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`JOURNAL OF
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`HEMATOLOGY
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`Volt ey
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`4 SEPTEMBER 2009
`
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`'ansplantation for
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`PGR2019-00002
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`

`blood
`
`JOURNAL OF
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`blood
`
`JOURNAL OF
`THE AMERICAN
`
`SDEEDUBF
`HEMATOLOGY
`
`24 SEPTEMBER 2009 - VOLUME 114, NUMBER 13
`
`CONTENTS
`
`COVER FIGURE
`
`It is unclear howneutrophils control Aspergillus species in healthy persons. Due to
`their large size, Aspergillus hyphae(red) cannot be phagocytosed by neutrophils
`(green), Bianchi et al showinthis issue that recently discovered NADPH
`oxidase-dependent microbicidal pathways through neutrophil extracellular traps
`(NETs) areefficient against Aspergillus conidia and hyphae in vitro, and that
`restoration of NET formation by gene therapy of NADPHoxidase—deficient
`X-linked chronic granulomatous disease aided clearing severe invasive Aspergillus
`nidulansinfection in vivo, underlining the role of functional NADPH oxidase in NET
`formation andantifungal activity. See the article by Bianchi et al on page 2619.
`
`INSIDE BLOOD
`
`
`2567
`
`NK antibody therapy: KIR-ative intent
`N. Shah and E. J. Shpall
`
`2568
`
`‘Tax-ing” the cancerstemcell
`G, Feuer
`
`2569
`
`~Missteps in “tango” for epigenometargeting
`K. D. Robertson and K. N. Bhalla
`
`2570
`
`HIV:getting to the heart of DARCness
`R. J.B. Nibbs
`
`2571
`
`‘Platelet integrin signaling: wherefore art thou?
`A.A. Schmaier and M. L.. Kahn
`
`2572
`
`~Superfactor B-gets atypical HUS
`E. M. Conway
`
`PERSPECTIVE
`2575 Defining prior therapy in myelodysplastic syndromes andcriteria for relapsed and
`refractory disease: implications for clinical trial design and enrollment
`M. A. Sekeres and D. P. Steensma
`
`REVIEW ARTICLE
`
`2581
`
`Allogeneic hematopoietic cell transplantation for chronic lymphocytic leukemia:
`readyfor prime time?
`J. Delgado, D. W. Milligan, and P. Dreger
`
`CLINICAL TRIALS AND
`OBSERVATIONS
`2589=Detailed analysis of p53 pathwaydefects in fludarabine-refractory chronic
`lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53
`mutation, p53-p21 dysfunction, and miR34ain a prospective clinicaltrial
`T. Zeng, S. Hiibe. T. Denzel. J. Mohr, D. Winkler, A. Biihler, A. Sarno. S. Groner, D. Mertens,
`
`R. Busch, M. Hallek, H. Doéhner, and S. Stilgenbauer
`
`ix
`
`BLOOD, 24 SEPTEMBER 2009 - VOLUME 114, NUMBERfis material was copied
`at the NLM and may be
`Subject US Copyright Laws
`
`CONTINUED ONx
`
`

`

`2598 Determining therise inBCR-ABL RNAthat optimally predicts a kinase domain
`mutation in patients with chronic myeloid leukemia on imatinib
`R. D. Press. 8. G. Willis, J. Laudadio, M. J. Mauro, and M, W. N. Deininger
`
`2606
`
`2617
`
`Donor, recipient, and transplant characteristics as risk factors after unrelated donor
`PBSC transplantation: beneficial effects of higher CD34* cell dose
`M.A. Pulsipher, P. Chitphakdithai, B. R. Logan, 8. F. Leitman, P. Anderlini, J. P. Klein,
`M. M. Horowitz, J. P. Miller, R. J. King, and D. L, Confer
`
`Brief report The importance of bone marrow examinationin determining
`complete response to therapyin patients with multiple myeloma
`C.E. Chee. S. Kumar, D. R. Larson, R.A. Kyle, A. Dispenzieri, M. A. Gertz, C. L. Colby, and
`S. V. Rajkumar
`
`GENE THERAPY
`
`
`=
`_
`
`2619
`
`Brief report Restoration of NET formation by gene therapy in CGD controls
`aspergillosis
`M. Bianchi, A. Hakkim, V. Brinkmann, U, Siler, R. A. Seger, A. Zychlinsky, and J, Reichenbach
`
`IMMUNOBIOLOGY
`
`
`
`2623
`
`Dynamic accumulation of plasmacytoid dendritic cells in lymph nodes is regulated
`byinterferon-[
`Y. Gao, B. Majchrzak-Kita, E. N. Fish, and J. L. Gommerman
`
`2632 Mesenchymal stromalcells cross-present soluble exogenous antigens as part of their
`antigen-presenting cell properties
`M. Frangois, R. Romieu-Mourez, 8. Stock-Martineau, M.-N. Boivin, J. L. Bramson, and
`J. Galipeau
`
`2639 Mast cells counteract regulatory T-cell suppression through interleukin-6 and
`OX40/OX40L axis toward Th17-cell differentiation
`S. Piconese, G. Gri, C. Tripodo, S. Musio, A. Gorzanelli, B. Frossi, R. Pedotti, C. E. Pucille. and
`M. P. Colombo
`
`2649
`
`Pneumoviruses infect eosinophils andelicit MyD88-dependent release of
`chemoattractant cytokines and interleukin-6
`K. D. Dyer, C. M. Percopo, E. R. Fischer, 5. J. Gabryszewski, and H. F. Rosenberg
`
`2657) Minimal requirement for induction of natural cytotoxicity and intersection of
`activation signals by inhibitory receptors
`Y. T. Bryceson, H.-G, Ljungeyren, and E, O. Long
`
`2667~—Preclinical characterization of 1-7F9, a novel human anti-KIR receptor therapeutic
`antibody that augments natural killer-mediated killing of tumorcells
`F. Romagneé, P. André, P. Spee, S. Zahn, N. Antossi, L. Gauthier, M. Capanni, L. Ruggeri,
`D, M. Benson Jr, B. W. Blaser, M.D. Chiesa, A. Moretta, E. Vivier, M. A. Caligiuri, A. Velardi.
`and N. Wagtmann
`
`x
`
`BLOOD,24 SEPTEMBER 2009 - VOLUME114, NUMBERS material was copied
`atthe NLM and may be
`Subject US Copyright Laws
`
`CONTINUEDONxi
`
`

`

`LYMPHOID
`NEOPLASIA
`
`2678
`
`Merreceptor tyrosine kinaseis a novel therapeutic target in pediatric B-cell acute
`lymphoblastic leukemia
`R. M.A. Linger, D. DeRyckere, L. Brandio, K. K. Sawezyn, K. M. Jacobsen, X, Liang,
`A. K. Keating, and D, K. Graham
`
`2688
`
`Deregulated expression of cytokine receptor gene, CRLF2,is involved in lymphoid
`transformation in B-cell precursor acute lymphoblastic leukemia
`L. J. Russell, M. Capasso, |. Vater, T. Akasaka, O. A. Bernard, M. J. Calasanz,
`T. Chandrasekaran, E. Chapiro, S. Gesk, M. Griffiths, D.S. Guttery, C. Haterlach, L. Harder,
`O. Heidenreich, J. Irving, L. Kearney, F. Neuyen-Khac, L. Machado, L. Minto, A. Majid,
`A. V. Moorman, H. Morrison, V. Rand, J. C. Strefford, C. Schwab, H. Tonnies, M. J. S. Dyer,
`R. Siebert, and C. J. Harrison
`
`2699
`AurorakinaseAis a target of Wnt/B-catenin involved in multiple myelomadisease
`progression
`J, Dutta-Simmons, Y. Zhang, G. Gorgun, M. Gatt, M. Mani, T. Hideshima, K. Takada,
`N. E. Carlson, D. E, Carrasco, YT. Tai, N. Raje, A. G. Letai, K. C. Anderson, and
`D. R, Carrasco
`
`2709
`
`2721
`
`2730
`
`Identification of cancer stemcells in a Tax-transgenic (Tax-Tg) mouse model of adult
`T-cell leukemia/lymphoma
`J. Yamavaki, T. Mizukami, K. Takizawa, M. Kuramitsu, He Momose, A. Masumi. Y. Ami,
`H. Hasegawa, W. W. Hall, H. Tsujimoto, 1 Hamaguchi. and K. Yamaguchi
`
`Therapeutic potential of an anti-CD79b antibody—drug conjugate, anti-CD79b-ye-
`MMAE,for the treatment of non-Hodgkin lymphoma
`D. Dornan, F. Bennett, Y, Chen. M. Dennis, D. Eaton, K. Elkins, D. French. M. A, T. Go,
`A. Jack, J. R. Junutula, H. Koeppen, J. Lau, J. MeBride, A. Rawstron, X. Shi, N. Yu. S.-F. Yu,
`P. Yue, B. Zheng. A. Ebens, and A. G. Polson
`
`Brief report A prospective study of serum soluble CD30 concentration and risk of
`non-Hodgkin lymphoma
`M. P. Purdue, Q. Lan, O. Martinez-Maza, M. M. Oken, W. Hocking, W.-Y. Huang, D. Baris,
`B. Conde, and N. Rothman
`
`MYELOID
`NEOPLASIA
`
`
`
`2733
`
`2744
`
`Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor
`3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against
`human AML cells
`W. Fiskus, Y. Wang, A. Sreekumar, K. M. Buckley. H. Shi, A. Jillella, C. Ustun, R. Rao,
`P. Fernandes, J. Chen, R. Balusu. S. Koul, P. Atadja, V. E. Marquez, and K.N. Bhalla
`
`RUNX1I regulates phosphoinositide 3-kinase/AKTpathway: role in chemotherapy
`sensitivity in acute megakaryocytic leukemia
`H. Edwards, C. Xie, K. M. LaFiura, A. A. Dombkowski. S.A. Buck, J. L. Boerner, J. W. Taub,
`
`LH. Matherly, and ¥. Ge
`
`Mutational spectrum at GATAL provides insights into mutagenesis and
`leukemogenesis in Down syndrome
`D.C. Cabelol, H. V. Patel, Q. Chen. H. van Remmen, L. Hl. Matherly, Y, Ge, and J. W. Taub
`
`x
`
`BLOOD,24 SEPTEMBER 2009 + VOLUME 114, NUMBER}. material wascopied
`atthe NLM and may be
`Subject US Copyright Laws
`
`CONTINUEDONxii
`
`

`

`2764
`
`Early epigenetic changes and DNA damagedonot predict clinical response in an
`overlapping schedule of 5-azacytidine and entinostat in patients with myeloid
`malignancies
`TLE. Pandy, J, G. Herman, P. Kerns, A. Jiemjit, E. A. Sugar, S.-H. Choi, A. S. Yang, T. Aucott,
`T. Dauses, R. Odchimar-Reissig, J. Licht, M. J. MeConnell, C. Nasrallah, M. K. H. Kim,
`W. Zhang, Y. Sun, A. Murgo, 1. Espinoza-Delgado, K. Oteiza, 1. Owoeye, L. R. Silverman,
`S. D. Gore, and H. E. Carraway
`
`PHAGOCYTES,
`GRANULOCYTES, AND
`MYELOPOIESIS
`
`
`2774
`
`Eosinophil viability is increased by acidic pH in a cAMP- and GPR65-dependent
`manner
`
`L. C. Kottyan, A. R. Collier, K. H. Cao, K. A. Niese, M. Hedgebeth, C. G. Radu, O. N. Wille,
`G. K. Khurana Hershey, M. E. Rothenberg, and N. Zimmermann
`
`2783
`
`The Duffy-null state is associated with a survival advantage in leukopenic HIV-
`infected persons of African ancestry
`H. Kulkarni, V. C. Marconi, W. He, M. L. Landrum, J. FP. Okulicz. J. Delmar, D. Kazandjian,
`J. Castiblanco, 8. S. Ahuja, E. J. Wright, R. A. Weiss, R. A. Clark, M. J, Dolan, and S. K. Ahuja
`
`PLATELETS AND
`THROMBOPOIESIS
`n704
`
`
`
`Distinct spatio-temporal Ca?* signaling elicited by integrin w2{1 and glycoprotein
`Vi under flow
`
`M, Mazzucato, M. R, Cozzi, M. Battiston, M. Jandrot-Perrus, M. Mongiat, P. Marchese,
`TO J. Kunicki, 2, M. Ruggeri, and L. De Marco
`
`
`THROMBOSIS AND _
`HEMOSTASIS a
`
`Enhancedefficacy of recombinant FVII in noncovalent complex with PEGylated
`liposome in hemophilia A mice
`J. Pan. Tf Liu, J-¥. Kim, D, Zhu, C, Patel, Z.-H. Cui, X. Zhang, J. O. Newgren, A, Reames,
`D. Canivel. G. Jesmok, G. F. Pierce, J, M. Sommer, and H, Jiang
`
`2812
`
`2819
`
`Thrombin induces the expression of oncostatin M via AP-1 activation in human
`macrophages: a link between coagulation and inflammation
`S.P. Kastl, W. 5. Speidl, K.M. Katsaros, C. Kaun, G. Rega, A. Assadian, G. W. Hagmueller,
`M. Hoeth. R. de Martin, Y. Ma, G. Maurer, K. Huber, and J. Wojta
`
`A novel binding site for ADAMTS1I3 constitutively exposed on the surface of
`globular VWF
`S. Zanardelli, A.C. K. Chion, E. Groot, P. J. Lenting, T. A.J. McKinnon, M.A, Laffan,
`M. Tseng, and D, A. Lane
`
`TRANSPLANTATION
`
`
`2829
`
`Selective depletionofalloreactive Tcells by targeted therapy of heat shock protein
`90: a novel strategyfor control of graft-versus-host disease
`C. Stuehler, S. Mielke, M. Chatterjee, J. Duell, S. Lurati, F Rueckert, H. Einsele. R. C. Bargou,
`and M.S. Topp
`
`xii
`
`BLOOD, 24 SEPTEMBER 2009 - VOLUME 114, NUMBER Ris mmaterialwascophed
`atthe NLM and may be
`Subject US Copyright Laws
`
`CONTINUEDONxiv
`
`

`

`
`
` VASCULAR BIOLOGY —
`2837 Hyperfunctional C3 convertase leads to complement deposition on endothelial cells
`andcontributes to atypical hemolytic uremic syndrome
`L. T. Roumenina, M. Jablonski, C. Hue, J. Blouin, J. D. Dimitroy, M.-A. Dragon-Durey,
`M. Cayla, W. H. Fridman, M.-A. Macher, D. Ribes, L. Moulonguet, L. Rostaing, 8. C. Satchel,
`P.W. Mathieson, C. Sautes-Fridman, C, Loirat, C. H. Regnier, L. Halbwachs-Mecarelli, and
`V. Fremeaux-Bacchi
`
`CORRESPONDENCE
`
`2846~—Role of TNFRSF13Bvariants in patients with common variable immunodeficiency
`N. Martinez-Pomar, N. Matamoros, D, Detkova, J. I. Arostegui, A. Alvarez, P. Soler-Palacin, A.
`Vidaller, T. Espanol, A, Sampalo, J. de Gracia, M. Hernandez, J. Yagiie, and N. Matamoros
`
`2848=Identification of monoclonal B-cell lymphocytosis among sibling transplant donors
`for chronic lymphocytic leukemia patients
`I, Del Giudice, PF. R. Mauro, M.S. De Propris, LD. Starza, D. Armiento, A. P. lori, G. Fernando
`Torelli, A. Guarini, and R. Poa
`
`2849=The role of CBFB in AMLI-ETO’s activity
`S. Park, NA. Speck, and J. H. Bushweller
`
`ERRATA
`
`2851
`Ambrogioet al. p130Cas mediates the transforming properties of the anaplastic
`lymphoma kinase. Blood. 2005; 106(12):3907-39 16.
`
`2851~—Nolte-’t Hoenet al. Activated T cells recruit exosomes secreted by dendritic cells via
`LFA-L. Blood. 2009;113(9):1977-1981.
`
`2852=Tai et al. CST promotes multiple myelomacell adhesion, clonogenic growth, and
`tumorigenicity via c-maf—mediated interactions with bone marrow stromal cells.
`Blood. 2009;113(18):4309-43 18,
`
`OTHER DEPARTMENTS
`
`xxiii Author Guide
`
`iib
`
`Classifieds
`
`SUBMISSION INSTRUCTIONS
`
`All manuscripts, including figures, should be submittedelectronicallyat
`hutp://submit.bloodjournal.org to Editor-in-Chief Cynthia Dunbar, MD. Before submitting your
`paper, review Blood '’s Author Guide at http://www.bloodjournal.org. If you need help during the
`submission process, contact the Editorial Office by phone at 202-776-0548 or via e-mail at
`editorial@hematology.org.
`
`xiv
`
`BLOOD, 24 SEPTEMBER 2009 - VOLUME114, NUMBER Tis material was copied
`atthe NLM and may be
`Subject US Copyright Laws
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`

`

` This material may be protected by Copyright law (Title 17 U.S. Code)
`
`IMMUNOBIOLOGY
`
`Preclinical characterization of 1-7F9, a novel human anti-KIR receptor
`therapeutic antibody that augmentsnatural killer—mediated killing of tumorcells
`Francois Romagné,' Pascale André,' Pieter Spee,* Stefan Zahn,? Nicolas Anfossi,' Laurent Gauthier,’ Marusca Capanni,?
`Loredana Ruggeri,? Don M. Benson Jr,‘ Bradley W.Blaser,’ Mariella Della Chiesa,° Alessandro Moretta,° Eric Vivier,®
`Michael A. Caligiuri,* Andrea Velardi,? and Nicolai Wagtmann?
`
`‘Innate-Pharma, Marseille, France; *Biopharmaceuticals Research Unit, Novo Nordisk, Maaloev, Denmark; “Department of Clinical and Experimental Medicine,
`University of Perugia, Perugia, Italy; ‘Ohio State University Comprehensive Cancer Center, Columbus; *Dipartimento di Medicina Sperimentale e Centro di
`Eccellenza per le Ricerche Biomediche, Universita degli Studi di Genova, V le Benedetto XV, Genova, Italy; and "Centre d'immunologie de Marseille-Luminy,
`Université de la Méditerranée, Inserm, Centre National de la Recherche Scientifique (CNRS), Marseille, France
`
`Inhibitory-cell killer immunoglobulin-like
`receptors (KIR) negatively regulate natu-
`ral killer (NK) cell-mediated killing of HLA
`class l-expressing tumors. Lack of KIR-
`HLA class | interactions has been associ-
`
`KIR2DL1, -2, and -3 receptors, and pre-
`vents their inhibitory signaling. The 1-7F9
`monoclonal antibody augmented NK cell—
`mediated lysis of HLA-C-expressing tu-
`mor cells,
`including autologous AML
`blasts, but did not inducekilling of nor-
`ated with potent NK-mediated antitumor
`mal peripheral blood mononuclear cells,
`efficacy and increased survival in acute
`suggesting a therapeutic windowfor pref-
`myeloid leukemia (AML) patients upon
`erential enhancement of NK-cell cytotox-
`haploidentical stem cell transplantation
`icity against malignanttargetcells. Admin-
`from KIR-mismatched donors. To exploit
`istration of 1-7F9 to KIR2DL3-transgenic
`this pathway pharmacologically, we gen-
`mice resulted in dose-dependent rejec-
`erated a fully human monoclonal anti-
`tion of HLA-Cw3-positive target cells. In
`body, 1-7F9, which cross-reacts with
`
`Introduction
`
`in
`an immunodeficient mouse model
`which inoculation of human NK cells
`
`alone was unable to protect against le-
`thal, autologous AML, preadministration
`of 1-7F9 resulted in long-term survival.
`These data show that 1-7F9 confers spe-
`cific, stable blockade of KIR, boosting
`NK-mediated killing of HLA-matched AML
`blasts in vitro and in vivo, providing a pre-
`clinical basis for initiating phase 1 clinical
`trials with this candidate therapeutic anti-
`body.(Blood. 2009;114:2667-2677)
`
`Natural killer (NK) cells play critical roles in host defense against
`infections and tumors, by secreting immunoregulatory cytokines
`and by killing infected or transformed cells. The activation of
`NK-cell effector
`functions is
`regulated by multiple types of
`activating andinhibitory receptors that recognizeligands expressed
`on potential
`target cells.'’ The balance between positive and
`negative signals transmitted via these NK receptors determines
`whether or not a target cell
`is killed by an NK cell? Activating
`receptors include NKp30, NKp44, NKp46, NKG2D, and DNAM-1,
`among others.’ Their ligands appear preferentially on stressed,
`transformed, or infected cells, but not on normal, healthy cells.
`Therefore, transformed or infected cells may provide activation
`signals and become sensitive to killing by NK cells, whereas
`healthy cells generally do not? NK-cell expression of CD16, the
`low-affinity Fe receptor, provides an additional mechanism by
`which NK cells may mediate antitumor effects via antibody-
`dependent cellular cytotoxicity (ADCC) toward antibody-coated
`target cells. Signaling through activating NK receptors is nega-
`tively regulated via inhibitory receptors, such as killer immuno-
`globulin (Ig)-like receptors (KIR), CD94/NKG2A, and leukocyte
`Ig-like receptor-], which recognize major histocompatibility
`complex (MHC)-class | molecules. Efficient NK cell-mediated
`killing occurs when target cells abundantly express stress- or
`transformation-inducedligands for activating NK receptors, and
`
`few or no MHCclass | ligands for inhibitory receptors, Cancer
`cells that
`retain expression of class | molecules may evade
`NK-mediated immunosurveillance.” Conversely.
`loss of MHC
`class | expression renders tumor cells more sensitive to killing
`by NK cells. which maybe associated with improved prognosis
`in sometypes of cancer.’
`The human KIR family comprises polymorphic Lg-like mol-
`ecules expressed on NK cells, and small subsets of CD8* and yd‘
`Teells. Individual KIR bind distinct subgroups of HLA class |
`allotypes. and are clonally expressed in NK cells. creating a
`repertoire of NK cells with specificities for different HLA class |
`molecules. KIR2DL and KIRS3DL have long cytoplasmic tails
`containing inhibitory signaling motifs, and 2 or 3 extracellular ly
`domains conferring specificity for HLA-C or HLA-A/Ballotypes,
`respectively. KIR2ZDL1 and KIR2DL2/3 recognize distinet HLA-C
`allotypes, based on polymorphisms at positions 77 and 80 in the ol
`domain of the HLA heavy chain." For example, KIR2DL1 binds
`HLA-Cw2,
`-4,
`-5, and -6, whereas KIR2DL2 and -3 bind to
`
`HLA-Cwl, -3, -7, and -8. Collectively, the inhibitory KIR2DL 1, -2,
`and -3 receptors recognize essentially all HLA-C allotypes.
`In
`contrast to KIR2DL receptors. KIR2DS and KIR3DS have short
`cytoplasmictails with activating potential.!!
`NK cells may coexpress multiple inhibitory KIRs, and/or other
`MHC class [-specific inhibitory receptors with different MHC
`
`
`
`Submitted February 23, 2009; accepted June 15, 2009. Prepublished online as
`Blood First Edition paper, June 24, 2009; DOI 10.1182/blood-2009-02-206532.
`
`An Inside Blood analysis of this article appears atthe front of 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.
`
`The online version ofthis article contains a data supplement.
`
`© 2009 by The American Society of Hernatology
`
`BLOOD, 24 SEPTEMBER 2009 « VOLUME 114, NUMBER13
`
`This material was copied
`at the NLM and may be
`Subject US Copyright Laws
`
`2667
`
`

`

`2668
`
`ROMAGNE etal
`
`BLOOD, 24 SEPTEMBER 2009 - VOLUME 114, NUMBER 13
`
`class [ specificities. Nevertheless, all people appear to also harbor
`some Nk cells that are inhibited by only one MHC class I
`allotype.'* Thus, the NK population as a whole may detect loss of
`even a single HLA class FE allotype, allowing killing of target cells
`deficient
`in only one or a
`few class 1 allotypes.’ The clinical
`relevance of such missing-self recognition has been demonstrated
`in patients with acute myeloid leukemia (AML), in which haploiden-
`tical stem cell transplantation (SCT) led to expansion and activa-
`tion of donor-derived, KIR-HLA class [-mismatched NK cells,
`resulting in NK cell-mediated antileukemia responses associated
`with reduced risk of relapse and increased survival rates, with no
`risk of graft-versus-host disease.'*'’ However, few patients with
`AML are candidates for transplant-based therapy, and thus, novel
`treatment options are urgently needed. To explore the feasibility of
`achieving similar NK-mediated antileukemia activity by a pharma-
`cologic approach that would circumvent
`the need for SCT, we
`generated fully human anui-KIR monoclonal antibodies (mAbs)
`that block the interactions of the 3 main inhibitory KIR2DL with
`their HLA-C ligands, thereby enhancing NK activity. In this study,
`we present the in vilro and in vivo characterization of 1-7F9, which
`formed the basis for selection of this mAb as candidate forclinical
`
`development.
`
`
`Methods
`
`Cell isolation
`
`All patient samples were collected and stored under institutional review
`board-approved protocols. Patients provided prior written informed con-
`sent in accordance with the Declaration of Helsinki, approved by the Ohio
`State University Comprehensive Cancer Center Instituional Review Board
`for use of samples.AML blasts were isolated from bone marrow aspirate at
`diagnosis and eryopreserved. Sample viability was more than 90%, Patients
`received induction and consolidation chemotherapy, and upon complete
`remission, peripheral blood mononuclear cells (PBMC) were obtained by
`Ficoll-Hypaque density gradient centrifugation, which were the source of
`autologous NK cells for functional assays with patient-derived target cells.
`For some experiments. NK cells from these patient samples, or
`from
`healthy donors (HLAclass F high resolution typed), were depleted of Tcells
`by negative CD3 immunomagnetic selection (Miltenyi Biotec), simulated
`with irradiated PBMC feeder cells, phytohemagglutinin (Biochrom), and
`500 U/mL interleukin (1L.)-2 (Chiron), and expanded for up to 2 months.!*
`Purity of NK cells was confirmed by flow cytometry, and was at least 85%
`CbD56'CD3
`lymphocytes in all experiments, NK-cell clones were gener-
`ated from PBMCof healthy donors.!"
`
`IgGl, and
`specitic for KIR2DLI/SL (clones EB6,
`Murine mAbs
`XAI41. IgM), KIR2DL2, -3/S2 (clones GLI83, IgG1, and Y249, IgM),
`KIR2DL1,
`-2. -3/S1,
`-S2,
`-S5 (clone DF200,
`IgGl), KIR2DS4 (clone
`FES172, lgG2a), HLA class [ (clone A6-136, IgM), and NKG2A (clone
`7199,
`IyG2b) have been described.“*"? Phycoerythrin (PE)-conjugated
`anti-KIR antibodies were from Beckman Coulter. Human IgG4 (17385;
`Sigma-Aldrich) was used as control tor 1-7F9,
`
`Flow cytometry
`
`Cell lines. A total of 10° cells was incubated at 4°C with | zg/mL puritied
`human (1-7F9) or murine (EB6, GLES3, and/or FES! 72) anti-KIR mAbs.
`followed by PE-conjugated mouse antichuman [gG4 (HP6025; Southern
`
`Biotechnology Associates) or PE-conjugated goat anti-mouse IgG (Beck-
`man Coulter). Percentages ofanti-KIR2D binding were calculated using the
`following formula: percentage of binding = MFI
`(mean fluorescence
`Se
`Intensity) C./MEBI C
`100, where C, is the anti-KIR2D concentrationin
`micrograms per milliliter (1 g/mL represents 100%).
`NKpopulations,
`“Three-color immunofluorescence was used to ana-
`lyze expression of KIR and NKG2A on CD56" NK cells. NK cells were
`incubated with allophycocyanin-conjugated anti-CD56 (IgG1) plus uncon-
`jugated antitNKG2A (lgG2b), followed by fluorescein isothiocyanate—
`conjugated goat antivmouse IgG2b antibodies (Southern Biotechnology
`Associates), in combination with PE-conjugated anti-KIR2DL2/3/S2 (clone
`GLIS3, lgG1), anti-KIR2DLA/S1 (clone EBOB, lgG1), or anti-KIR3DLI/S1
`(clone 427, [gG1).
`Blood samples were incubated at
`Whole blood staining.
`lemperature with PE-conjugated 1-7F9 and combinations of cell
`specific mAbs, as indicated,
`Binding assays. Fusion proteins consisting ofthe extracellular part of
`KIR2DLI attached to Fe part of murine or human IgGl
`(designated
`KIR2DL1-mFeand -hFe, respectively) were incubated with human (1-7F9)
`or mouse (DF200 or GLI83) anti-KIR mAbs for 30 minutes on ice.
`LCL721.221-Cw4 cells were added and incubated for
`| hour on ice.
`
`room
`type-
`
`washed, and incubated with allophycocyanin-conjugated F(ab')> goat
`anti-human IgG or anti-mouse IgG, and analyzed by flow cytometry.
`CDIO7/1FN-y assay. Cells were processed, as described.“? Brietly,
`NK cells or PBMCwere incubated for 4 hours at 37°C in the presence of 5 jz£M
`monensin (Sigma-Aldrich) and fluorescein isothiocyanate-conjugated anti-
`CD107a and -b mAbs, with or without anti-KIR mAbs, and with or without
`target cells. After incubation, cells were washed in phosphate-buffered
`saline and 2mMEDTA (ethylenediaminetetraacetic acid) and stained for
`extracellular markers, fixed, permeabilized with IntraPrep reagent (Beck-
`man Coulter), labeled with anti-[FN-y, and analyzed by flow cytometry.
`
`Cytotoxicity assays
`
`KIR2D-transfected BWZcell lines.“ YTScells transfected with KIR2DL I+!
`(YTS-2DL1), and 721.221 B-EBYcells transfected with HLA-Cw3and -4
`have been described.““ The 721.221 cells expressing both HLA-Cw3 and
`HLA-Cw4 molecules were venerated by sequential
`transfection of
`pCDNA.3-Cw3 and pRSV.5-Cw4, and sorting for high-level HLA-Cw3
`and -Cw4 expression by flow cytometry of cells stained with soluble
`KIR2DL1 and -3.
`
`Cell lines
`
`Antibodies
`
`Fully human anti-KIR mAbs were venerated by immunization of KM mice
`(Medarex) bearing human genomic IgG loci, with BW5417 cells stably
`transfected with KIR2DL1, followed by 3 booster immunizations with the
`soluble, extracellular part of KIR2DL3 produced in’ Escherichia coli.
`Antibodies were screened for binding to recombinant. soluble KIRs by
`enzyme-linked immunosorbent assay, and positive clones were tested for
`binding 10 YTS-KIR2DL1 cells by flow cytometry. Selected hybridomas
`were subcloned until stable lines were obtained.
`
`Lysis of 721.221-Cw3 and -4 target cells by NK-cell lines or clones was
`evaluated in 4-hour°'Crrelease assays, as described.** Lysis ofAML blasts
`by autologous, IL-2-activated NK cells isolated from the same patient in
`remission Was evaluated by standard *'Cr release assay, or by the Topro-3
`iodide/PKH-26 flow cytometry—based assay.*? In the latter,
`target cells
`labeled with the membrane dye, PKH-26 (Sigma-Aldrich), were coincu-
`bated with NK cells at 37°C tor 4 hours, and then placed on ice. At the
`beginning of incubation,
`|-7F9, control human [gG4 (hlgG4), or a mixture
`of mouse ant-KIR F(ab"): fragments was added, each at 30 jzg/mL final
`concentration. A total of 10 jb of the viability dye To-Pro3 iodide
`(1/10 000 dilution, Molecular Probes) was addedto each well immediately
`before flow cytometric analysis. Percent specific lysis was determined, as
`described.’ Cytotoxicity byfresh, patient-derived NK cells toward autolo-
`gous AML bhists was measured as granzyme B release, using enzyme-
`linked immunospot (ELISPOT) technique, as described.’ Cytotoxicity
`results using this methodology correlate closely with results of chromium
`release andflow cytometric—based methods with the advantage of requiring
`manyfewer cells. Briefly, effector NK cells were incubatedfor 4 hours with
`1-7F9 or hleG4 isotype control, A total of 5 * 10" targ