

cells from patients were exposed to DAC (500 nM) or 5-azacytidine (2 mM) for 48 hours before incubation with AML blasts coated with BI 836858 or BI 836847 for the

ADCC assay (10 mg/mL, E:T 5 3:1; A) or CD107a assay (5 mg/mL, E:T 5 2:1; B). Shown are representative data of autologous NK cells and AML primary blasts from 2

patients. **P , .01, ***P , .001. ns, not significant.





P, .0001); 4.5-fold higher at 1mg/mL (95%CI, 1.9-10.6; P5 .003),and 2.3-fold higher at 0.1 mg/mL (95% CI, 1.0-5.5; P 5 .106)(Figure 2A). Furthermore, NK cells that were cocultured with BI836858–coated AML blasts exhibited significantly increased de-granulation, as evidenced by upregulation of CD107a expressioncompared with those cocultured with BI 836847–coated AML blasts,indicating the potent induction of NK activation by BI 836858 againstAML blasts (7.3-fold higher; 95% CI, 3.2-16.9; P , .0001; n 5 8)(Figure 2B).

In vitro pretreatment of AML blasts with DAC or 5-azacytidine

does not inhibit BI 836858–mediated ADCC or autologous

NK-cell activation

To evaluate whether BI 836858 can be used in combination withDAC or 5-azacytidine, we pretreated primary AML blasts withDAC or 5-azacytidine and evaluated the efficacy of BI 836858–induced autologousNK-cell activation asmeasured by induction of

CD107a and ADCC. Representative data from 2 patients of a totalof 8 patients assessed are shown in Figure 3. As shown in Figure 3,pretreatment of AMLblasts withDAC or 5-azacytidine up to 48 hoursin vitro did not significantly alter BI 836858–induced autologousNK-cell degranulation or BI 836858–induced ADCC by autolo-gous NK cells compared with untreated target cells.

Pretreatment of both AML blasts and allogeneic NK

effector cells with DAC or 5-azacytidine does not inhibit

BI 836858–mediated ADCC or NK-cell activation

In patients treated with DAC or 5-azacytidine, NK cells and AMLblasts are expected to be simultaneously exposed to these agents.This warranted evaluation of the effect of pretreatment of both NKcells and blast cells with DAC or 5-azacytidine on BI 836858–mediated NK-cell activation and ADCC. Due to the relatively lownumbers of autologous NK cells that could be obtained fromthawed apheresis samples, we used allogeneic NK cells when there

A

0

20

40

60

80

0

20

40

60

80

100

Untreated Decitabine 5-azacytidineBlast & NK treatment: Untreated Decitabine 5-azacytidine

% c

ytot

oxic

ity

% c

ytot

oxic

ity

AML blast 1 AML blast 2

***** ***

**** ***

ns

ns ns ns ns

ns

No Ab

BI 836858

050

100150

200250300350

0

50

100

150

200

0102030405060708090

0

50

100

150

0

50

100

150

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

0

25

50

75

100

125

102

103

104

105

102

103

104

105

0

50

100

150

200

102

103

104

105

0

25

50

75

100

0

25

50

75

100

102

103

104

105

0

50

100

150

200

102

103

104

105

102

103

104

105

B

Untreated

BI836847 BI836858

Decitabine

BI836847 BI836858

5-azacytidine

BI836847 BI836858

40.4%0.9% 0.8% 39.5% 1.3% 46.4%

1.6% 40.5% 1.1% 33.4% 26.7%

CD107a

AML blast 1

AML blast 2

Blast & NK treatment

1.7%

0102030405060708090

0

25

50

75

100

125

BI836847

Figure 4. Pretreatment of both AML blasts and NK effector cells with DAC or 5-azacytidine does not inhibit BI 836858–mediated ADCC or NK-cell activation. Both

NK cells from healthy donors and AML blasts were exposed to DAC (500 nM) or 5-azacytidine (2 mM) for 48 hours before AML blasts were coated with BI 836858 or BI 836847

and coincubated for the ADCC assay (10 mg/mL, E:T 5 3:1; A) or CD107a assay (5 mg/mL, E:T 5 2:1; B). Shown are representative data of NK cells from 2 donors against 2

AML primary blasts. **P , .01, ***P , .001. ns, not significant.





were inadequate numbers of autologous NK cells. Dead blasts dueto DAC or 5-azacytidine treatments were excluded and the NK-cellADCC activity and NK-cell activation mediated by BI 836858 orcontrol antibody BI 836847 were evaluated using viable blasts andNK cells from healthy donors. Primary AML blasts coated with BI836858 significantly increased NK-cell activation when comparedwith control antibody; prior treatment of both target and effectorcells with DAC or 5-azacytidine did not affect BI 836858–mediated ADCC and NK-cell activation (Figure 4). These resultssuggest that short-term in vitro culture of AML blasts withDAC or 5-azacytidine does not result in significant immunemodulation.

Upregulation of NKG2DL in AML patients treated with DAC

Because DAC-mediated changes require epigenetic modulation,we speculated that short-term culture of blasts with DAC wasnot adequate to observe epigenetic changes. Hence, we sought toevaluate samples from patients who received DAC on clinicaltrials (OSU-0336; OSU-0717). DAC (20 mg/m2) was adminis-tered daily IV from days 1 to 10 as part of a 28-day cycle. Patientsunderwent bone marrow biopsies pre-DAC, days 1, 4, 11, and 28.Blast percentages in pre- and post-DAC samples were assessed;blasts percentages ranged from 22% to 94% in the pre-DACsamples and ranged from 11% to 46% in the post-DAC samples.We evaluated NKG2DL expression by messenger RNA on days 1,4, 11, and 28 in 18 patients. We noted expression of at least 1NKG2DL (ULBP-1, ULBP-2, ULBP-3, MICA, or MICB) in all ofthe samples at day 28 but not on days 4 or 11 post-DAC treatment(Figure 5; supplemental Table 3). ULBP1 expression increased by2.17-fold (95% CI, 1.25-3.73; P 5 .0069) on day 28 of the first

cycle of DAC treatment. ULBP-2 expression increased by 2.88-fold(95% CI, 1.54-5.37; P 5 .0016) and ULBP-3 expression increased2.04-fold (95% CI, 1.2-3.45; P 5 .0101) at this same time point.There was a trend toward increased expression ofMICA (P5 .0547),although it was not statistically significant. Overall, expression ofNKG2DLs was significantly higher on day 28 compared with day 1(Figure 5; supplemental Table 3).

BI 836858–mediated ADCC is increased in primary AML blasts

on day 28 post-DAC compared with pre-DAC blasts

Wewanted to evaluatewhetherDAC treatment increased susceptibilityof AML blasts to ADCC mediated by NK cells through BI 836858.Hence, we evaluated BI 836858–mediated ADCC on bone marrowsamples obtained fromAML patients’ pre- and post-DAC treatment.As shown in Figure 6, we consistently noted an increase in BI836858–mediated cytotoxicity in day 28 samples compared withpre-DAC samples (P , .0001) when healthy donor NK cells wereused as effectors (there were insufficient autologous NK cells toperform these assays). Each subpanel in Figure 6 shows ADCC ondays 1, 4, and 28 of DAC treatment. Higher E:T ratios (25:1) wereassociated with higher ADCC; E:T ratios of 6:1 still resulted insignificant BI 836858–specific cytotoxicity.

Blocking of NKG2DL on day 28 samples abrogates

BI 836858–mediated ADCC

We hypothesized that increased expression of NKG2DL was one ofthe mechanisms whereby DAC-treated samples showed higherBI 836858–mediated ADCC. Consistent with this hypothesis, block-ing of DAC-induced ULBP-3 in a day 28 post-DAC treatment sample

0

2

4

6

8

10

12

Day1 Day4 Day11 Day28

Day1

Day4

Day11

Day28

Norm

alize

d RN

A fo

ld c

hang

e

ULBP-1*

0369

121540

45

50

55

60


Norm

alize

d RN

A fo

ld c

hang

e

ULBP-2

**

0

20

12

9

6

3

22

24


Norm

alize

d RN

A fo

ld c

hang

e

ULBP-3

***

0

10

8

6

4

2

12


Norm

alize

d RN

A fo

ld c

hang

eMICA

n.s.

0

10

8

6

4

2

12


Norm

alize

d RN

A fo

ld c

hang

e

MICB

****

A B

C D E

Figure 5. Evaluation of NKG2DL expression in patients treated with DAC for 10 days. Expression analysis of NKG2DL by RT-PCR on AML blasts from patients treated

with DAC. In DAC-treated patients, NKG2DL expression is significantly higher at the end of cycle 1 (day 28) compared with day 4 or day 11 of treatment. (A-E) Expression of

ULBP-1, ULBP-2, ULBP-3, MICA, and MICB (*P5 .0069, **P5 .0016, ***P5 .0101, n.s. P5 .0547, ****P5 .03). Upregulation of NKG2DL during DAC treatment was noted;

expression of DNAM ligands (PVR and Nectin-2) was not observed.





(Figure 7A) with ULBP-3 blocking antibody resulted in reduced BI836858–mediatedADCCcomparable topre-DACtreatment (Figure7B).

Discussion

Our studies showpharmacological activity of the novel, Fc-engineered,CD33 mAb BI 836858 against primary AML blasts, supporting theclinical application of this antibody in AML. BI 836858 inducesNK-cell–mediated ADCC that is higher than that of HuM195, apreviously developed, non–Fc-engineered anti-CD33 antibody.CD33 is an extensively studied candidate antigen that can serve asa therapeutic target for antibodies due to its frequent expression onAML blasts.4,28 Clinical trials have confirmed the validity of CD33as a viable target for the treatment of AML.29 The antibody-drugconjugate GO has shown efficacy in older AML patients. In additionto GO, another agent in development was lintuzumab (HuM195), ahumanized mAb (IgG1) directed against CD33.30 HuM195 showedsigns of clinical efficacy in phase 1 trials.11 However, a randomizedmulticenter phase 3 trial showed that, although safe, the addition ofHuM195 to salvage chemotherapy did not result in a statisticallysignificant improvement in response rate or survival in patients withrefractory/relapsed AML.12

ADCC is recognized as an important factor in the efficacy of othertherapeutic mAbs such as obinutuzumab, recently approved for use inchronic lymphocytic leukemia.31 CD33 is a rapidly internalizing cellsurface antigen,7 which may result in suboptimal ADCC activity ofmAbs that bind it. An NK-engaging mAb is likely to provide superiorADCC if its internalization when bound to the target is minimal.BI 836858 was developed as an Fc-engineered mAb with en-hanced affinity to human FcgRIIIa, leading to significantly higherNK-cell–mediated ADCC on cell lines and primary AML cellscompared with HuM195. Initial internalization experiments haveshown that BI 836858 and BI 836854, the non–Fc-engineeredversion of BI 836858, do not differ in their internalization behavior,resulting in comparable CD33 levels retained over time on the cellsurface (not shown). It is noteworthy, however, that BI 836858causes less reduction of cell surface CD33 than HuM195, whichbinds to a different epitope of CD33. This may contribute to theobserved differences in internalization kinetics, resulting in higherlevels of CD33 retained on the cell surface after incubation withBI 836858 compared with HuM195, which may further enhancethe ADCC efficacy of BI 836858 when administered in vivo. Here,we demonstrate the ability of BI 836858 to induce NK-cell acti-vation and potent NK-cell–mediated ADCC against AML primaryblasts even at low E:T ratios, underscoring its potential for treatmentas a single agent in AML.

Day 1

Day 4

Day 2

8

Day 1

Day 4

Day 2

8

Day 1

Day 4

Day 2

8

% c

ytot

oxic

ity**

–20

20

40

60

0

BI836858 BI836847 no Ab

E:T 25:1

Day 1

Day 4

Day 2

8

Day 1

Day 4

Day 2

8

Day 1

Day 4

Day 2

8

** E:T 12:1

% c

ytot

oxic

ity

–20

20

40

60

0

BI836858 BI836847 no Ab

E:T 6:1

Day 1

Day 4

Day 2

8

Day 1

Day 4

Day 2

8

Day 1

Day 4

Day 2

8

% c

ytot

oxic

ity

**

0

20

40

60

BI836858 BI836847 no Ab

No effectors

Day 1

Day 4

Day 2

8

Day 1

Day 4

Day 2

8

Day 1

Day 4

Day 2

8

% c

ytot

oxic

ity

0

20

40

60

BI836858 BI836847 no Ab

A B

C D

Figure 6. Higher BI 836858–mediated ADCC in day 28 post-DAC blasts compared with pre-DAC samples. Summary of ADCC data from 7 patients. As effectors, NK

cells from healthy donors were used. Negative controls are no antibody (no Ab) and isotype-matched antibody BI 836847 (BI47). Different E:T ratios are shown with 25:1 (A),

12:1 (B), 6:1 (C), and no effectors (D).





In addition to antibody-based immunotherapy, hypomethylat-ing agents have shown efficacy in AML. The cytidine nucleosideanalogs DAC and 5-azacitidine are putative epigenetic modula-tors and their role in AML has been extensively studied.32-34

Besides DNA-hypomethylating and apoptosis-inducing activi-ties that may contribute to clinical response, some studies sug-gest that DAC and 5-azacytidine have immunomodulatoryactivity.35,36 In addition to effects on NK cells, DAC enhancesupregulation of ligands that increase susceptibility to NK-mediatedADCC. To elucidate the effect of these 2 drugs in the setting ofBI 836858–based immunotherapy, we performed experiments inwhich either the AML blasts (target cells), NK cells (effector cells),or targets and effectors together were pretreated with these 2 drugsand BI 836858–mediated ADCC activity and NK-cell activa-tion were evaluated. AML primary cells exposed to DAC or5-azacytidine exposure remain sensitive to BI 836858–induced,NK-cell–dependent ADCC. In patients that received DAC, weconsistently noted higher BI 836858–mediated ADCC at day 28after start of DAC treatment compared with pre-DAC and this timepoint coincided with upregulation of NKG2D ligand(s) in primaryleukemia samples from DAC-treated patients. We sought toevaluate the mechanism by which higher ADCC was observedpost-DAC compared with pre-DAC. Although short-term in-cubation of AML blasts with DAC in vitro did not modulateNKG2DL expression (data not shown), upregulation of thesemolecules in 28-day post-DAC samples from in vivo–treated AMLpatients suggested NKG2DL expression may be epigenetically

modulated and may take longer than what can be observed inshort-term cultures ex vivo. In order to prove there was a cause-and-effect relationship, we performed blocking experiments andshow that antibodies blocking binding of NKG2DL receptordecreased BI 836858–mediated ADCC. This suggests a biologicalrationale to combine BI 836858 with DAC or 5-azacytidine intreatment of AML patients; a clinical trial evaluating the com-bination of DAC and BI 836858 is expected to begin accrualthis year (Clinicaltrials.gov; NCT 02632721). These findings mayhave implications in the combination of Fc-engineered mAbsand hypomethylating agents or NK-cell–based therapies in themanagement of AML.

Oneof the limitations ofour studyhas been lackof serumsamples tomeasure soluble NKG2DL. Although blasts express NKG2DL, theyare also known to shed these factors as a mechanism to evade immunesurveillance.37 Furthermore, studies are necessary to measure solubleNKG2DL in patients receivingDAC to evaluatewhether shedding alsois increasedwithDAC.Another limitation of the study is the paucity ofavailable autologousNKcells and availability of blasts todetect surfaceexpression of NKG2DL. Recent publications point to a qualitativedefect in NK cells obtained from AML patients; an evaluation of 15patients showed that NK cells at diagnosis show downregulation ofactivating receptors, upregulation of inhibitory receptors, reduceddegranulation as evidenced by CD107a expression, and reducedcytotoxicity against autologous blasts.16 In 12 of the 15 patients whoachieved remission, functionality of NK cells was restored, suggestingthat AML blasts exert inhibitory effects on NK-cell function. Another

MICA MICB ULBP-1 ULBP-3ULBP-2Co

unt

A

B

0

80

100 101 102 103

% c

ytot

oxic

ity

–10

None

Isotype

ULBP-3

Block Day 1 Day 28

–

–

–

–

+

–

–

–

+

–

–

–

–

+

–

–

–

+

0

10

20

30

40

50

Coun

t0

80

100 101 102 103

Coun

t

0

10

20

30

40

50

100 101 102 103

Coun

t

0

20

40

60

100 101 102 103

Coun

t

0

20

40

100 101 102 103

Figure 7. Blocking of NKG2DL receptor shows decrease in BI 836858 ADCC in post-DAC samples. (A) Surface expression of NKG2DL in patients who received DAC.

The dark histograms show expression of MICA, ULBP-1, ULBP-2, and ULBP-3 at baseline, and the gray histograms show expression on day 28 post-DAC. (B) Blocking of

surface expression of NKG2DL shows decrease in BI 836858–mediated ADCC in post-DAC samples. Surface expression of ULBP-3 was blocked in day 1 and day 28

samples. BI 836858–mediated ADCC was lower is samples where ULBP-3 was blocked.



http://Clinicaltrials.gov



limitation of this study is that these experiments were performedin unfractionatedmarrow samples.CD34 selection ofmarrow samplesmay have altered the findings; the low cell numbers precludedperforming experiments on CD34-sorted cells. We show that DACincreases transcriptional induction ofNKG2DL; limited availability ofsamples from patients who received DAC precluded elaborateanalyses of protein and surface expression of NKG2DL. Detailedprospective, phenotypic analyses of NK cells and AML blasts inpatients receiving hypomethylating agentswouldbe useful to assessthe role of hypomethylating agents in modulating NK-cell functionand transcriptional regulation of NKG2DL.

In summary, we show that the novel, Fc-engineered, CD33 anti-body BI 836858 has activity against primary AML blasts. We alsoshow that DAC pretreatment renders the blasts more susceptible toBI 836858 and NK-mediated ADCC. These findings provide astrong rationale for the use of BI 836858 for AML treatment eitheras a single agent or in combination with the standard of care usingDAC or 5-azacytidine.

Acknowledgments

This work was supported in part by grants from the NationalInstitutes of Health National Cancer Institute (CA135332,CA095426, and CA068458), Pelotonia, the intramural researchprogram of the Ohio State Comprehensive Cancer Center (IRP46050-501876) and the Lauber AMLResearch Fund. The authorsgratefully acknowledge The Ohio State University Leukemia

Tissue Bank Shared Resource for providing patient samples(P30CA016058).

Authorship

Contribution: S.V. and S.H. are co-first authors of the manuscript andequally contributed to the study’s conception, design, and analysis,interpretation of data, and writing of the manuscript; C.C., B.G.,and R.M. contributed to the design of the studies, and datacollection and interpretation; V.G. provided cell lines; X.M. helpedwith statistical analysis and data interpretation; G.L., S.P.W., J.Y.,M.A.C., W.B., and G.M. contributed to the study’s conception;K.-H.H., R.K., C.K., P.J.A., B.R., and E.B. contributed to studydesign; D.B. and D.M.L. provided access to samples and helped inwriting themanuscript; N.M. contributed to the study’s conception,design, and analysis, interpretation of data, writing of the manuscript,overall study supervision, and funding; and all authors reviewed themanuscript.

Conflict-of-interest disclosure: R.K., C.K., P.J.A., K.-H.H.,and E.B. are employees of Boehringer Ingelheim RCV, Vienna,Austria. B.R. is an employee of Boehringer Ingelheim PharmaGmbH, Biberach/Riss, Germany. The remaining authors declareno competing financial interests.

Correspondence: Natarajan Muthusamy, Division of Hematology,Department of Internal Medicine, The Ohio State University Compre-hensive Cancer Center, 455E, 410 West 12th Ave, Columbus, OH43210; e-mail: [email protected].

References

1. Oran B, Weisdorf DJ. Survival for older patientswith acute myeloid leukemia: a population-basedstudy. Haematologica. 2012;97(12):1916-1924.

2. Siegel R, Naishadham D, Jemal A. Cancerstatistics, 2013. CA Cancer J Clin. 2013;63(1):11-30.

3. Estey E. Acute myeloid leukemia andmyelodysplastic syndromes in older patients.J Clin Oncol. 2007;25(14):1908-1915.

4. Dinndorf PA, Andrews RG, Benjamin D, RidgwayD, Wolff L, Bernstein ID. Expression of normalmyeloid-associated antigens by acute leukemiacells. Blood. 1986;67(4):1048-1053.

5. Bernstein ID. Monoclonal antibodies to themyeloid stem cells: therapeutic implications ofCMA-676, a humanized anti-CD33 antibodycalicheamicin conjugate. Leukemia. 2000;14(3):474-475.

6. Linenberger ML. CD33-directed therapy withgemtuzumab ozogamicin in acute myeloidleukemia: progress in understanding cytotoxicityand potential mechanisms of drug resistance.Leukemia. 2005;19(2):176-182.

7. Walter RB, Appelbaum FR, Estey EH, BernsteinID. Acute myeloid leukemia stem cells and CD33-targeted immunotherapy. Blood. 2012;119(26):6198-6208.

8. Petersdorf SH, Kopecky KJ, Slovak M, et al.A phase 3 study of gemtuzumab ozogamicinduring induction and postconsolidation therapy inyounger patients with acute myeloid leukemia.Blood. 2013;121(24):4854-4860.

9. Burnett AK, Russell NH, Hills RK, et al. Additionof gemtuzumab ozogamicin to inductionchemotherapy improves survival in older patientswith acute myeloid leukemia. J Clin Oncol. 2012;30(32):3924-3931.

10. Hills RK, Castaigne S, Appelbaum FR, et al.Addition of gemtuzumab ozogamicin to inductionchemotherapy in adult patients with acute myeloidleukaemia: a meta-analysis of individual patientdata from randomised controlled trials. LancetOncol. 2014;15(9):986-996.

11. Raza A, Jurcic JG, Roboz GJ, et al. Completeremissions observed in acute myeloid leukemiafollowing prolonged exposure to lintuzumab:a phase 1 trial. Leuk Lymphoma. 2009;50(8):1336-1344.

12. Feldman EJ, Brandwein J, Stone R, et al. PhaseIII randomized multicenter study of a humanizedanti-CD33 monoclonal antibody, lintuzumab,in combination with chemotherapy, versuschemotherapy alone in patients with refractory orfirst-relapsed acute myeloid leukemia. J ClinOncol. 2005;23(18):4110-4116.

13. Romain G, Senyukov V, Rey-Villamizar N, et al.Antibody Fc engineering improves frequencyand promotes kinetic boosting of serial killingmediated by NK cells. Blood. 2014;124(22):3241-3249.

14. Gleason MK, Ross JA, Warlick ED, et al.CD16xCD33 bispecific killer cell engager (BiKE)activates NK cells against primary MDS andMDSC CD331 targets. Blood. 2014;123(19):3016-3026.

15. Chretien AS, Le Roy A, Vey N, et al. Cancer-induced alterations of NK-mediated targetrecognition: current and investigationalpharmacological strategies aiming at restoringNK-mediated anti-tumor activity. Front Immunol.2014;5:122.

16. Stringaris K, Sekine T, Khoder A, et al. Leukemia-induced phenotypic and functional defects innatural killer cells predict failure to achieveremission in acute myeloid leukemia.Haematologica. 2014;99(5):836-847.

17. Rohner A, Langenkamp U, Siegler U, Kalberer

CP, Wodnar-Filipowicz A. Differentiation-promoting drugs up-regulate NKG2D ligandexpression and enhance the susceptibility ofacute myeloid leukemia cells to natural killercell-mediated lysis. Leuk Res. 2007;31(10):1393-1402.

18. Tang KF, He CX, Zeng GL, et al. Induction ofMHC class I-related chain B (MICB) by 5-aza-29-deoxycytidine. Biochem Biophys Res Commun.2008;370(4):578-583.

19. Lonberg N, Taylor LD, Harding FA, et al. Antigen-specific human antibodies from mice comprisingfour distinct genetic modifications. Nature. 1994;368(6474):856-859.

20. Heider KH, Kiefer K, Zenz T, et al. A novel Fc-engineered monoclonal antibody to CD37 withenhanced ADCC and high proapoptotic activity fortreatment of B-cell malignancies. Blood. 2011;118(15):4159-4168.

21. Kossman SE, Scheinberg DA, Jurcic JG, JimenezJ, Caron PC. A phase I trial of humanizedmonoclonal antibody HuM195 (anti-CD33) withlow-dose interleukin 2 in acute myelogenousleukemia. Clin Cancer Res. 1999;5(10):2748-2755.

22. Alter G, Malenfant JM, Altfeld M. CD107a as afunctional marker for the identification of naturalkiller cell activity. J Immunol Methods. 2004;294(1-2):15-22.

23. Groh V, Wu J, Yee C, Spies T. Tumour-derivedsoluble MIC ligands impair expression of NKG2Dand T-cell activation. Nature. 2002;419(6908):734-738.

24. Geert V, Molenberghs G. Linear Mixed Models forLongitudinal Data. New York, NY: Springer; 2000.

25. Hsu J. Multiple Comparisons: Theory andMethods. Boca Raton, FL: CRC Press; 1996.



mailto:[email protected]



26. Zhuravleva MA, Trandem K, Sun PD. Structuralimplications of Siglec-5-mediated sialoglycanrecognition. J Mol Biol. 2008;375(2):437-447.

27. Buhring HJ, Sures I, Jallal B, et al. The receptortyrosine kinase p185HER2 is expressed on asubset of B-lymphoid blasts from patients withacute lymphoblastic leukemia and chronicmyelogenous leukemia. Blood. 1995;86(5):1916-1923.

28. Peiper SC, Ashmun RA, Look AT. Molecularcloning, expression, and chromosomallocalization of a human gene encoding the CD33myeloid differentiation antigen. Blood. 1988;72(1):314-321.

29. Jurcic JG. What happened to anti-CD33 therapyfor acute myeloid leukemia? Curr Hematol MaligRep. 2012;7(1):65-73.

30. Sutherland MK, Yu C, Lewis TS, et al. Anti-leukemic activity of lintuzumab (SGN-33) in

preclinical models of acute myeloid leukemia.MAbs. 2009;1(5):481-490.

31. Rogers KA, Jones JA. Obinutuzumab for thetreatment of chronic lymphocytic leukemia. DrugsToday (Barc). 2014;50(6):407-419.

32. Fenaux P, Mufti GJ, Hellstrom-Lindberg E, et al.Azacitidine prolongs overall survival comparedwith conventional care regimens in elderlypatients with low bone marrow blast count acutemyeloid leukemia. J Clin Oncol. 2010;28(4):562-569.

33. Kantarjian HM, Thomas XG, Dmoszynska A,et al. Multicenter, randomized, open-label,phase III trial of decitabine versus patientchoice, with physician advice, of eithersupportive care or low-dose cytarabine for thetreatment of older patients with newly diagnosedacute myeloid leukemia. J Clin Oncol. 2012;30(21):2670-2677.

34. Quintas-Cardama A, Ravandi F, Liu-Dumlao T,et al. Epigenetic therapy is associated with similarsurvival compared with intensive chemotherapy inolder patients with newly diagnosed acute myeloidleukemia. Blood. 2012;120(24):4840-4845.

35. Schmiedel BJ, Arelin V, Gruenebach F, Krusch M,Schmidt SM, Salih HR. Azacytidine impairs NKcell reactivity while decitabine augments NK cellresponsiveness toward stimulation. Int J Cancer.2011;128(12):2911-2922.

36. Kopp LM, Ray A, Denman CJ, et al. Decitabinehas a biphasic effect on natural killer cell viability,phenotype, and function under proliferativeconditions. Mol Immunol. 2013;54(3-4):296-301.

37. Hilpert J, Grosse-Hovest L, Grunebach F, et al.Comprehensive analysis of NKG2D ligandexpression and release in leukemia: implicationsfor NKG2D-mediated NK cell responses.J Immunol. 2012;189(3):1360-1371.





online March 24, 2016 originally publisheddoi:10.1182/blood-2015-11-680546

2016 127: 2879-2889

Borges, Bjoern Rueter, Karl-Heinz Heider, Guido Marcucci and Natarajan MuthusamyDonna Bucci, David M. Lucas, Jianhua Yu, Michael A. Caligiuri, William Blum, Paul J. Adam, EricLozanski, Xiaokui Mo, Veronica Groh, Susan P. Whitman, Renate Konopitzky, Christian Kössl, Sumithira Vasu, Shun He, Carolyn Cheney, Bhavani Gopalakrishnan, Rajeswaran Mani, Gerard mediated natural killer ADCC against AML blasts

−Decitabine enhances anti-CD33 monoclonal antibody BI 836858

http://www.bloodjournal.org/content/127/23/2879.full.htmlUpdated information and services can be found at:

(1651 articles)Myeloid Neoplasia Articles on similar topics can be found in the following Blood collections

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

Copyright 2011 by The American Society of Hematology; all rights reserved.of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society


http://www.bloodjournal.org/content/127/23/2879.full.html

http://www.bloodjournal.org/cgi/collection/myeloid_neoplasia

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests

http://www.bloodjournal.org/site/misc/rights.xhtml#reprints

http://www.bloodjournal.org/site/subscriptions/index.xhtml





decitabine enhances anti-cd33 monoclonal antibody bi … · 2017-04-13 · regular article myeloid...

Documents