Blood.
`v. 123, no. 5 (Jan. 30 2014)
`Genera! Collection
`W1 BL661
`2014-02-21 14:52:37
`
`Ve)0)=i P<)
`NUMBER 5
`CUNTNari
`
`ee rncma
`increasedbonejporosity and
`ec MittaollonsLYa
`
`Eosinophils in airways provide
`potentantiviral protection
`(p 609, p 743)
`
`Worldwide anemia prevalence
`contributes to global burden
`of disability (p 611, p 615)
`
`Exosomes from stored
`red cell units are
`immunostimulatory (p 687)
`
`Teme)AULT LI
`secretion of GDF15 enhances
`myeloma cell proliferation
`(p 725)
`
`UNIV. CHICAGO EX. 2011
`
`
`<=, INDEXED
`
`Ail | |MI
`
`Mm
`
`mW
`
`hy
`
`43128087
`
`- 1
`
`Genome & Co. v. Univ. of Chicago
`PGR2019-00002
`
`

`

`601
`
`30 JANUARY 2014 » VOLUME 123, NUMBER 5
`
`CONTENTS
`
`COVER FIGURE
`
`
`
`Targeted deletion of mouse Tmod3 leads to embryonic lethality with anemia dueto
`
`defects in definitive erythropoiesis in the fetal liver. Native erythroblastic island
`formation was impaired in Tmod3-‘~ fetal livers, with Tmod3 required in both
`erythroblasts and macrophages. Tmod3-mediated actin remodeling may be required
`for erythroblast-macrophage adhesion as well as coordination ofcell cycle with
`differentiation, and F-actin assembly and remodeling during erythroblast enucleation.
`This image shows erythroblast-macrophage islands in wild-type fetal livers, which
`were stained with Alexa Fluor-488 phalloidin (green) for F-actin, rabbit anti-Tmod3
`(red) for Tmod3, and Hoechst 33258 (blue) for nuclei. See the article by Sui et al
`on page 758.
`
`INSIDE BLOOD
`
`602
`
` 603
` lai
`
`Enucleate or replicate? Ask the cytoskeleton
`Stefano Rivella
`
`Is rituximab oneforall ages and each sex?
`Thomas M. Habermann
`
`Less strength and morefractures for MGUS bones
`Evangelos Terpos and Meletios A. Dimopoulos
`
`Free
`blood app
`
`605
`
`606
`
`Delta-1 provides pleasant stem cell environs
`Louis M. Pelus and Pratibha Singh
`
`Inhibitory FeyRUb and CD20internalization
`lan Dransheld
`
`608 Hanging tough: CMV-specific CD8* Tcells in CLL
`John C, Riches and John G. Gribben
`
`609
`
`611
`
`28 dayslater: eosinophils stop viruses
`vaige Lacy
`
`Anemia: a comprehensive global estimate
`Sant-Rayn Pasricha
`
`BLOOD WORK
`
`613
`BCGitis induced inflammatory granuloma of the bone marrow
`Zhaodong Xu and Virginia R. Roth
`
`614 Green neutrophilic inclusions could be a sign of impending death!
`Tarek Jazaerly and Ali M. Gabali
`
`PLENARY PAPER
`
`615
`A systematic analysis of global anemia burden from 1990 to 2010
`Nicholas J. Kassebaum, Rashmi Jasrasaria, Mohsen Naghavi,Sarah K. Wulf, Nicole Johns.
`Rafael Lozano, Mathilda Regan, David Weatherall, David P. Chou, Thomas P. Eisele,
`
`Seth Ro Plaxman, Rachel L. Pullan, Simon J, Brooker, and Christopher J. L. Murray
`
`REVIEW ARTICLE
`625
`Oxidative stress in angiogenesis and vascular disease
`Young-Woong Kim and Tatiana V. Byzova
`
`a
`
`BLOOD, 30 JANUARY 2014 + VOLUME 123, NUMBER 5
`
`This material was copied
`at the NLM and may be
`Subject US Copyright Laws
`
`CONTINUED ON vi
`
`

`

`CLINICAL TRIALS AND
`OBSERVATIONS
`
`
`Mutation of NRASbut not KRASsignificantly reduces myelomasensitivity to
`632
`single-agent bortezomib therapy
`|
`George Mulligan, David |. Lichter, Alessandra Di Bacco, Stephen J, Blakemore,
`Allison Berger. Erik Koenig, Hugues Bernard, William Trepicchio, Bin Li, Rachel Neuwirth,
`Nibedita Chattopadhyay. Joseph B, Bolen, Andrew J. Dorner, Helgi van de Velde, Deborah Ricci,
`Sundar Jagannath, James R, Berenson, Paul G. Richardson, Edward A. Staditmauer,
`Robert Z, Orlowski, Sagar Lonial, Kenneth C. Anderson, Pieter Sonneyeld, Jesus F. San Miguel,
`Dixie-Lee Esseltine, and Matthew Schu
`Suboptimal dosing of rituximab in male and female patients with DLBCL
`Michael Pfreundschuh, Carsten Miiller, Samira Zeynalova, Evelyn Kuhnt, Martin H. J. Wiesen,
`Gerhard Held. Tanja Rixecker, Viola Poeschel, Carsten Zwick, Marcel Reiser, Norbert Schmitz.
`and Niels Murawskt
`
`640
`
`647
`
`Brief Report
`Altered cortical microarchitecture in patients with monoclonal gammopathyof
`undetermined significance
`Joshua N. Farr, Wei Zhang. Shaji K. Kumar, Richard M. Jacques, Alvin C. Ng,
`Louise K. MeCready, 5. Vincent Rajkumar, and Matthew T, Drake
`
`HEMATOPOIESIS AND
`STEM CELLS
`
`650
`
`
`
`Blood stemcell fate regulation by Delta-1-mediated rewiring of IL-6 paracrine
`signaling
`Elizabeth Csaszar, Weijia Wang, Tatiana Usenko, Wenlian Qiao, Colleen Delaney,
`Irvin D. Bernstein, and Peter W. Zandstra
`
`IMMUNOBIOLOGY
`
`659
`
`
`Inhibitors of apoptosis proteins (IAPs) are required for effective T-cell
`expansion/survival during antiviral immunity in mice
`lan E. Gentle, Isabel Moelter, Nadja Lechler, Sarah Bambach, Smiljka Vucikuja, Georg Hicker,
`and Peter Aichele
`
`669
`
`678
`
`Inhibitory FeyRIb (CD32b) becomes activated by therapeutic mAb in both cis and
`trans and drives internalization according to antibodyspecificity
`Andrew T. Vaughan, Chisako Iriyama, Stephen A. Beers, Claude H. T. Chan, Sean H, Lim,
`Emily L. Williams, Vallari Shah, Ali Roghanian. Bjorn Frendeus, Martin J. Glennie,
`
`and Mark S. Cragg
`
`Anti-KIR antibody enhancementof anti-lymphoma activity of natural killer cells as
`monotherapyand in combination with anti-CD20 antibodies
`Holbrook E. Kohrt, Ariane Thielens, Aurelien Marabelle, dit Sagiv-Barli, Caroline Sola,
`Fabien Chanuc, Nicolas Fuseri, Cécile Bonnafous, Debra Czerwinski, Amanda Rajapaksa,
`Erin Waller, Sophie Ugolini, Eric Vivier, Frangois Romagneé, Ronald Levy, Mathieu Bléry,
`and Pascale André
`
`687
`
`Exosomes fromred blood cell units bind to monocytes and induce proinflammatory
`cytokines, boosting T-cell responses in vitro
`Ali Danesh, Heather C. Inglis, Rachael P. Jackman, Shiquan Wu, Xutao Deng, Marcus O, Muench,
`John W. Heitman, and Philip J. Norris
`
`LYMPHOID NEOPLASIA
`
`
`697
`Potent antimyelomaactivity of the novel bromodomaininhibitors 1-BETIS1 and
`I-BET762
`Aristeidis Chaidos, Valentina Caputo. Katerina Gouvedenou, Binbin Liu, Maria Marigo,
`Mohammed Suhail Chaudhry, Antonia Rotolo, David F. ‘Tough, Nicholas N. Smithers,
`Anna K. Bassil, Trevor D. Chapman, Nicola R. Harker, Olena Barbash, Peter Tummino,
`Niam Al-Mahdi, Andrea C. Haynes, Leanne Cutler, BaoChau Le, Amin Rahemtulla, Irene Roberts,
`Maurits Kleijnen, Jason J. Witherington, Nigel J. Parr, Rab K. Prinjha, and Anastasios Karadimitris
`
`vi
`
`BLOOD, 30 JANUARY 2014 + VOLUME 123, NUMBER oOote ial
`ee
`rial was copi
`at the NLM and may be
`Subject US Copyright Laws
`
`CONTINUED ONviii
`
`

`

`706
`
`717
`
`725
`
`A novel small molecule inhibitor of deubiquitylating enzyme USP14 and UCHLS
`induces apoptosis in multiple myeloma and overcomes bortezomibresistance
`Ze Tian, Padraig D'Arcy, Xin Wang, Arghya Ray, Yu-Tau Tai. Yiguo Hu, Ruben D. Carrasco,
`Paul Richardson, Stig Linder, Dharminder Chauhan, and Kenneth C. Anderson
`
`CMV-specific CD8* T-cell function is not impaired in chronic lymphocytic leukemia
`G. Doreen te Raa, Maria Fernanda Pascutti, Juan J. Gareia-Vallejo, Emilie Reinen,
`Ester B. M. Remmerswaal, Incke J, M. ten Berge, René A. W. van Lier, Eric Eldering,
`Marinus H. J. van Oers, Sanne H. ‘Tonino, and Arnon P, Kater
`
`Growth differentiating factor 15 enhances the tumor-initiating and self-renewal
`potential of multiple myelomacells
`Toshihiko Tanno, Yiting Lim, Qiuju Wang, Marta Chesi, P. Leif Bergsagel, Geoff Matthews,
`Ricky W. Johnstone, Nilanjan Ghosh, Ivan Borrello, Carol Ann Huff, and William Matsui
`
`MYELOID NEOPLASIA
`
`
`734 MUCI-Concoprotein promotes FLT3 receptor activation in acute myeloid
`leukemia cells
`Suiyang Liu, Li Yin, Dina Stroopinsky, Hasan Rajabi, Alexandre Puissant, Kimberly Stegmaier,
`David Avigan, Surender Kharbanda, Donald Kufe, and Richard Stone
`
`PHAGOCYTES,
`GRANULOCYTES, AND
`MYELOPOIESIS
`
`
`743 Activated mouse eosinophils protect against lethal respiratory virus infection
`Caroline M. Percopo, Kimberly D. Dyer, Sergei
`I. Ochkur, Janice L. Luo, Elizabeth R. Fischer,
`James J. Lee, Nancy A. Lee, Joseph B. Domachowske, and Helene F. Rosenberg
`
`PLATELETS AND
`THROMBOPOIESIS
`
`
`753
`Brief Report
`High-level transgene expression in induced pluripotent stem
`megakaryocytes: correction of Glanzmann thrombasthenia
`Spencer K, Sullivan, Jason A. Mills, Sevasti B. Koukouritaki, Karen K. Vo, Randolph B. Lyde,
`ti, Lisa M. Sullivan, Yuhuan Wang, Siddharth Kishore,
`Prasuna Paluru, Guoha Zhao, Li Zhe
`Eyad Z. Gharaibeh, Michele P. Lambert, David A. Wilcox, Deborah L. French, Mortimer Poncz,
`and Paul Gadue
`
`cell-derived
`
`el
`
`e-Blood
`Time-resolved characterization of cAMP/PKA-dependent signaling reveals that
`platelet inhibition is a concerted process involving multiple signaling pathways
`Florian Beck, Jorg Geiger, Stepan Gambaryan, Johannes Veit, Mare Vaudel, Peter Nollau,
`Oliver Kohlbacher, Lennart Martens, Ulrich Walter, Albert Sickmann, and René P. Zahedi
`
`
`RED CELLS, IRON, AND
`Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid
`ERYTHROPOIESIS
`758
`terminal differentiation in the fetal liver
`Zhenhua Sui, Roberta B. Nowak, Andrea Bacconi, Nancy E. Kim, Hui Liu, Jie Li,
`Amittha Wickrema, Xiu-li An, and Velia M. Fowler
`
`THROMBOSIS AND
`HEMOSTASIS
`
`
`Polyphosphate suppresses complement via the terminal pathway
`768
`Jovian M, Wat, Jonathan H, Foley, Michael J. Krisinger, Linnette Mae Ocariza, Victor Lei,
`Gregory A. Wasney, Emilie Lameignere, Natalie C. Strynadka, Stephanie A. Smith,
`James H. Morrissey, and Edward M. Conway
`
`77
`
`A meta-analysis of genome-wide association studies identifies ORM1 as a novel gene
`controlling thrombin generation potential
`Ares Rocanin-Arjo, William Cohen, Laure Carcaillon, Corinne Frere, Noémie Saut, Lue Letenneur,
`Martine Alhenc-Gelas, Anne-Marie Dupuy, Marion Bertrand, Marie-Christine Alessi,
`Marine Germain, Philipp S. Wild, Tanja Zeller, Francois Cambien, Alison H. Goodall,
`Philippe Amouyel, Pierre-Yves Scarabin, David-Alexandre Trégouét, Pierre-Emmanuel Morange,
`and the CardioGenics Consortium
`
`vill
`
`BLOOD, 30 JANUARY 2014 - VOLUME 123, NUMBER 5
`This material was copied
`
`CONTINUED ON x
`
`

`

`TRANSPLANTATION _
`
`
`
`786
`
`Plasma CXCL9elevations correlate with chronic GVHD diagnosis
`Carrie L. Kitko, John E. Levine, Barry E. Storer, Xiaoyu Chai, David A. Fox, Thomas M. Braun,
`Daniel R. Couriel, Paul J. Martin, Mary E. Flowers, John A. Hansen, Lawrence Chang.
`Megan Conlon, Bryan J. Fiema, Rachel Morgan, Prae Pongtornpipat, Kelly Lamiman,
`James L.. M. Ferrara, Stephanie J. Lee, and Sophie Paczesny
`
`CORRESPONDENCE
`
`
`794
`
`Correcting 2 more myths regarding transplants for AML in second remission
`Robert Peter Gale and Hillard M. Lazarus
`
`794 Association of a single-nucleotide polymorphism in the SH2B3 gene with
`JAK2V617F-positive mycloproliferative neoplasms
`Elodie Lesteven, Marie Picque, Carole Conejero Tonetti, Stéphane Giraudier, Nadine Varin-Blank,
`Laura Velazquez, Jean-Jacques Kiladjian, Bruno Cassinat, and Fanny Baran-Marszak
`
`796
`
`Hepatitis E transmission by transfusion of Intercept blood system-treated plasma
`Lisette Hauser, Anne-Marie Roque-Afonso, Alexandre Beylouné, Marion Simonet,
`Bénédicte Deau Fischer, Nicolas Burin des Roziers, Vincent Mallet, Pierre Tiberghien,
`
`and Philippe Bierling
`
`ERRATA
`798
`Duh EJ. Sema3Aresists retinal revascularization. Blood, 201151 17(22):5785-5786.
`
`798
`
`798
`
`Hole PS, Darley RL, Tonks A. Do reactive oxygen species play a role in myeloid
`leukemias? Blood. 2011;117(22):5816-5826.
`
`Hsu LL. Hydroxyurea makes inflammation “just right”? Blood. 20123119(8):
`1796-1798.
`
`
`OTHER DEPARTMENTS
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`Regular Article
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`IMMUNOBIOLOGY
`
`Anti-KIR antibody enhancementofanti-I
`ymphomaactivity of
`naturalkiller cells as monothera
`py and in combination with
`anti-CD20 antibodies
`Holbrook E. Kohrt,' Ariane Thielens,? Aurelien Marabelle,'Idit Sagiv-Barfi,' Caroline Sola, Fabien Chanuc,*
`Nicolas Fuseri,* Cécile Bonnafous,® Debra Czerwinski,’ Amanda Rajapaksa,’ Erin Waller, Sophie Ugolini,? Eric Vivier,"
`Frangois Romagné,* Ronald Levy,' Mathieu Bléry,? and Pascale André?
`‘Departmentof Medicine, Division of Oncology, Stanford University, Stanford, CA;
`Cancérologie de Lyon, Centre Léon Bérard, Université de Lyon, Lyon, France; and
`UMe2, Pare scientifique et technologique de Luminy, Marseille, France
`
`“Innate Pharma, Marseille, France: “Centre de Recherche en
`“Centre d'immunologie de Marseille-Luminy, Aix Marseille Université
`
`
`
`
`Blockadeofinhibitory KIRs
`with MHCclass| antigens on
`lymphomacells by anti-KIR
`antibodies augments NK-cell
`spontaneouscytotoxicity.
`In combination with anti-CD20
`5
`;
`mAbs,anti-KIR induces
`:
`enhanced NKcell-mediated,
`tisdmnabdeneneient
`r
`~~
`p
`:
`cytotoxicity against lymphoma.
`
`
`
`*
`
`Naturalkiller (NK) cells mediate antilymphoma activity by spontaneous cytotoxicity and
`antibody-dependentcell-mediated cytotoxicity (ADCC) whentriggered byrituximab,an anti-
`CD20 monoclonalantibody (mAb) used to treat patients with B-cell lymphomas.The balance
`of inhibitory and activating signals determines the magnitude of the efficacy of NK cells
`by spontaneous cytotoxicity. Here, using a killer-cell immunoglobulin-like receptor (KIR)
`transgenic murine model, we show that blockadeof the interface of inhibitory KIRs with
`major histocompatibility complex (MHC)class| antigens on lymphomacells by anti-KIR
`antibodies prevents a tolerogenic interaction and augments NK-cell spontaneous cytotox-
`icity. In combination with anti-cD20 mAbs, anti-KIR treatment induces enhanced NK-
`:
`Bo
`oe
`;
`cell-mediated,rituximab-dependent cytotoxicity against lymphomain vitro and in vivo in
`KIR transgenic and
`-
`g
`syngeneic murine lymphoma models. Theseresults support a therapeu
`;
`and
`tic strategy of combinationrituximab and KIR blockade throughlirilumab,illustrating the
`potentialefficacy of combining a tumor-targeting therapywith an NK-cell agonist, thus
`stimulatingthe postrituximab antilymphoma immuneresponse. (Blood. 2014;123(5):678-686)
`
`Immune checkpoint blockade represents a promising cancerther
`_ apy—cells. The activation of NK-cell effector function is regulated by_
`
`
`that aimsto restore an efficient antitumoral response mediated by multiple types ofactivating and inhibitory receptors,
`including KIR,
`I
`é
`‘
`4
`aE
`.
`o
`#3
`*m
`endogenous immunecells.' Antibodies to CTLA-4, an inhibitory
`that recognize ligands expressedonpotential larget cells, The balance
`receptor that dampens T-cell receptor (TCR) signaling, enhance
`between positive and negative signals transmitted via these NK
`immune cell function by blocking a negative regulator, CTLA-4
`receptors determines whether or not a target cell is killed by an NK
`shares CD80(B7.1) and CD86 (B7.2) as ligands with the TCR
`‘aly?
`:
`it
`F
`j
`i
`ce
`cell.”
`In addition,
`lack of KIR-HLA class I interactions has been
`costimulatory receptor CD28. The intracellular signals transduced
`associated with potent NK-mediated antitumor efficacy and i>
`by the TCR, CD28, and CTLA-4 determine the outcome of T-cell
`creased survival in acute myeloid leukemia (AML) patients upo?
`activation.” The therapeutic concept of immunomodulation Ww
`ds
`haplo-identical stem cell
`transplantation from KIR mismatched
`validated bythe approval of anti-CTLA-4 ipilimumabin metastatic
`donors.” To exploit
`this pathway pharmacologically,
`the fully
`melanoma, thus increasing overall survival.!* Other inhibitory re-
`human mAb anti-KIR 1-79 (IPH2101) was first generated,” and
`ceptors of T-cell function, such as PD-1 and LAG-3.
`‘
`are being
`then a recombinant version of this mAb wias developed with
`targeted by therapeutic monoclonal antibodies (mAbs) in clinical
`a stabilizedhinge(lirilumab), |-7F9 andlirilumab mAbscross-react
`andpreclinical development.!"!
`with KIR2DLI, ~L2, and -L3 receptors and impair their inhibitory
`As a corollary to targeting negative regulators of T cells, we
`signaling by preventing their binding to HLA-C. Invitro, anti-KIR
`hypothesizedthat the killercell immunoglobulin-like receptor (KIR)
`mAbs augmented NK-cell-mediated lysis of HLA-C~expressing
`family of natural killer (NK) cell negative regulators would represent
`tumor cells,
`including autologous AML. blasts and autologous
`a novel andactive class of immunotherapy.” Indeed, NK cells play
`,
`‘
`‘
`:
`CD138" multiple myelomacells,” In addition, splenocytes from
`critical roles in host defense against infections and tumors by secreting
`mayor histocompatibility complex class | (MHC-I)-deficient mice
`immunoregulatory cytokines and by killing infected ortranslormed
`expressing HLA-Cw3wererejectedin 20 hours from Rag! KO mice
`
`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.
`
`Introduction
`
`Submitted August 2, 2013: accepted October 14, 2013. Prepublished online as
`BloodFirst Edition paper, December 10, 2013; DOI 10.1182/blood-2013-08-
`519199.
`
`H.E.K., A.T., ALL, M.B., and P.A. contributed equally to this study.
`The online version ofthis article contains a data supplement.
`
`678
`
`© 2014 by The American Society of Hematology
`
`BLOOD, 30 JANUARY 2014 « VOLUME 123, NUMBER 5
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`

`

`BLOOD, 30 JANUARY 2014 + VOLUME 123, NUMBER 5
`
`expressing KIR2DL3 with increasing doses of 1-7F9, demonstrat-
`ing that
`in vivo blockade of KIR HLA class I interactions could
`mediate rejection of HLA-C-expressing cells.” In mice, the Ly4?
`receptors have functions similar to human KIRs andbindto murine
`H-2 (MHC-1) molecules. We demonstrated a beneficial effect of
`blocking H-2-Ly49 interactions in vivo in combination with
`lenalidomide in rejecting MHC-I-positive tumor cells.!" A phase |
`clinical trial in elderly patients with AML was performed with an
`escalating-dose of 1-7F9. Results demonstrated that
`the 1-7F9
`mAb injections were safe and could block KIR for long periodsof
`time (more than 2 weeks at
`| and 3 mg/kg) withlimited adverse
`effects, !*
`Morethan a decadeprior to the approval ofipilimumab,a murine-
`human chimeric immunoglobulin G1 (IgG1) antibody against CD20
`Called rituximab was approved and has since become @ standard
`treatment for patients with B-cell lymphomas. Althoughrituximab
`has multiple mechanismsofaction, antibody-dependent cell-mediated
`Cytotoxicity (ADCC) is of particular importance. Neutralizing anti-
`bodies that prevent
`the Fe-FeR-y interaction abrogate the B-cell-
`depleting and antilymphomaactivity in vitro’? and in vivo in murine
`models.'*"° In clinical practice, FeyREA polymorphism with a higher
`affinity for IgG] is associated with a higher response rate.!”"'® Because
`the response rate to rituximab among patients with relapsed/retractory
`lymphoma may be approximately halfthat of patients previously
`untreated, we investigated whether NK-cell immunomodulation by
`Combination of blockade ofinhibitory KIR by lirilumab and stimu-
`lation via FeyRIM byrituximab could enhance antilymphomaefficacy
`'N preclinical models.
`- Here we presentresults that support the monotherapy activity of
`lirilumab in a KIR2DL3 transgenic lymphoma model and ofanti-
`Ly49c/| F(ab’)> ina CS7BL/6syngeneic lymphoma model. In both
`models tested, combination ofeither of those with an anti-CD20
`mAb significantly improves the antitumorefficacy of anti-CD20 mAb
`alone. Thesefindings provide rationale fora clinicaltrial in lymphoma
`Mvestigating this novel combination of an anti-KIR mAbtotlt the
`balance of inhibitory andstimulatory NK signals favoring spontane-
`OUS cytotoxicity and to further enhancetheefficacy ofrituximab by
`augmentation of ADCC.
`
`
`
`KIR BLOCKADE ENHANCES RITUXIMAB ACTIVITY
`
`679
`
`for animal experimentation. Experiments using C57BL/6 mice were ap-
`proved by Stanford Laboratory Animal Care and were conducted in
`accordance with Stanford University Animal Facility and National Institutes
`of Health guidelines.
`
`Therapeutic antibodies and reagents
`
`-L3, -S1, -S2
`-L2,
`Anti-KIR mAbis a fully human 1gG4 anti-KIR2DL1,
`producedin the CHOcell line (lirilumab/IPH2102/BMS-986015).” Anti-CD20
`mAb is a chimeric human IgGl (rituximab [MabThera]; Roche). Phy-
`coerythrin-conjugated lirilumab was produced at
`Innate Pharma. F(ab')>
`fragments of anti-Ly49C/ (5E6) mAbs were provided by Innate Pharma.
`The CAT-13.6E12 hybridoma cell
`line was purchased from Deutsche
`Sammlung von Mikroorganismen und Zellkulturen. These cells were used to
`produce the anti-huCD20 CAT-1 3 IgG2a mAb.Irrelevant antibodies, with
`the sameisotypes as lirilumab (hlgG4) and rituximab (chimeric [gG1), 5%
`normal mouse serum, and 4D 11 (Ly49G2, a mAb against Ly49G2 specific
`for H2-Dd) were used as controls,
`
`In vitro NK functional assays
`
`To evaluate NK-cell cytotoxicity against EL4-huCD20, chromium release was
`performedas described.'” Percent lysis was determined after 4-hourcultures of
`purified NK cells at variable effector:target cell ratios with *'Cr-labeled lym-
`phomacells in mediaalone, or with anti-Ly49C/I (SE6; LO g/mL) alone, anti-
`CD20 mAb (CAT-13; 10 jxg/mL) alone, or the combination of anti-Ly49C/l
`and anti-CD20 mAbs(both at [0 jzg/mL). To evaluate NK-cell degranulation,
`CD107a mobilization wasassayedas detailed previously. "Cells were cultured
`for interferon-y (IFN-y) assessment by using conditions similar to those
`detailed above forassessmentof cytotoxicity, Supernatant was analyzedfor
`IFN-y by enzyme-linked immunosorbent assay, In vitro assays were per-
`formed with 3 independent samples.
`
`Tumortransplantation and antibody therapy
`In tumoral challenge experiments, 0.5 % 10" BL4-huCD20lymphoma tumor
`cells in 50of phosphate-buffered saline were injected retro-orbitally into
`wild-type C57BL/6 mice. In 300 L of phosphate-bulferedsaline, 10! 221
`cells were injected intravenously into Rag! KO-Tg KIR mice. Anti-CD20
`mAbs (250jug per injection) and 200 jug of F(ab’)> fragments ofanti-Ly49C/1
`were administered byintraperitoneal injections, whereas all human mAbs were
`administered by intravenous injection. Tumor growth (EL4-huCD20 tumor
`model) andsurvival (221 tumor model) were monitoredfor 90to 150 daysafter
`tumoral challenge. Tumorgrowth was measured bycaliper twice a week and
`expressed as length times width in square centimeters. Mice were euthanized
`when tumorsize reached 4 cnr or when tumorsites ulcerated.
`
`Material and methods
`
`Cell lines and mice
`
`Depletion of CD8 T cells, NK cells, and macrophages
`Anti-CD8 mAb (hybridoma 2.43), anti-Asialo-GMI mAb (Wako Chem-
`icals), andliposomal clodronate (Weissman Laboratory, Stanford, CA) were
`Human B-cell fine LOL 721.221 was transduced with lentiviral particles
`used to deplete CD8 T-cell, NK-cell, and macrophage activity in vivo in
`“ncoding either HLA-Cw3 (221 HLA-Cw3) or enhancedgreen fluorescent
`C57BL/6 mice, respectively. Depleting anti-CD8 mAb (S00 jug) and anti-
`Meee: as a control (22 1), from which sequences were cloned into
`Asialo-GM1 mAb(50 pL) were injected intraperitoneally on day —1, day 0 of
`bimanasa plasmid, EL4 murine T|ymphomacellswere transduced with
`tumorinoculation, and every 5 days thereafter for 4 weeks. Liposomal clodronate
`pithach § 2() pouipemnentaty‘DAY (EL4-huCD20).Both cell
`lines were
`wasinjected retro-orbitally on day ~ 2 at a dose of 200 wL, on day 0 of tumor
`tented my RI Mie) medium (Invitrogen ite Technologies) supple-
`inoculation, and every 4 days thereafter for 4 weeks at a dose of 100 wi. The
`100 srr Ma * heat-inactivated fetal call serum (HyC lone Laboratories),
`depletion conditions were validated by flowcytometry of blood showing more
`Life Heil 2 penicillin and 100 pe/n. streptomycin (both from Invitrogen/
`than 95% depletion of CD8 Tcells, 90% depletion of NK cells, and 90%
`Binooks eens 2 mM L-glutamine (Sigma-Aldrich), and | mM sodium
`depletion of macrophages.
`Ciiiaee tgran-Aldrich), with the addition ot 50uM 2-mercaptoethanol
`In RagIKO-Tg KIR mice, NK-cell depletion was achievedbyinjecting
`37°C e
`drich) for EL-4 only. Cells were grown in suspension culture at
`100 jg of anti-NK 1.1 mAb (PK 136) intravenously every 10 days, starting
`In 5% COs,
`| day prior to treatment of 221 cells.
`tistics 10-week-old male and female RaglKO Tg KIR2DL3
`Lacilities ‘i 8 KIR) mice were bred and maintained at Innate Pharma animal
`ona DRPaLetia pathogen-free conditions. Rag! KO Tg KIR2DL3 mice
`old Weicaks a background were previously described. Eight- to 10-week-
`iis oa eatBLA mice were purchased from Charles River and were
`Csttae cae Laboratory Animal Facility at the Stanford University Medical
`Innate Pk expenments using Rag! KO-Tg KIR mice were approved by the
`arma ethics committee in accordance with the Europeandirective
`
`Antibodies for flow cytometry
`The following mAbs to mouse antigens were used: CDS fluorescein iso-
`thiocyanate (FITC) (Becton Dickinson [BD|/BDBiosciences (San Jose, CA),
`CD107a FITC (BD), CD4 FITC (BD), CD3peridinin chlorophyll protein complex
`(BD), NKL.1 allophycocyanin (BD), and P4/80 allophycocyanin (eBio-
`science, San Diego, CA), Stainedcells were collected on a FACSCalibur
`
`

`

`680
`
`KOHRTet al
`
`BLOOD, 30 JANUARY 2014 - VOLUME 123, NUMBER 5
`
`90
`
`:
`
`|
`
`2
`2
`=
`
`ee
`
`750
`
`500
`
`3
`250
`=
`>
`Bi
`=
`>
`E
`“i
`sa
`>
`0
`
` 400
`
`80
`2 70
`
`>2
`
`x 60
`2
`=
`=
`oo
`= 30
`20
`10
`a
`
`EL4-hucb20
`Anti-CD20CATI3.
`AntiLy49Cil F(ab),
`
`-
`-
`-
`
`+
`-
`-
`
`+
`-
`+
`
`+
`+
`-
`
`+
`+
`+
`
`EL4-huco20
`Anti-CD20CATIZ.
`Anti-Ly49Cil Flab),
`
`He
`+
`t+
`-
`- - +
`=
`=
`+
`=
`
`+
`+
`+
`
`Effector: Target Ratio
`
`Figure 1. Anti-Ly49C/l F(ab’), increases anti-CD20 mAb-mediated NK-cell degranulation and cytotoxicity. (A) NK cells isolated and purified from C57BL/6 mice were
`analyzed for degranulation by CD107a mobilization after 24-hour culture with 5 conditions: medium alone; human CD20° murine lymphomacellline (EL4-huCD20)at a 1:1 ratio of
`tumor:NK cells; tumor and anti-CD20 mAb (CAT13; 10 j.g/mL); tumor and anti-Ly49C/ F(ab’), (5E6; 10 jag/mL); or tumor, antiCD20 mAb (10 j.g/mL), and anti-Ly49C/ F(ab").
`(10 pg/mL). *P—0109. (B) Enzyme-linked immunosorbent assay (ELISA) results measuring IFN-y following a 4-hour culture of purified murine NK cells in medium alone; EL4-
`huCD20at a 1:1 ratio of tumor:NK cells; tumor and anti-CD20 mAb (CAT13; 10 j.g/mL); tumor andanti-Ly49C/l F(ab’), (10 j.g/mL); or tumor, anti-CD20 mAb (10 jxg/mL), and anti-
`Ly49C/l F(ab’). (10 g/mL)