Blood.
`
`v. 123. no. 5 (Jan. 30 2014]
`Genera! Collection
`W1 BL661
`2014-02-21 14:52:37
`
`VOLUME 1'23
`numeens
`30 JANUARY 2014
`
`MGUS patients have
`increased bone porosity and
`fracturerisk (1.1 603, P 547}
`
`EosinOphils'In alrways provide
`Patent antiviral protection
`(p 609,1) 743)
`
`Worldwide anemia prevalence
`contributes to global burden
`ol disability (p 611, p 615)
`
`Exosomes from stored
`red cell units are
`immunostimulatory (p 687)
`
`Bone marrow stromal
`secretion of GDF15 enhances
`myeloma cell proliferation
`(p 725)
`
`
`
`Genome & Co. v. Univ. of Chicago
`PGR2019-00002
`
`SISRC
`
`III
`
`Jlljlllll14}
`
`32.
`
`:1'"E
`
`4312808?
`
`gm” I NDEXED
`an” 3202239
`
`UNIV. CHICAGO EX. 2011
`
`

`

`
`
`
`
`30 JANUARY 2014 - VOLUME 123. NUMBER 5
`
`CONTENTS
`
`COVER FIGURE
`
`
`
`'l'argeled deletion of mouse 'l'motlfi leads to embryonic lethality with anemia due to
`
`defects in definitive erythropoiesi.‘ in the fetal liver. Native crylliroblastie island
`formation was impaired in 'i‘iriiridj
`‘1‘ fetal livers. with 'l'inod] required in both
`erythroblasts and macrophages. 'I‘modS-meilialed aetin remodeling may he required
`for erylhrohlast-imieriiphage adhesion as well as eoortiinalion of cell cycle with
`tlilT’erentialion. and li-aetin assemhly and remodeling (luring erythroblasl enuelealion.
`This image shows erylliroblasl-macrophage islands in wild-type l'etal livers. which
`were stained with Alexa li‘luor»488 phalloiilin (green) for F-actin. rabbit anti-'I‘mod]
`(red) for 'l‘modfi, and l-Ioechst 33258 (blue) for nuclei. See the article by Sui et al
`on page 758.
`
`INSIDE BLOOD
`
`601
`
`Enucleale or replicate? Ask the cyloskelelon
`Stefano Rivella
`
`602
`
`ls rituximab one for all ages and each sex‘.’
`‘I'Iiomas M. Ilahermann
`
`603
`
`Less strength and more l'raclurm for MGI‘S bones
`I"I\'-.ingc|ns 'I'el'pos and Melctius A. Dilutipoulus
`
`505 Delta-l provides pleasant stem cell environs
`Louis M. Pains and Pi'atihha Singh
`
`506
`
`Inhibitory li‘eyllllh and (1)2" intermilization
`Ian lh‘anslieitl
`
`608 Hanging tough: CNN-specific l‘l)8+ T cells in C”.
`John ('. Riches and Julio (i. tirihhen
`
`609
`
`611
`
`28 days tater: eosinopliils stop viruses
`’iligi‘ Lacy
`
`Anemia: a comprehensive global estimate
`Sum-Ra)" Ptlsl'ielia
`
`_
`_
`BLOOD WQBK
`613
`B(.‘(iitis induced inflammatory granuloma ol' the hone marrow
`Khamlnng XLI and Virginia R. Ruth
`
`___
`
`614 Green neutropllilic inclusions eoultl be a sign of impending death!
`'l‘uI'ek .Iamci'ly and Ali M. (hihali
`
`PLENAFIY PAPER
`
`615
`A systematic analysis of global anemia hurtlen from 19‘)" to lelll
`Nicholas J. Kussehalnn. Rnshlni .Iusr'nsill'izl. Mnhscn Nughnrimfitu'nh K. Willi. Nit'nlt’ .thH-K
`Rafael Liminn. l\-1.‘Ilhild.‘| Regan. ”mid \\-'L':ii|1cl':lli. I).'I\'itl
`l’. (‘liuti‘ 'I‘lmmtis l'.
`IiisclL‘.
`
`Seth R. l-‘Jasman. Rachel |.. I’ullain. Sinmn .I. Iii-ookei'. amt (‘Iirislophei' J. L. Murray
`
`REVIEW ARTK—ZLE
`625
`{)sidatire stress in angiogenesis and vascular disease
`‘i'uuog-Wonne Kim and 'l'aliana V. Hymn-:1
`
`__
`
`\I‘
`
`BLOOD. 30 JANUARY 2014 - VOLUME 123. NUMBER 5
`
`This materialwas copied
`atthe NLM and maybe
`Subject USCDpyright Laws
`
`CONTINUED ON Hi
`
`

`

`CLINICAL TRIALS AND
`OBSERVATIONS
`
`
`
`RAS but not KRAS significantly reduces myelonia sensitivity to
`632 Mutation of N
`single-agent liortemnlili therapy
`Lienree Mulligan. David I. Lichter. Alessantha |)i Bacco. Stephen J. lilukcmm-c.
`Allison Beredll I-Lrik Koenig. illigues Bernard. William 'I'rcpicehio. li'm Li‘ Rachel Nulminh.
`Nil‘lctlllil [‘1lullttpiltlllyil)‘1”“:th Ii. Bolen. Andrew J, Denier. ilelei van (le Velde. Deborah Ricei.
`.‘iundar .lauannath, James R. liercnsun. Paul (E. Richardson. litlward A. Slacltlnauer.
`Ruben '/.. ("1““ka sugar Lonial, Kennelh ('. Anderson. l’ieler Sonncveld, .lesiis Ii San Miguel.
`Dixie-Lee Lisseltine. and Matlhew Sehu
`
`640
`
`Suboptimal dosing of ritnximah in male and female patients with DLIKII.
`‘ar‘elcll Miiiler. Sahara Zeynalnva. lively" Kulint. Marlin H. J. Wiescn
`Michael l’l'reumlschuh. (
`Rixeeker. Viola l’oesellel. {'arsten Zwick. Marcel Reiser. Norhcrl Sclnnitr
`(icrhani llelll. "l'anja
`and Niels Murawdxi
`
`547
`
`Brief Report
`Altered cortical mieroarchitecture in patients with monoclonal gammopathv of
`undetermined significance
`.‘ihaji K, Kunial'. Richard M. Jacques. Ali-in (‘. Ng.
`.loshna N. Farr. Wei Khang.
`Louis-e K. Met‘reatlv. .‘i. Vincent Rajkumar. and Malthew '['_ Drake
`
`
`'
`_
`550
`Blood stem cell fate regulation by Delta-l—inedialed rewiring of Ill-(i paracrine
`signaling
`lilieahclh ('saszzir. Weijia Wang. Tatiana Uscnko. Wenlian Qiao. (‘ollcen [Jelanev
`ins-in l). Bernstein. and Peter W. Zanilstra
`
`_
`
`HEMATOPOIESIS AND
`STEM CELLS
`
`IMMUNOBIDLOGY
`
`
`
`659
`
`669
`
`Inhibitors of apoptosis proteins (lAl’Sl are required for el’l'ectivc 12“,"
`expansioni'survival during antiviral immunity in mice
`tan l1. (lenlle. Isabel Mueller. Nadia Lecliler. Sarah Bambaell. Sniiljka Vucikuja 0mm lliieker
`and Peter Aichelc
`
`Inhibitory lie-vRIlb {CDSZhJ becomes activated by therapeutic mAh in both eis and
`trans and drives internalimtion according to antibody specific“).-
`.r\l1tll'L'\\
`'I'. Vaughan. t‘hisako lt'l)’i'lil'l:l. Slephen A. Beers. (‘Iaatie l-|_ 'l‘, chm .‘iean ll.
`limily l,. Williznns. \r'allari Shah, Ali Rnglialiian. lijnrn lirentleos. Martin J. tilennie.
`and Mark 5. Claire
`
`|.im.
`
`678 Anti-KIR antibody enhancement of anti-lymphoma activityr of natural killer cells as
`monotherapy and in combination with anti-Clntl antibmlies
`ilnlhrnok l-L. Kuhrl, Ariane 'l‘hielens. Aurelie-n Marabelle. idil Sagiv-liarli. (.‘aroline Sulgh
`liabicn (‘h:n|uc. Nicolas I’uscri. t‘ecile lionnal'ous. llUl1F=l(‘ZUI'\i-'iliski. Amanda Ritiatiaksa.
`
`I'Lrin Waller. Sophie llgnlini. liric Vivier. l-‘rauenis Romagne. Ronahl Lye-y. Malliieu ”My
`and Pascale mun-c-
`
`687
`
`lflsosomes from red blood cell units bind to monocytes and induce proinflannnatorv
`eylokincs, boosting T-cell responses in vitro
`‘
`Ni l'lanesh. Ileatlierf’. Inglis. Rachael I'. Jaekinan. Shiquan Wu, Xutao Deng. Marcus 0. Mneneh.
`.lltllll W. lleitnian. and Philip J. Norris
`
`LYMPHOID NEOPLASIA
`597
`Potent antilnyeloma activity of the novel hrolnodomain inhibitors l-lllfl'l‘lSl and
`l-lilfi'l'WiZ
`i\l'lhlt‘itlir~ (humus. Valentina ('aputo. Katerina (iom-etlennu. li'mhin Lia. llaI'ia Marigo.
`Mohammcd Suhail (‘hauilllrv Antonia Rotnln. David 1".
`'l‘nugh. Nicholas N.
`.‘iniilhers.
`Anna K. liassil. 'l'revor l). Chapman. Nicola R. llarkcr, [Menu ital-hash. l'cter 'l'unnninn.
`Niani Al'Millltll. Andrea L‘. Haynes. Leanne ('uticr. liaot 'han Le. Alain Rahemtolla. Irene Roberts.
`\‘lii'
`'.".
`i- '.
`-
`'-
`i
`u rils Munich. Jason J. \N lllicrinrrinn. ngcl J. Pair. Rali K.
`l’I'in_|ha. and Anastasius Karatliinilris
`
`vi
`
`.
`.
`BLOOD. 30 JANUARY 2014 - VOLUME 123. NUMBIER 5
`i5materlaiwas copied
`at the ML” and may be
`Subject USCapvright Laws
`
`CONTiNUED ON “ill
`
`

`

`7’05
`
`717
`
`A novel small molecule inhibitor oi‘deuhiquitylating enzyme USPM and UCl-ll.5
`induces apoptosis in multiple myeloma and overcomes horteaomih resistance
`'/.e 'l‘ian. l’atli'aig D‘Arcy. Xin Wang. Arghya Ray. ‘i’u—'l}.u 'I'ai. Yiguo Ila. Ruhcn I}. (‘airascm
`l’aul Richardson, Slip Linder. Dharmlnder [lamina]. and Kennelh ('. Anderson
`
`CMV-speeilic Cl)8+ 'l‘-ceil function is not impaired in chronic lymphocytie leukemia
`(i. Doreen 1e Raa. Maria |-'ernanda l’ascalli. Juan J. Garcia-Vallejo. limilic Reinen.
`léslei' it. M. Remmei'swaal.
`Inelx'e .l. M. Ien lierge. Rene A. W. van |..ier. liric lilderinp.
`Marinas ii. .I. can (leis. Sarine Ii. 'l'onino. and Anion l’. Kaler
`
`725 Growth differentiating factor I5 enhances the tumor-initiating and self-renewal
`potential of multiple myeloma cells
`‘l'osliiliiko 'l‘anno. Yiling Lim. Qiuiu Wang. Marla Chest. I’. tell llergsagel. (leol‘l' Matthews.
`Ricky W, John-none. Nilaliian {'iliosli.
`lvan liol'rello. Carol Ann Hull. and William Malstli
`
` MYELOlD NEOPLASIA
`734 MUC l-C oncoprotein promotes [*‘LT3 receptor activation in acute myeloid
`leukemia cells
`Suiyang Lin. 1.l Yin. Dina Siroopinsliy. llasan Rajabi. Alexandre
`David Avigan. Surcnder Kliarhanda. Donald Kul'c. and Richard Stone
`
`l’oissanl. Kimberly Slegmaler.
`
`PHAGOCYTES,
`GRANULOCYTES' AND
`MYELOPOIESIS
`
`
`743 Activated mouse eosinophils protect against lethal respiratory virus infection
`
`|. (Jcllkur. Janice l.. Luo. iiliaahelli R. Fischer.
`Caroline M. l’el‘eopo. Kimherly I). IJyer. Sergei
`James J. Lee. Nancy A, Lee. Joseph B. Doniachowskc. and i-lelenc l". Rosenberg
`
`PLATELETS AND
`
`THHOMBOPOIESIS
`
`
`753
`Brief Report
`High-level transgene expression in induced pluripotent stem cell—derived
`megakaryocytes; correction of (ilanamann thronihasthenia
`Spencer K. Sullivan. Jason A. Mills. Sevasli It, Kotlkourilaki. Karen K. Vo. Randolph B. Lydc.
`ll. Lisa M. Sullivan. Yuliuan Wang. Siddharlh Kishoie.
`l’i'asuna I’alnru. (iuoha Khan. Li Ill;
`|.. French. Morlimet' l’oncx.
`liyad K. (lhai'aiheh. Michele P. Lamherl. David A. Wilcox.
`l.}chora}i
`and Paul (iadtlc
`
`91
`
`c~lilrmd
`'l‘ime-resolved characterization of cA M Pi“PKA-dependent signaling reveals that
`platelet inhibition is a concerted process involving multiple signaling pathways
`Slepan Liamharyan. Johannes Veil. Marc Vamlel. Peter Nollau.
`I‘lorian Heck. .liii'g (ieigcr.
`Vahedi
`[)licer Kohihaclicr. Lennart Martens. Ulrich Waller. Aihen Sickmann. and Rene l’.
`
` RED CELLS. IRON. AND
`EHYTH ROPOlES'S
`758
`TrapomodutinJ-iiull mice are embryonic lethal with anemia due to impaired erythroid
`terminal differentiation in the fetal liver
`Klienliua Sui. Roberta it. Nowak. Andrea Itaeconi. Nancy [5. Kim. llal Lin. Jie Li.
`Amiliha Wickrema. Xin-li An. and Velia M. lr'owlcr
`
` THFIOMBOSIS AND
`HEMOSTAS'S
`Tr'BB
`l’olyphosphate suppresses complement via the terminal pathway
`Jovian M. Wat. Jonalhan ll, Foley. Michael J. Krisiiigci'. Lilmcllc Mae ()cariza. Victor Lei.
`Gregory A. Wasney. l-Lmilic Lanieignei'e. Natalie (I Slrynadka. Stephanie A. Sinilli.
`James II. Mm'rissey. and l-Zdivard M. Conway
`
`777
`
`A meta-analysis of genome-wide association studies identities ()RM] as a novel gene
`controlling lhronihin generation potential
`Ares Rocanin-Ario. William ('ohen. Laure (‘ai'calllorL (‘orinne I‘i'L'i'e. Noémie Saul. |.uc Lclemieui'.
`Marline Allienc-(ielax. r\llllt."MEll'lU IJLipuy. Marion liei‘ll'anil. Marie-Christine Alessi.
`Marine (Jermain. l’liilipp 5'. Wild. 'l‘anja Keller. Francois (‘amliicIL Alison ll. (inodall.
`l’liilippe Amouyei. Pierre—Yves Scarahin. David-Alexandre 'l'i'egnuL‘l. l’icrre-linunamlel Morange.
`and die (‘anliotienics Consortium
`
`vili
`
`BLOOD. 30 JANUARY 2014 - VOLUME 123. NUMBER 5
`This material was copied
`
`CONTINUED ON K
`
`

`

`TRANSPLANTATION
`
`__
`
`
`
`786
`
`l'lasma CKFL‘) elevations correlate with chronic (Will) diagnosis
`(‘urrie |._ Killm. John |-.. Levine. Barry IE. Store-r. Xiaoyn Chan. David A. Fox. 'l‘holnus M. liraun.
`Daniel R. L'ouriel. Paul J. Martin. Mar}- [-J. Mowers. John A. l-Iunsen. Lawrence Chung.
`Megan [‘onlon. Bryan J.
`|-'ie.mu. Rachel Morgan. Prue l’onglornpipal. Kelly l,:unim:tn.
`James L. M.
`l-errura. Stephanie J. Lee. and Sophie l’:tL‘;:‘.esI1_~,r
`
`CORRESPONDENCE
`
`794
`
`Correcting 2 more myths regarding transplants for AMI. in second remission
`Rohcl't l’eler (iale and llillilrti M. Lil/.Ell'lts
`
`794
`
`1’96
`
`Association] of a single-nucleotide polymorphism in the SHZB} gene with
`.MKEWHIV-positive myeloprolife alive neoplasms
`lilodie l.L‘\'lL'\'l'II, Marie Pie-Line. (‘urole ('onejeru 'l'onelti. Stephanie (.iilaudier. Nadine Varinvl'tlnnk.
`l.alll‘u Velazquez. Jean-Jacques Kiitldjitln. Bruno {‘assinttl. and l-‘tmny lial'anuMnl'szak
`
`Hepatitis IC transmission by transfusion of Intercept blood system—treated plasma
`Lisetle llauser. .a\nne-!\«1arie RntltIe-Al‘onso. Alexandre Iteyionne. Marion Simonel.
`Ittiltetliete Dean |-'ise|1er. Nicolas Burin des Roxie-rs. Vincent Mallet. Pierre 'I‘il'nel'gliien.
`
`and Philippe Him-ling
`
`EFlFlATA
`
`793
`
`”Ill! EJ- Sema 3A resists retinal revasecularization. Blood. 201m 1792157855786.
`
`YQB
`
`793
`
`Ilole PS. [)arley Rl., Tanks A. [)0 reactive oxygen species play a role in myeloid
`leukemias? Blood. 2m l;l 1712358166826.
`
`”fill LL- Hydroxyurea makes inflammation “just righl"? Wood. 2012:] [9(8):
`1796-1798.
`
`
`OTHEFl DEPARTMENTS
`xxiii Authurfluide
`
`ib
`
`Classifieds
`
`S_UB_MI_SSIQN tNSTRUCTIONS
`
`All manuscripts. including figures. should he submitted electronically at
`hllptflsuhn:it.hlimiliuurualorg to lidilor-in-Chiel' Bob Lowenherg. Ml). Phl). Before sulnnitting your
`paper.
`review Binmt's Author Guide at httpu’t’hltmdj:nurnal.hentau:logylihrm'y.orgr’sitciauLhorsi'
`nulliorytlide..\hl|ni.
`ll' you need help during the sul‘nnission process. contact the Editorial ()l‘lice by
`phone at 202-776—0548 or via e—mail at edil:in:IIt“-henuuologynrg
`
`x
`
`BLOOD. 30 JANUARY 2014 - VOLUME 123. NUMBER 5
`
`

`

`
`
`
`Free
`
`btood app
`
`Mont!""._.inurtmi of The ritttt’t'lrct‘flt .S‘ocr'c-fir offlentrttoi'ogt'
`[print
`lSFs'N noun-tail. onliire ISSN lfiltt‘tttlltlt.
`is
`ptrhlishcd Weekly every 'I'Iurr‘sday. except
`lor'
`the last
`week in December. til times \t-eeklyt. plus the AS”
`
`Annual Mcetir .-_ Alisa-acts in November. by the American
`Society ol' Hematology infill]. Jill! I. Street. NW. Suite
`‘Jtlil. Washington. IX‘ ltlflh. Printed in the l 'ttiled Slates ol-
`America. Periodicals pistage paid at Washington. l)(‘. and
`additional Itlailing ol'liccs. Him»! is a registered It'adclttut'k
`ol' The American Stricty ol' Hematology.
`
`Postmaster; Send ch
`Ie-ot'--.rdt|ress intonnation to
`Monti.
`lil‘ili'itrtr'l of trite .-’ttta't'it'tut Sor't't‘tr of Hcttttrtttirts‘t'.
`Suhscription titliec. Ittll
`I. Street. NW. Suite l.‘ttl'tl.
`Washington. Itt' jlttlfitr.
`Canadian regulations: I’uhlications Mail Agreement
`JUIHKU-t't. Return irridelivcrahle t‘anadian ad-
`
`No:
`
`dresses to: Circulation Dept. or |)l’t.iM. Wood Walker
`Road. Windsor. UN N‘JA til}.
`
`Manuscript submissions
`(‘onsnll
`the Author tittide printed in each isstlL‘ ol
`tit'ood iand posted on the “clone at wit-ti-.ltioodjotn'nal.
`orgt herm- submitting your manuscript online at Irttpit't
`stllunil.Itiootljoirr'rralorg.
`
`2014 subscription rates
`l digital. Individual
`Individual suhsci'iplions Include print
`tvith I‘SA shipping:
`“a | .tttttt: Individuals with lndia
`shipping: SI .37”: Individuals with other international
`shipping: H.350.
`Institutions print
`'- digital tivith USA shippingr‘. Tier I:
`$l.httfi. Tier 3:‘iiitiltlt.'i‘ier}:$l.h’2ll.'1'ier-l and 'I‘icrfi:
`contact ASH tor a quote.
`Institutions print + digital rvvith India shipping}: Tier I:
`Si .975. Tier 1'. $31K)“. Tier 3: film“. Tier 4 and Tier 5:
`contact ASH tor a quote.
`ttvith other irtlcrnalittltill
`Institutions prirrt
`t digital
`“linemen Tit-i- r: stsss. our 2: stain. Tier .1: slate.
`
`'I‘iel'4 and Tier 5: contact A3“ for a quote.
`Institutions digital oniy'. 'I let
`i: Fii.~lttt|. Tier 2'. MAI-ii.
`Tier .i: 5111115.
`
`Price for singletbactt issues: "545 t t‘SA r. $52 twitlt
`lndia shippingt. $5“ [with other international slrippitlt-‘L
`Paysper-view: hull-test onlinc access to all articles
`is free one year ai'ter puhlicttliorr. Norrstthsct'ihet's tttay‘
`purchase 24-hour access to the hill test ol' any article
`pirhlislrcd in
`the preceding l1 months through the
`pay-per» view option on the Blood website.
`
`Subscription information. claims.
`and changes of address
`Customer senile-3 for subscribers: 16653234560 (US and
`Canada): 1-202-292-0280 (outside US and Canada): fax:
`1-202~292-6010: &mail:btoodsubs@hematologyorg. ('or-
`respondencc
`regarding suhscr'iplions and changes
`ol'
`address should he addressed to Mme! Journal Subscrip-
`Iiorr Ullice. Still
`l. Street. NW. Suite ‘Nlll.
`\‘ir’ashirigttut.
`|)(‘ loom (‘lrecks should he made payable to “Mint
`Stilrseriptions."
`ASH Mcttthcrs should send their change-ol'—addrcss
`inlormation to the Mill Menihership IJcparunenl. 302i
`I. Street. NW. Suite titttt. Washington. |)(' Jilttio,
`All change«ti-address notices should he sent at least
`it wcelis hcl'ore the tirst issue is to he mailed to the new
`address. I’r'ovitle holit old and new addresses.
`Claims: ('laims must he submitted a ithirr -1 months of
`
`the pttlllicalion date to months for international \trhscrihcr'st.
`
`The American Society of Hematology
`2021 L Street. NW. Suite 900
`
`Washington. DC 20036
`
`Internet
`Blood Home Page: \s'svti .hluiodjirur'nalairg.
`ASH Home Page: \v\vw.Ileniatologyorg.
`Manuscript Submission: sultrrrit.|tloot|_'|orir'nal.sarg.
`
`Copyright
`('opyr'iglit 6'} 2tl|4 hy 'l'he i'\nter'icart Society ol'llenratol-
`ogy. All rights reserved. No pan of this publication may
`he reproduced tsec exception hclowt. stored in a re-
`tr'icsal system. translated. or transmitted in any form or
`hy any rrreairs ttmv or hereafter knoun. electronic or
`mechanical. without permission iii writing l'r'om the
`Publisher. The American Society of llematology. Ad-
`dress l'or' cta'r'cspoudencc: Blood l’uhlislrirrg [)I'lice. Ettll
`I. Street. NW. Suite ‘Jtlil. Washington. DC Elltljo.
`Important notice: Author's retain certain nonexclusive
`copyrights. For l‘urtlrer intorrnation on rights and permis-
`sions. see unwhitmdjtattr'nalail'gitnisctrightssltttttl.
`The copyright owner consents that copies ol'ar'ticles may
`he made for personal or iotcmal use. or for the personal or
`internal use ot' speeilic clients. for tltose registered with the
`{‘opyrighl (‘lcarancc Center.
`Inc {322 Rosewood Drive.
`Dartvcts. MA {it‘ll}:
`t‘flitl 750—8400: W\\'\\'.L‘ttp)-Tig|tl.
`Court. This consent is given on the condition that the copier
`pay the stated tier-copy tees through the Copyright Clearance
`Center. lnc. tor roprr'trg inn-oral that permitted or .S‘cr'tr'orti-
`it)? and H38 ot' the US (hpi'rtletrt Lure (Fair it's-ct. This
`consent does not extend to other kinds of copying. such as
`copying for general distrihution. l'ot'tulvertising or promotional
`purposes. for creating new collective works. or for resale. For
`those kinds ol‘ pin-poses. permission must he sought than the
`Publisher {see above}.
`
`Advertising representation
`To place orders for product advertisements. classifieds.
`or commercial reprints. contact Kevin Dunn. Fanning--
`hanr Associates. Itttt {lld 'I‘arppan Road. ()ld Tappan. NJ
`tt'i't’r'i'fi,
`'l‘eicplronc: Elliot—4170;
`l'ax: EtlI-Tti?—iitltifi;
`e-rnail: kdtrnrI(“-‘cunrrassocorn.
`
`Disclaimer
`
`The ideas and opinions expressed in Blood do not necessarily
`rcllect those of The American Society ol' 1 lcnraloiogy or the
`union. of films}. Publication ol' an advertisement or other
`pnrtlucl Inentiori
`in Maui should not he construed as an
`endorsement of the product or the manul'actur'er"s claims.
`Readers are encouraged to c: intact the manufacturer with at Iy
`questions about the features or limitations of the products
`mentioned. The American Society of l-lematology does not
`assume arty rcsrsrnsihility for any injury andtor' damage to
`persons or property arising from or related to arty rise ol' the
`material contained in this periodical. The reader is advised to
`check the appropriate medical
`litenrtnrc and the product
`inlor'mation currently provided by the nrarrulitcturet' ot'each
`drug to lie administered to verily the dosage. the method and
`duration of administration. or c:rntr'aindications.
`It
`is the
`n-sluinsihility ol' the treating physician or other health care
`professional.
`relying on his or her independent experi-
`ence and knowledge of the patient.
`to determine drug
`dosages and the best treatment for the patient.
`
`Indexing 8. abstracting
`.VletiiL'tts.
`Blood is
`indexed and :thstt'actctl by [tides
`liscerpta Medica. (‘urrcrrt L‘ontcnlsi’ljl'e Sciences. ('tir-
`rent (‘ontentsr’t'lirrical Medicine. Science t'itation index.
`SE'ISI-LARt‘ll. Automatic .‘s'nliject
`(‘itatiorr Alert.
`ISIt
`lilthl-LI). and BIOSIS.
`
`

`

`Regular Article
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`IMMUNOBIOLOGY
`
`Anti-KIR antibody enhancement of anti-lymphoma activity of
`natural killer cells as monotherapy and in combination with
`anti-CD20 antibodies
`
`Holbrook E. Kohrt.‘ Ariane Thielens.2 Aurelien lvtarabelle.“3 ldit Sagiv—Barti.1 Caroline Sola.2 Fabien (Shanon.2
`Nicolas Fuseri,2 Cécile Bonnatous? Debra Gzertlviriski,1 Amanda Rajapaksa.‘ Erin Waller.‘ Sophie Ugolini.3 Eric \t’ivier.4
`Francois Flomagne.2 Ronald Levy.1 Mathieu Blery.2 and Pascale titlndré2
`
`‘Department of Medicine. Division of Oncology. Stanford University. Stanlord. CA;
`Cancerologio de Lyon. Centre Leon Berard. Université de Lyon. Lyon. France; and
`UM2. Parc scienlilique el Iechnologique de Luminy. Marseille. France
`
`ytnnale Pharma. Marseille. France; ”Centre rte Recherche en
`JCentre d'lmmunologie de MarseilIe-Luminy. Air Marseille Universilé
`
`
`
`
`- Blockade of inhibitory Kle
`with MHC class I antigens on
`lymphoma cetls by 3,“me
`antibodies augments NK-cell
`spontaneous cytotoxicity.
`In combination with anti-CD20
`mAbs anti-KlFi induces
`_
`-
`grhanlfideEefiiignri‘edlated‘
`l‘l UXllTl
`- 8
`.
`‘
`_
`cytotoxtcny against lymphoma.
`
`
`
`'
`
`Natural killer {NK} celts mediate antilymphoma activity by spontaneous cytotoxicity and
`antibody-dependent cell-mediated cytotoxicity {ADCC) when triggered by rituximab, an a"“'
`0020 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. The balance
`of inhibitory and activating signals determines the magnitude of the efficacy at NK cells
`by spontaneous cytotoxicity. Here, using a killer-cell immunoglobulin-Iike retoolitlti'r {Kim
`transgenic murine model. we show that blockade ot the intertace of inhibitory Kilns-trillth
`mat-or histocompatibility complex tiltiHC) class I antigens on lymphoma cells by anti-KlFl
`antibodies preyents a tolerogenlc interaction and augments NK-cell spontaneous cn°t°*'
`icity. In combination wrth anti-(3020 mAbs. anti-KIR treatment induces enhaHCEd NF‘
`cell-mediated. ritualmab-dependent cytotoxicity against lymphoma in vitrc and in vivo In
`KIFt transgenic and syngeneic murine lymphoma models. These results supporta therapeu'
`tic strategy of combination rituximab and KIR blockade through Iirilumab. illustrating "‘9
`potential etficacy ot combining a tumor-targeting therapy with an NK-cell agonist. thus
`
`Introduction
`
`lmtiitme checkpoint blockade represents a promising cancerlhcrapy
`that ailiis to restore an cl'lieient antttuliioral response mediated by
`endogcnous intiiiulic cells. Antibodies to ( ILA-4. all Inhibitory
`receptor that dampens 'l'—eel| receptor t'I‘CRt signaling. enhance
`immune cell function by blocking a negative regulator. ("l‘LA-4
`shares ("080 (Bi. l) and (1)86 H3732} it-‘i
`ligillltl-‘i Willi llte TCR
`costimulatory receptor (1)23. The intracellular signals Iraiisduced
`by the 'l‘CR. ('[)28. and ("l‘LA—Li determine the outcoliic ol'
`'l‘—ce|l
`activation.“ The therapeutic concept of irrimuliornodulation was
`validated by the approval ol‘aliti—("l'LA—él ipiliinulnab
`in metastatic
`.
`.
`-
`I.t
`.
`.
`.
`liiclanoma. thus increasing overall survival.
`()thcr inhibitory rc-
`ceptors ol' '|'-cel|
`l'tiiiction. such as Pl)—l and LAG—3.
`are being
`targeted by therapeutic monoclonal antibodies thst in clinical
`and preclinical devclopmelil. ""‘S
`As a corollary to targetilig negative regulators til T cells. we
`hypothesized that the killer cell irninuiiriglobtilin—like receptor tKlRt
`I'amily ol‘naltiral killer (NK] cell negative regulators w
`.
`.
`.
`.
`otild represent
`a novel and active class ol imliitlnotherapy.
`' Indeed. NK cells play
`critical roles in host delenseagainst inl'cctions and Illinois by
`secreting
`ausloniicd
`iniliitinoregulatoiy cytokines and by killing inl'ected or ti'
`
`Submitted August 2. 2013: accepted October 14.2013. Prepublished online as
`Blood First Edition paper. December to. 2013: DOI 10.1182tblood-2013-08-
`519199.
`
`H.E.K.. A.T.. Hi... MB. and PA. contributed equally to this study.
`The online version of this article contains a data supplement.
`
`cells. The activation ol' NK—ccll e l'l'cctor l'tmction is regulated by
`multiple types ol‘ activating and ilihibitol},r receptors. including KIR-
`that recognize ligands expressed on potential target cclls.1 he balance
`between positive and negative signals transmitted via these NK
`receptors deterlitines whether or not a target cell is killed by an NK
`cell.T In addition.
`lack of KlR—l-liA class I interactions has been
`associated with potent NK—rnedialed antitumor efficacy and ill'
`creased survival iii acute niycloid leukemia tAMl.t patients upt‘”
`liaplo—idcntical stem cell
`transplantation I'rom KIR titismatchctI
`doliors.x 'l‘o exploit
`this pathway pliarmacologieally.
`the l‘ully
`.
`.
`‘3
`I
`human iii/\b anti-Kilt
`l—7l5‘) tll’l-lthilt w
`as lost generated.
`«Ind
`It
`tell a recoliibinalit version ol‘ this mAb was developed will!
`a stabilized hinge tlirilumabt. l-i'l‘") arid lirihimab mAbs cross—rearI
`with KIRZI.)I.I.
`--|.2. and AL} receptors alld impair their inhibitor!
`signaling by preventing their binding to HI .A-('. [It vitro. anti-KlR
`t‘lti’\i‘ts augitielited NK-eell-tttcdiated lysis of lll,A—(‘
`—expl't'$$i"£1
`tumor cells.
`Winding illltologous AMI. blasts and tltlloltig‘UU"
`(‘DIRX‘ multiple niycloma eclls.""” in addition. splcnocytcs i‘rom
`"mint hifilttL‘orlipatibility complex class I tMH(.‘-l]—delieiclil micc
`C’ii‘I‘L'ti-iing l-ll.AA('w3 Were rejected in it) hours from Ragl K0 "1"“:
`
`
`The publication costs ol this article were defrayed in part by page Charge
`payment. Therefore. and solely to indicate this fact.
`this articie is hereby
`marked "advertisement" in accordance with 18 USC section 12’34.
`
`(D 2014 by The American Society of Hematology
`
`era
`
`BLOOD. 30 JANUARY 2014 - VOLUME 123. NUMBER 5
`
`

`

`BLOOD. an JANUARY 2014 - VOLUME 123. NUMBER 5
`
`KIFl BLOCKADE ENHANCES RITUXIMAB ACTIVITY
`
`679
`
`expressing K|R2DL3 with increasing doses of l—7F‘). dcntonstrat'
`mg lliat
`in vivo blockade of KIR IILA class I interactions could
`mediate rejection of HLA-(‘—esprcssing cells."'II In mice. the W49
`rec“Phil‘s have functions similar to human [(le and biiid to marine
`”*2 (MHC—I) molecules. We demonstrated a beneficial effect ol‘
`blocking H—Z-l.y49 interactions in vivo in combination with
`lettalidomidc in rejecting MHC—l—posltive tumor cellsm A phase I
`clinical trial irt elderly patients witli AM]. was performed with an
`escalating—dose of L71“). Results demonstrated that
`the l—TF‘J
`I1.1m,liljcctions were safe and could block KIR for long periods of
`time (more than 2 weeks at
`l and 3 mgi’kgl Willi limited 3"“ch
`affects.”
`More than a decade prior to the approval ol‘ipilimtunab. a murine-
`htlnian chimeric inimiinoglobulin GI (lgGll :lnlil‘ml)’ “Ei'l'l-‘l CD2”
`Ci'llCll I'ilttsimab was approved and has since heconie a standard
`”Cilltttent for patients witlt B-cell lymphomas. Although riiuximah
`has multiple mechanisms ot'aetion. aritibody-dcpcndcn| CL‘ll'lnt-‘dlillm
`Cytotoxicily tAIJC‘C) is of particular importance. Nettlmliaiug anti-
`bodies that prevent
`the Fe—FeR-y interaction abnoglllc the [3'03“—
`ilcplcting arid antilymphorna activity in vitro” and in vivo itt Itttll‘illc
`Inotlets. "" "' In clinical practice. chRllt/t polynmriihism with a higher
`alliniiy fur lgGl is associated with a higher response rated?” Because
`the response rate to ritusimab among patients with relapsedhcl‘ractoty
`ly'llllhoma may be approximately halt‘ that of patients previously
`l~|tt|t‘eatetl. we investigated whether NK-cell iinntunomodulation h)”
`L‘t'ttnbination of blockade of inhibitory KIR by lirilurnab aiid stimu—
`lilhttlt via Fclell by rituximab could enhance aritilymphomael'lieacy
`m Preclinical models.
`.
`. Here we present results that support the monotherapy activity of
`“ml“"ilh in a KIRZDL3 transgenic lymphoma model and of anti-
`LYNCH I-‘tab’tg in a C5TBU6 syttgeneie lymphoma model. In botli
`models tested. combination of either of those with an anti—("1330
`mAh significantly improves the antitamorefficacy ot‘anti—(‘DZU INNt
`alone. These findings provide rationale for a clinical trial in lymphoma
`IHVestigaling this novel combination of an anti-KIR ntAb to tilt the
`hillilttcc of inhibitory and stimulatory NK Slgllill-‘i lavoring 51'l’ll'llimc'
`“US C)ltotosicity and to further enhance the ell—tum)“ ”l "lllml'm'b by
`llilgmentation ol‘ ADCC.
`
`
`
`Material and methods
`
`Cell lines and mice
`
`line I.(‘[. 721.22l was ti'ansdtieed with lentiviral particles
`Huomn B-ccll
`mm‘ll'lls‘ L‘Illher INA-Cw?! (221 I'IIAAL‘wM or enhanced greeir fluorescent
`¥:i::::;lljciil-'l’l as a control {22 I." from which sequences were cloned into
`hmmm' Elli?“ Plilfiltlld. HA mtII'Inc 'l lymphoma cells were transduced with
`L'Llltirmd" J] complementaryDNA [IEI.4’I1|I('Id?iIl.‘liltlll cell
`lines were
`"Ivtited
`l" Rl rllt’ll-lti-lll medium tlrivtli'rigetif[.r|e [eehnologiest supple-
`lll'll Llfri‘lllh Ill ril- heat-Inactivalcd letal call scrum tl-IyL lone I.;Il1ttrillllll'tt.‘.\'l,
`l-il'c'l‘CL-i
`. penicillin and liltl ugfiiil. streptomycinIlhoth lnorn Invttrogcnl‘
`l‘l'l'ttv-u .IETMWCSJ' 3 niM i.-glutainrnc {Sigrnavpldrtcht arid | niM sodium
`{minor-{HI Ignia-Aldrlrchi. with the addition til 50pr J-Inercaptoethanol
`.l'l'tii" :
`Flr'clu loi'
`I‘.I.—-l- only. (‘ells were grown tti suspension culture at
`"15% ((1..
`lRillltllgl‘CliIhrm ltI-n-eek-tiltl male and female Rag] KO 'i‘g KIRBIHJ
`lllcilltim ._
`t1 Kl-R-t once were bred and maintained at Innate I’hai'nia animal
`0,. u (.gj'lgltl‘wt's l‘t'lllltgL‘n-llcc maintains. Ragl so] lg shunts Itltt‘t.‘
`llld l-fljm. {Hi h"Vl‘llI'ttnlntl were previously described.
`Iztght— to Ill-week-
`hltllsed ‘11; fi'l'lilio riirce were purchased troin (liarlcs lover und were
`('L'Ittcr- Inuit: Laboratory Allttltlttl I‘actlity at the .‘ilarilord Ilnrvcrstty Medical
`[Imam l’h- L-“l"t-'|'l|-IIL‘nIs using Ragl K()-- [g KIR mice were approved by the
`stt‘lttn cllircs committee in accordance with the lzttropean directive
`
`lispci'iments using (5“on mice. were ap-
`for animal csperirricntation.
`proved by Stanlor‘d Laboratory Animal ('arc and were conducted in
`accordance with Stanford University Animal Facility and National liistitutes
`of | Iealth guidelines.
`
`Therapeutic antibodies and reagents
`
`-I,2. Li, -SI_ 81
`Anti-KIR limb is a fully human IgG4 anti-KIR'lIJLI.
`produced in the (‘l K) cell line tliriltltnahi'l I’I-II‘. IIJZIBMS JJlioil IS ti.u Ariti-CDEU
`limb is a chimeric human lgt‘il
`tritusirnab lh-Iab'l‘hcral'. Roclici. Pm.
`coerytliriIi-conjugated I'u'iluinab was produced at
`lnriate I’liarma.
`l-‘tab'it
`fragments of :inti-I.y4‘JL‘i"I tfiliol IttNtti' were provided by lnnalc l’harma.
`The ('A'Iillolili hybridoma cell
`line was purchased front
`Iletttsche
`Sanmtlung von Milttoorganismcn iitid Vellkulturcn. These cells were ttscd to
`produce the :inti-bu(‘l)2tl (“111$ Igtila mob. Irrelevant antibtalies. with
`the same isoiypes as lirilitinab thlg(34i and rituxiuiab tcliiriieric lgGl i. 5'}
`normal mouse serum. and 4|}l | {Ly-I‘JGZ. a ntAb against Lynltltjl specific-
`l'or ll’i-lhlt were used as controls.
`
`in vitro NK tunctional assays
`
`To evaluate NK-cell cylotoxicity against [Ll .4-huCl)3tl. chromium release was
`performed as described. I" Percent lysis was delcnnincd after “I -hoar cu lturcs of
`pitrilicd NK cells at variable ellectoritarget cell ratios with ‘ '(‘r-labcled lym-
`phoma cells III media alone.or with anti—l .y4¢J(‘ll tfilib: lllughtil.taloric.anti-
`(1)1" mAI-i t('AT—l_'l: Ill ughnl.) alone. or the combination of aritivl.y-I‘)Cl'l
`and nnti-(‘DZtl mobs [both at
`IllI uglinld. 'I'oevaluate NK-cell dcgranulation.
`(‘l ) l ll'ia mobilization was assayed as detailed previously . "' ' ( ‘clls were cultured
`for interferon-yI [IliN-rvt assessment by using conditions siriiilar to those
`detailed above for nsscssriicnt of cytotoxicity. Supernatant was analyzed for
`IIiN-y h). enxynie-lirilted immunosorbent assay. In vitro assays were per-
`formed with 3 independent samples.
`
`Tumor transplantation and antibody therapy
`
`In turnonil challenge experiments. [1.5 X ill“ lilAvliut'lfltl lymphoma tumor
`cells in fill pl. of phosphatc-buflcrcd saline \vcrc injcctcd rctro—orhitallv into
`wild-type (‘Sililfli mice. Irt Lilli] pd. ol‘ Iiluispirate—buffered saline.
`Ill:f Ell
`cells were injected intravenously into RagthL’l'g KIR mice. Arrli—(‘D'Ztl
`mAbs (350 pg per itijcctiortt and Bill! pg of I‘tah'ty fragments of antivl.y4‘JC'fl
`were administered by intrapaitoncal injections. whereas all human niAbs were
`administered by intravenous injection. Ttiriior growth tl‘llA—liuCllltl tumor
`ntodclt and survival {23! tumor modelt were monitored for hill to lfitl (Ito's after
`tumornl challenge. 'I'unior giowth was incasuncd by caliper twice a week trial
`expressed as length times width in sultan: ccntiriietcis. Mice were euthanimt
`when tuntor size rcaclted -I cut" or when tumor sites ulcerated.
`
`Depletion of ODS T cells. NK cells, and macrophages
`
`Anti-CUB tnAb thybridonia 2.43]. anti-Asian-(iMI mAb tWaito ('liern-
`icalsl. and liposontal elotlronatc thissrnan Laboratory. Stanford. Cm were
`used to deplete CUR 'l'—ce1|. NK‘ccll. and macrophage activity in \-'ivo in
`(‘fiTBIJo mice. respectively, Depicting autilClhl mAb tillll pgl and anti-
`Asialo—GMI inAb tilt at.) were injected intraperiloncally on day — 1. day [l of
`tumor inoculation. and every 5 days thereafter for 4 weeks. Liposomal clodrorialc
`was injected i