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`Page 10664 =
`
`=e Cancer
`Research |
`
`Page 10861 Abcee eel ge
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`
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`bh Receptor Kinases
`MeCTP me LC|
`URese ita
`Page 10815
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`OSes)
`Supports ATM
`Function after
`SW)Coydemeats
`
`Miltenyi Ex. 1009 Page 1
`
`

`

`
`
`Cancer Research
` A Journal of the American Association for Cancer Research
`Volume 66 - Number 22
`November 15, 2006 + Pages 10639-11084
`
`
`
`
`A Causal Role for the Human TumorAntigen Preferentially
`Expressed Antigen of Melanomain Cancer. Mirjam T. Epping
`genic: René: Beriiards wssesewivicnsmcsra winecuacesenenueuiuanRS 10639
`
`Oncogenes, Trousseau Syndrome, and Cancer-Related Changes
`in the Coagulome of Mice and Humans.Janusz Rak, Joanne L. Yu,
`James Luyendyk, and Nigel Mackmansssessssssssssesesessescsssccseesesseerseseeseneesens 10643
`
`Caveolin-1, Mammary Stem Cells, and Estrogen-Dependent
`Breast Cancers.Federica Sotgia, Hallgeir Rui, Gloria Bonuccelli,
`Isabelle Mercier, Richard G. Pestell, and Michael P. Lisanti..sssssssssscssssssssese 10647
`
`Meeting Report
`
`Markers and Tissue Resources for Melanoma: Meeting Report.
`Dorothea Becker, Martin C. Mihm, Stephen M.Hewitt, Vernon K. Sondak,
`
`Jane W. Fountain, and Magdalena Thurin.............
`sseseeeeees 10652
`
`Priority Reports
`
`TMPRSS2:ERG Fusion by Translocation orInterstitial Deletion
`Is Highly Relevant in Androgen-DependentProstate Cancer,
`But Is Bypassed in Late-Stage Androgen Receptor-Negative
`Prostate Cancer. Karin G. Hermans, Ronald van Marion, Herman van Dekken,
`GuidoJenster, Wytske M. van Weerden, and Jan Trapman ...s.sessssessesesserees 10658
`
`Epigenetic Inactivation of the HOXA Gene Cluster in Breast
`Cancer, Petr Novak, Taylor Jensen, Mare M. Oshiro, Ryan J. Wozniak,
`Marcella Nouzova, George S. Watts, Walter T. Klimecki, Christina Kim,
`para Bernard Wi, Bubechercissccswesimuaumieneuecaeansercibarcmauineauysdae 10664
`
`p53 Attenuates Cancer Cell Migration and Invasion through
`Repression of SDF-1/CXCL12 Expression in Stromal Fibroblasts.
`Neta Moskovits, Alexander Kalinkovich,Jair Bar, Tsvee Lapidot,
`Rae NEOSH1G OTEopsnnswssicnsensoncansncasnveactvedssxusivbuaniisdvassckeuvusesiaveuidsisnnatvancbionds 10671
`
`HumanArrest Defective 1 Acetylates and Activates B-Catenin,
`Promoting Lung CancerCell Proliferation.Ji-Hong Lim, Jong-Wan Park,
`Bid LANG-SOGK: Chiltissicscssseecusiseavecsccacscnecnciveaersssassnnyiantanaesuaiseannieives 10677
`
`Molecular Biology, Pathobiology, and Genetics
`
`Decreased NKX3.1 Protein Expression in Focal Prostatic Atrophy,
`Prostatic Intraepithelial Neoplasia, and Adenocarcinoma:
`Association with Gleason Score and Chromosome8p Deletion.
`Carlise R. Bethel, Dennis Faith, Xiang Li, Bin Guan,Jessica L. Hicks,
`Fusheng Lan, Robert B. Jenkins, Charles J. Bieberich,
`and Angelo M. De Marz.....ssescsssssossssscsesscnessanercanevsanessenenscuesecuesennesecessees 10683
`
`LKB1 Is Recruited to the p21/WAF1 Promoterby p33 to Mediate
`Transcriptional Activation. Ping-Yao Zeng andShelley L. Berger....+. 10701
`
`NewPotential Ligand-Receptor Signaling Loops in Ovarian Cancer
`Identified in Multiple Gene Expression Studies, Giancarlo Castellano,
`James F. Reid, Paola Alberti, Maria Luisa Carcangiu, Antonella Tomassetti,
`and Silvana! CANCVALE ssorseevesencesarssennsersssussnszsvecusutinaeavantes setbeeaaaetevsoreverasas 10709
`
`Improved Tumor Control through Circadian Clock Induction by
`Seliciclib, a Cyclin-Dependent Kinase Inhibitor. Ida lurisci,
`ElisabethFilipski, Jens Reinhardt, Stéphane Bach, Athos Gianella-Borradori,
`Stefanolacobelli, Laurent Meijer, and Francis L6Vi....tsssessssssssssessessseseseeses 10720
`
`Up-regulation of Expression of the Ubiquitin Carboxyl-Terminal
`Hydrolase L1 Gene in HumanAirwayEpithelium ofCigarette
`Smokers.BrendanJ. Carolan, Adriana Heguy, Ben-Gary Harvey,
`Philip L. Leopold, Barbara Ferris, and Ronald G. Crystal....ccssseccsoressenerereese 10729
`
`Molecular Mechanismsof RET Receptor-Mediated Oncogenesis
`in Multiple Endocrine Neoplasia 2B.Taranjit §. Gujral, VinayK. Singh,
`ZongchaoJia, and Lois M. Mulligan..sssssessssscsssseseerssensseneseneessdeceit 10741
`
`Mutantp53 Protects Cells from 12-O0-Tetradecanoylphorbol-13-
`Acetate-Induced Death by Attenuating Activating Transcription
`Factor 3 Induction. Yosef Buganim, Eyal Kalo, Ran Brosh, Hila Besserglick,
`Ido Nachmany, Yoach Rais, Perry Stambolsky, Xiaohu Tang, Michael Milyavsky,
`Igor Shats, Marina Kalis, Naomi Goldfinger, and Varda Rotter .sessssseseesssees 10750
`
`Chronic Protein Kinase B (PKB/c-akt) Activation Leads to Apoptosis
`Induced by Oxidative Stress-Mediated Foxo3a Transcriptional
`Up-regulation. Ankie G.M. van Gorp, Karen M. Pomeranz, Kim U. Birkenkamp,
`Rosaline C-Y. Hui, Eric W-F, Lam, and PaulJ. Coffer c.c.sscsssessssssssessosensanaees 10760
`
`Epigenetic Regulation of Tumor Endothelial Cell Anergy: Silencing
`of Intercellular Adhesion Molecule-1 by Histone Modifications.
`Debby M.E.L Hellebrekers, Karolien Castermans, Emmanuelle Viré,
`Ruud P.M.Dings, Nicole T.H. Hoebers, Kevin H. Mayo,
`Mirjam G.A. oude Egbrink, Grietje Molema, Francois Fuks,
`Manon van Engeland, and Arjan W. Griffioen .....csssssssssssessseeeseveabetrelaaranis10770
`
`Cell, Tumor, and Stem Cell Biology
`
`Imaging Mitogen-Activated Protein Kinase Function in Xenograft
`Models of Prostate Cancer. RomylaIlagan,Jill Pottratz, KimLe,
`Liqun Zhang, Steven G. Wong, Raul Ayala, Meera lyer, Lily Wu,
`Sanjiv &, Gambhir, and Michael Carey ..ssssessecesssessesessserseeesatsesersesenresenses 10778
`
`A Novel SV40 TAg Transgenic Modelof Asbestos-Induced
`Mesothelioma: Malignant Transformation Is Dose Dependent.
`Cleo Robinson,Ivonne van Bruggen, Amanda Segal, Melissa Dunham,
`Amanda Sherwood, Frank Koentgen, Bruce W.S. Robinson,
`
`CETERACHAU AC LORE.ansvetse
`tleaneesh sgovnnratnnsenipadstenssatuusestavnekstonaséaosutapieneeveres 10786
`
`Telomere-Driven Karyotypic Complexity Concurs with p16'NK4a
`Inactivation in TP53-Competent Immortal EndothelialCells.
`Victoria W. Wen, Kaida Wu, Sheik Baksh, Rebecca A. Hinshelwood,
`Richard B. Lock, Susan J. Clark, Malcolm A.S. Moore.
`md Karen L: MacKenzie sissssiscsasstisisscasnccednnesaviseeororonenensstaqneenscenensnvevanaces 10691
`
`Mass Spectrometry-Based Metabolic Profiling Reveals Different
`Metabolite Patterns in Invasive Ovarian Carcinomas and Ovarian
`Borderline Tumors. Carsten Denkert, Jan Budczies, Tobias Kind,
`Wilko Weichert, Peter Tablack,Jalid Sehouli, Silvia Niesporek,
`Dominique Kénsgen, Manfred Dietel. and Oliver Fiehn .....sssssssseseneesssseseee 10795
`
`Miltenyi Ex. 1009 Page 2
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`Miltenyi Ex. 1009 Page 2
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`}1
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`}
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`|
`
`Contents (Continued)
`
`Oxidative Stress Induces Premature Senescence by Stimulating
`Caveolin-1 Gene Transcription through p38 Mitogen-Activated
`Protein Kinase/Sp1-Mediated Activation of Two GC-Rich Promoter
`Elements. Arvind Dasari, Janine N. Bartholomew, Daniela Volonte,
`and Ferruccio Galbiati -s.cssesresernesssereeserseneensasensessnsesanareesneeenneeeneraraeretse 10805
`
`A Genome-Wide Screen Reveals Functional Gene Clusters in the
`
`Cancer Genomeand Identifies EphA2 as a Mitogen in Glioblastoma.
`FenghuaLiu, Peter J. Park, Weil Lai, Elizabeth Maher, Arnab Chakravarti,
`Laura Durso,Xiuli Jiang, Yi Yu, Amanda Brosius, Meredith Thomas,
`Lynda Chin, Cameron Brennan, Ronald A. DePinho, Isaac Kahane,
`Rona §. Carroll, Peter M. Black, and Mark D. Johnson...cesssscssescssscsscessessnees 10815
`
`Oncogenic Serine-Threonine Kinase Receptor-Associated Protein
`Modulates the Function of Ewing Sarcoma Protein through a Novel
`Mechanism.Govindaraj Anumanthan,Sunil K. Halder, David B. Friedman,
`and Pran‘K; Datta ccsasvsssisesssnticcnaiscasassnnuiieaddwidaianlitinsbaiscindanisinie 10824
`
`Cyclooxygenase-2 Inhibition Suppresses «,(8, Integrin-Dependent
`Oral SquamousCarcinomaInvasion. Maria L. Nystrom, Diana McCulloch,
`Paul H. Weinreb, Shelia M. Violette, Paul M. Speight, John F. Marshall,
`lan R. Hart, and Gareth J. Thomas ...sccsscccsscscssersssccesanreseeesenssseneesenseseneoaes 10833
`
`MicroRNA Responsesto Cellular Stress. Carmen J. Marsit,
`Karen Eddy, and Katl T, Kelsey s-csssssasessssccssecssesatessevisseudecadesodasseusuassveurece 10843
`
` Experimental Therapeutics, Molecular Targets,
`
`and Chemical Biology
`
`Cytosine Methylation Profiles as a Molecular Marker in Non-Small
`Cell Lung Cancer. Mathias Ehrich, John K. Field, Triantafillos Liloglou,
`George Xinarianos, Paul Oeth, Matthew R. Nelson, Charles R. Cantor,
`ceetan 10911
`andDirk van den BOOM csssessrersssererenssseesrserssernsaresstesens
`
`Inhibition of Prostate Tumor Growth and Bone Remodeling by
`the Vascular Targeting Agent VEGF, ,,/rGel. Khalid A. Mohamedali,
`AnnT. Poblenz, Charles R, Sikes, Nora M. Navone, Philip E. Thorpe,
`Bryant G. Darnay, and Michael G, Rosenblum ..secsrsssesesesereesssensssssserseseeree LOGLD
`
`Osteoclasts Direct Bystander Killing of Bone Cancer.
`MargaretL. Ramnaraine, Wendy E. Mathews, James M. Donohue,
`Christine M. Lynch, MichaelJ. Goblirsch, and Denis BR. Clohisy .......+ssssssssssse 10929
`
`Reversal of Drug Resistance in Breast Cancer Cells by
`Transglutaminase 2 Inhibition and Nuclear Factor-«B
`Inactivation. Dae-Seok Kim, Sung-Soo Park, Byung-Ho Nam,
`In-Hoo Kim, and So0-You! Kim ssssssssscssesescssssessscsssessssesenssasnssssssssssesasorss 10936
`
`The Internalization and Degradation of Human Copper
`Transporter 1 following Cisplatin Exposure. Alison K. Holzer
`
`and Stephen B. Howell....ccccsssssscossscssssseconesentenassesenenenssensensonssasionse
`
`SpontaneousTransformation of Cultured Mouse Bone
`Marrow-Derived Stromal Cells, Yi Fu Zhou, Marta Bosch-Marce,
`Hiroaki Okuyama,Balaji Krishnamachary, Hideo Kimura, Li Zhang,
`David L. Huso, and Gregg L. Semenzasrssssessessesenseesesesscssseessensessesesssneses 10849
`
`A Mechanism-Based Combination Therapy ReducesLocal
`Tumor Growth and Metastasis in an Orthotopic Model of
`Prostate Cancer.Boleslav Kosharskyy, Nicolas Solban, Sung K. Chang,
`Imran Rizvi, Yuchiao Chang, and Tayyaba Hasan.....ssesssessssesensersnsessnersesers 10953
`
`Metabolic Imaging by Hyperpolarized °C Magnetic Resonance
`Imagingfor In vivo Tumor Diagnosis. Klaes Golman, René in't Zandt,
`Mathilde Lerche, Rikard Pehrson, and Jan Henrik Ardenkjaer-Larsen ......+ 10855
`
`A Sequential Blockade Strategy for the Design of Combination
`Therapies to Overcome Oncogene Addiction in Chronic
`Myelogenous Leukemia. Rong Chen, Varsha Gandhi,
`and William Plunkett....cccscsesessssssssscssssessesessnsessnessseerseneasresarsssessesseseseners 10959
`
`Inhibition of Transforming Growth Factor-B1 Signaling Attenuates
`Ataxia Telangiectasia Mutated Activity in Response to Genotoxic
`Stress. Julia Kirshner, Michael F. Jobling, Maria Jose Pajares,
`Shraddha A. Ravani, AdamB, Glick, Martin J. Lavin, Sergei Koslov,
`Yosef Shiloh, and Mary Helen Barcellos-Holf....ssssessssesssessssssseecrosnerssseseee 10861
`
`Reassessmentof Id1 Protein Expression in Human Mammary,
`Prostate, and Bladder Cancers Using a Monospecific Rabbit
`Monoclonal Anti-Id1 Antibody. Jonathan Perk, Ignacio Gil-Bazo,
`Yvette Chin, Paola de Candia,JohnJ.S. Chen, Yuntao Zhao, Shirley Chao,
`Wai Cheong, Yaohuang Ke, Hikmat Al-Ahmadie, William L. Gerald,
`Edi Brogi, and Robert Benezra.....csssssssssosssssssessscsscsessesesssensessessessseseesaeneses 10870
`
`Up-regulation of Heat Shock Protein 27 Induces Resistance to
`17-Allylamino-Demethoxygeldanamycin through a Glutathione-
`Mediated Mechanism.Andrea K. McCollum, Cynthia J. TenEyck,
`Brian M. Sauer, David O. Toft, and Charles Erlichman.....s.cssssssssessesessessenes 10967
`
`Biological Properties of IDN5174, a New Synthetic Camptothecin
`with the Open Lactone Ring. Giovanni L. Beretta, Giovanna Petrangolini,
`Michelandrea De Cesare, Graziella Pratesi, Paola Perego, Stella Tinelli,
`MonicaTortoreto, Massimo Zucchetti, Roberta Frapolli, Ezia Bello,
`Carla Manzotti, Gabriele Fontana, Ezio Bombardelli, Arturo Battaglia,
`Cristian Samori, and Franco ZuUNINO..s.000 ere0976
`
`Phosphorylation by DNA-Dependent Protein Kinase Is Critical
`for Apoptosis Induction by Insulin-Like Growth Factor Binding
`Protein-3. Laura J. Cobb, Bingrong Liu, Kuk-WhaLee,
`nenien 10878
`And Pinchas COWEN siscsaicananasci
`
`Generation of a Stable Antioxidant Response Element-Driven
`Reporter Gene Cell Line and Its Use to Show Redox-Dependent
`Activation of Nrf2 by Cancer Chemotherapeutic Agents.
`Xiu Jun Wang, John D, Hayes, and C. Roland Wolf..ssssssessesrssensesessnsernses10983
`
`In vivo Activity of the Cleaved Form of Soluble Urokinase Receptor:
`A New Hematopoietic Stem/Progenitor Cell Mobilizer. CarmineSelleri,
`Nunzia Montuori, Patrizia Ricci, Valeria Visconte, Antonio Baiano,
`Maria Vincenza Carriero, Bruno Rotoli, GuidoRossi, and Pia Ragno.......... 10885
`
`Oncostatin M (OSM) Cytostasis of Breast TumorCells:
`Characterization of an OSM Receptor B-Specific Kernel.
`Nicholas Underhill-Day and John K. Heath....sssssssseseseessneeseneessneesssensererss10891
`
`Immunology
`
`CD28 Costimulation Provided through a CD19-Specific Chimeric
`Antigen Receptor EnhancesIn vivo Persistence and Antitumor
`Efficacy of Adoptively Transferred T Cells, Claudia M. Kowolik,
`MaxS. Topp, Sergio Gonzalez, Timothy Pfeiffer, Simon Olivares,
`Nancy Gonzalez, David D, Smith, Stephen J. Forman, Michael C. Jensen,
`and LaurenceJ.N. CoOper....ssesseecsseressseessnseasessavansseceesearsseneaeeseaeaviseersasars 10995
`
`Gonadotropin-Releasing Hormone Promotes Ovarian CancerCell
`Invasiveness through c-Jun NH,-Terminal Kinase-Mediated
`Activation of Matrix Metalloproteinase (MMP)-2 and MMP-9.
`Lydia W.T. Cheung, Peter C.K. Leung, and Alice S.T. WOng.....-srssssesserseeees 10902
`
`Enhanced Antitumor Response by Divergent Modulation of
`Natural Killer and Natural Killer T Cells in the Liver.
`
`JeffJ. Subleski, Veronica L. Hall, Timothy C. Back, John R. Ortaldo,
`and Robert H. Wiltrout ..ccccccccsseeersssesssessseesensAINMHUE LOUD.
`
`Miltenyi Ex. 1009 Page 3
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`Miltenyi Ex. 1009 Page 3
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`

`

`Contents (Continued)
`
`Functional CXCR4-Expressing Microparticles and SDF-1
`Correlate with Circulating Acute Myelogenous LeukemiaCells.
`Alexander Kalinkovich, Sigal Tavor, Abraham Avigdor, Joy Kahn,
`AlexanderBrill, Isabelle Petit, Polina Goichberg, Melania Tesio,
`Neta Netzer, Elizabeth Naparstek, Izhar Hardan, Arnon Nagler,
`Igor Resnick, Alexander Tsimanis, and Tsvee Lapidot .....ssssessssesssssssssssses11013
`
`Endocrinolo
`
`Ciz1, a Novel DNA-Binding Coactivatorof the Estrogen
`Receptor a, Confers Hypersensitivity to Estrogen Action.
`Petra den Hollander, Suresh K. Rayala, Dawn Coverley,
`and Rakesh Kumarscsinsveescussovssesussceeentscnsndssteossiseaiseaiionssneserssarseeseeee LOZ
`
`An Inherent Role of Integrin-Linked Kinase-Estrogen
`Receptor @ Interaction in Cell Migration.Filippo Acconcia,
`Bramanandam Manavathi, Joseph Mascarenhas, Amjad H. Talukder,
`Gordon Mills, and Rakesh Kumar ...ssssssssssersssessssseersenrsenenseesavevanansnaiaane 11030
`
`Steroid Receptor Coactivator-3 and Activator Protein-1
`Coordinately Regulate the Transcription of Components of
`the Insulin-Like Growth Factor/AKT Signaling Pathway.
`Jun Yan, Cheng-Tai Yu, Mustafa Ozen, Michael Ittmann,Sophia Y. Tsai,
`atid Ming-Jer TSah ciscsisscseuvascawsscoceseiicssasscssscesasccssinseaccccdcbaastecsventiavceseceiety 11039
`
`Receptorfor Activated C Kinase 1 cRACK]1and Src Regulate
`the Tyrosine Phosphorylation and Function of the Androgen
`Receptor. Sarah Kraus, Daniel Gioeli, Tomas Vomastek, Vicki Gordon,
`anid: Michiel J: WeDer cissssecneesicssvansieeceigenpsesiuoieiesaesubectebacsmeesanstaneiuaunsssbiaen 11047
`
`Cancer Research will no longer print author and subject indexes for each yearly
`volume. The majority of our readers report that they now useonline search
`
`capabilities to find articles (http://cancerres.aacrjournals.org).
`
`Clinical Research
`
`Molecular Imagingof Proliferation in Malignant Lymphoma.
`Andreas K. Buck, Martin Bommer, Stephan Stilgenbauer, Malik Juweid,
`Gerhard Glatting, Holger Schirrmeister, Torsten Mattfeldt, Djurdja Tepsic,
`Donald Bunjes, Felix M. Mottaghy, BerndJ. Krause, Bernd Neumaier,
`Hartmut Dohner, Peter Maller, and Sven N. Reske ...esssssssssssssssssssssnessssesses 11055
`
`Epidemiology and Prevention
`
`DNA Repair and Cell Cycle Control Genes and the Risk of
`Young-Onset Lung Cancer. Stefano Landi, Federica Gemignani,
`Federico Canzian, Valérie Gaborieau, Roberto Barale, Debora Landi,
`Neonilia Szeszenia-Dabrowska, David Zaridze, Jolanta Lissowska,
`Peter Rudnai, Eleonora Fabianova, Dana Mates, Lenka Foretova,
`Vladimir Janout, Vladimir Bencko, Lydie Gioia-Patricola, Janet Hall,
`Paolo Boffetta, Rayjean J. Hung, and Paul Brennan ..ssssessesseserssnssseerseneoeers 11062
`
`Persistent Human Papillomavirus Infection Is Associated
`with a Generalized Decrease in Immune Responsiveness
`in Older Women.Alfonso J. Garcfa-Pifieres, Allan Hildesheim,
`Rolando Herrero, Matthew Trivett, Marcus Williams, Ivannia Atmetlla,
`Margarita Ramirez, Maricela Villegas, Mark Schiffman. Ana Cecilia Rodriguez,
`Robert D. Burk, Mariana Hildesheim, Enrique Freer, José Bonilla,
`Concepcion Bratti, Jay A. Berzofsky, and Ligia A. Pinto..scsresssssessssseneees 11070
`
`Germ-Line Genetic Variation in the Key Androgen-Regulating
`Genes Androgen Receptor, Cytochrome P450, and Steroid-5-a-
`Reductase Type 2 Is Important for Prostate Cancer
`Development.Sara Lindstrom, Fredrik Wiklund, Hans-Olov Adami,
`Katarina Augustsson Balter, Jan Adolfsson, and Henrik Gronberg.......s0s000 11077
`
`Miltenyi Ex. 1009 Page 4
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`Miltenyi Ex. 1009 Page 4
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`
`
`Research Article
`
`CD28 Costimulation Provided through a CD19-Specific Chimeric
`Antigen Receptor Enhances /n vivo Persistence and Antitumor
`Efficacy of Adoptively Transferred T Cells
`Claudia M. Kowolik, Max S. Topp,’ Sergio Gonzalez, Timothy Pfeiffer,’
`Simon Olivares, Nancy Gonzalez,’ David D. Smith,’ Stephen J. Forman,’
`Michael C. Jensen,“ and Laurence J.N. Cooper”
`
`Divisions of ‘Molecular Medicine, "Cancer Immunotherapeutics and Tumor Immunology, *Hematology and Hematopoietic
`Cell Transplantation, and ‘Pediatric Hematology/Oncology and ‘DepartmentofBiostatistics, Beckman ResearchInstitute
`and City of Hope National Medical Center, Duarte, California; ‘Medizinische Klinik und Poliklinik II, Bayerische
`Julius-Maximillian Universitat Wirzburg, Wiirzburg, Germany; and "Division of Pediatrics, University of Texas
`M.D. Anderson Cancer Center, Houston, Texas
`
`Abstract
`
`T cells, the genetically modified T cells showed antigen-specific
`binding, and target cell lysis in vitro, but limited in vivo antitumor
`Chimeric antigen receptors (CAR) combine an antigen-
`efficacy (9). One restriction of a CAR that activates T cells solely
`binding domain with a CD3-¢ signaling motif to redirect T-
`through chimeric CD3-¢ is that engagementof this signaling motif
`cell specificity to clinically importanttargets. First-generation
`does notfully activate the genetically modified T cells, resulting in
`CAR,such as the CD19-specific CAR (designated CD19R), may
`compromised antigen-dependent interleukin-2 (IL-2) production,
`fail
`to fully engage genetically modified T cells because
`cell proliferation, and survival. This is
`in contrast
`to the
`activationis initiated by antigen-dependent signaling through
`physiologic activation of T cells via the endogenous af T-cell
`chimeric CD3-¢, independentof costimulation through acces-
`receptor by antigen-presenting cells (APC), which leads to both a
`sory molecules. We show that enforced expression ofthe full-
`primary signal initiated by the engagement of CD3-¢ and a second
`length costimulatory molecule CD28 in CD8*CD19R*CD28~
`signal provided by ligation of costimulatory receptors, such as
`T cells can restore fully competent antigen-dependent T-cell
`CD28 (10-12). Binding of CD28 on activated antigen-specific T
`activation upon binding CD19* targets expressing CD80/CD86.
`cells to its ligands CD80/CD86 on APCs or tumorcells is essential
`Thus, to provide costimulation to T cells. through a CD19-
`for (a) producing IL-2 (13-15), (b) preventing anergy (16),
`(c)
`specific CAR,
`independent of binding to CD80/CD86, we
`promoting survival through up-regulation of antiapoptotic pro-
`developed a second-generation CAR (designated CD19RCD28),
`teins (17, 18), and (d) prevention of replication senescence (19).
`which includes a modified chimeric CD28 signaling domain
`fused to chimeric CD3-C. CD19R* and CD19RCD28* CD8*
`Because B-lineage malignancies, such as acute lymphoblastic
`T cells specifically lyse CD19* tumor cells. However,
`the
`leukemias, vary or lack expression of CD28 ligands (20-22), and
`CDI9RCD28* CD8*Tcells proliferate in absence of exogenous
`because ex vivo propagation of our genetically modified T cells
`typically leads to loss of expression of endogenous CD28 (8),
`recombinant human interleukin-2, produce interleukin-2,
`propagate, and up-regulate antiapoptotic Bcl-X;, after stimu-
`infused T cells expressing first-generation CARs may be prone
`lation by CD19tumorcells, For thefirst time, we show in vivo
`incomplete T-cell activation,
`leading to truncated in vivo
`that adoptively transferred CD19RCD28* T cells show an
`persistence, compromising the therapeutic success of this adoptive
`improved persistence and antitumor effect compared with
`immunotherapy.
`CDI9R* T cells. These data imply that modifications to the
`Onetarget for redirected T-cell specificity is CD19, a cell surface
`CAR can result in improved therapeutic potential of CD19-
`molecule that
`is
`restricted to B cells and their malignant
`specific T cells expressing this second-generation CAR. (Cancer
`counterparts (23). In this study, we investigated whether we could
`Res 2006; 66(22): 10995-1004)
`provide CD28-mediated costimulation to genetically modified
`T cells through a CD19-specific CAR. Using CD8*CD28~ T cells
`expressing the first-generation CD19-specific CAR (designated
`CD19R), we show that enforced expression of full-length CD28
`transgene in CD19R* T cells could restore CD19-dependent
`costimulation through introduced CD28. To provide fully compe-
`tent T-cell activation through the CD19-specific CAR, which could
`be used to target tumors lacking CD80/CD86, we generated a
`second-generation CD19-specific CAR (designated CD19RCD28),
`by fusing a CD19-specific scFv and modified human Fc region to
`the transmembrane and modified intracellular signaling domains
`of CD28 and CD3-¢. We compared CD19R* and CD19RCD28* T
`cells in vitro and in vivo and show augmented persistence of
`adoptively transferred CD19RCD28”T cells in a nonobese diabetic,
`severe combined immunodeficient
`(NOD/scid) mouse model,
`which results in an increased clearance of established CD19*
`tumor.
`
`
`
`reeeee
`
`Introduction
`
`Adoptive transfer of T cells expressing chimeric antigen
`receptors (CAR) into oncology patients is a promising approach
`to eradicate tumor cells. Initially, CARs designed for adoptive
`immunotherapy were generated by connecting an extracellular
`antigen-binding domain to a transmembrane domain anda single
`intracellular signal transduction domain, such as CD3-¢ or FcRy
`(1-8). When thesefirst-generation CARs were expressed in primary
`
`Requests for reprints: Laurence J.N. Cooper, Division of Pediatrics, University of
`Texas M.D. Anderson Cancer Center, Unit 907, 1515 Holcombe Boulevard, Houston, TX
`77030. Phone: 713-563-3360; Fax: 713-563-0604; E-mail: jncooper@mdanderson.org.
`©2006 American Association for Cancer Research.
`doi:10,1158/0008-5472.CAN-06-0160
`
`www.aacrjournals.org
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`10995
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`Miltenyi Ex. 1009 Page 5
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`Miltenyi Ex. 1009 Page 5
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`www.aacrjournals.org
`thiL-2
`
`Stimulatorcells
`CTLA-4 Ig
`
`Figure 1. Generation and characterization of CD19R*CD28* T cells.A, lysis of
`CD19* leukemia lines (RS4, JM-1, and SUP-B15) by CD19R*CD28™ T-ceil
`clone by 4-hour CRA. Absenceoflysis of CD19~ K562 cells shows specificity of
`killing through CD19R CAR.8,cell surface expression by flow cytometry of
`CD28 on CD19A*CD8* T-cell clone (i) before and(ii) after transduction with
`retrovirus expressingfull-length CD28 transgene. C, IL-2 in culture medium was
`analyzed after 36 hours of coculture of 2 10° T cells and Daudi responder
`tumorcells at 1:1 ratio, D, T-cell proliferation by 9H-thymidine incorporation
`measured at 90 hours after coculture at 1:1 ratio with 2 x 10° mitomycin
`C-treated Daudi stimulator cells supplemented with 10 units/mL rhIL-2 and
`10 pg/mL CTLA-4 Ig as described.
`
`Quantitative reverse transcription-PCR. T cells were cocultured in a
`12-well plate with irradiated (1 Gy) Daudi or SUP-B15 cells at a ratio of 1:5
`(responder/stimulator). The cells were harvested after 0, 2, and 24 hours,
`and RNA for quantitative PCR assays was isolated using the RNeasy kit
`(Qiagen, Valencia, CA). The SuperScript III Platinum SYBR green One-step
`qRT-PCRkit (Invitrogen) was used to reverse transcribe and amplify 100 ng
`of total RNA. Samples were reverse transcribed for 30 minutes at 50°C and
`
`Miltenyi Ex. 1009 Page 6
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`Cancer Res 2006; 66: (22). November15, 2006
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`10996
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`Cancer Research
`
`
`
`%SpecificLysis
`
`25:1
`Effector :
`
`CD19R*
`
`—- RS4
`-O- JM-1
`—A- SUP-B15
`—@ K562
`
`10
`10°
`o¢
`CD8-FITC
`
`§ CD8*CD19R*CD28*
`
`3&a&%P
`
`e
`
`O cpstcp19R*CD28"
`a CDstcD19R*CD28*
`
`Materials and Methods
`
`DNA constructs. CDI9R transgene has been previously described (8).
`The CD19RCD28 transgene was assembled by PCR using splicing by overlap
`extension.
`It was composed of a CD19-specific single-chain variable
`fragment (scFv) derived from the murine FMC63 monoclonal antibody
`(23) fused to a modified (CPSC — CPPC;ref. 24) human IgG4 hinge and Fc
`region fused to residues 153 to 179 ofintracellular human CD28 molecule,
`which included the transmembrane and a modified (RLLH — RGGH;
`ref. 25) intracellular domain, fused to residues 31 to 142 of the human
`cytoplasmic CD3-§ chain (Fig. 2A). The CDI9RCD28 and CD19R transgenes
`were inserted into the plasmid ffLucHyTK-pMG(26) to create the plasmids
`CD19RCD28/ffLucHyTK-pMG (Fig. 2B) and CD19R/ffLucHyTK-pMG,
`respectively. A bifunctional hRluc-Zeocin fusion gene that coexpressed
`the Renilla luciferase (hRiuc) and zeomycin resistance genes (zeo) was
`cloned into peDNA3.1 (Invitrogen, Carlsbad, CA) to generate plasmid hRluc:
`Zeocin-pcDNA3.1.
`Cell culture and genetic modification. T cells were isolated from
`primary peripheral blood mononuclear cells (PBMC), numerically expanded,
`and genetically modified by electroporation as previously described
`(8, 27). Briefly, expansion cultures were established using 10° T cells,
`30 ng/mL. anti-CD3 (OKT3; Ortho Biotech,Raritan, NJ), 5 x 10” y-irradiated
`allogeneic PBMC (3,500 cGy), and 10” y-irradiated allogeneic lymphoblastoid
`cell lines (LCL; 8,000 cGy) in 50 mL culture-medium containing 10% FCS
`(Hyclone, Logan, UT) or 10% human AB serum. Recombinant human IL-2
`(thIL-2; Chiron, Emeryville, CA) was added at 25 to 50 units/mL every
`48 hours, beginning on day 1 of each 14-day expansioncycle. T-cell clones
`were obtained by platingat limiting dilution in 96-well plates as previously
`described (8). Transduction of a CD8°CD28™CD19R* T-cell clone was
`accomplished on day 2 after OKT3-mediated stimulation and 24 hours of
`culturing in phosphate-deficient medium (Invitrogen, Carlsbad, CA)
`supplemented with 50 units/mL rhIL-2. The washed T cells were
`resuspendedin retroviral supernatant (collected from puromycin-resistant
`Phoenix GALV packaging cells) encoding the full-length human transgene
`(28), supplemented with 50 units/mL rhIL-2 and 8 g/mL polybrene, spunat
`1,000 x g for 1 hour at 32°C, and incubated for 23 hours. T cells were then
`washedand cultured in culture medium containing 50 units/mL rhIL-2. The
`leukemia cell lines SUP-B15, RS4, JM-1, Daudi, and CD19”CD80™CD86—
`K562 cells were obtained from the American Type Culture Collection
`(Manassas, VA). The EBV-transformed LCL was kindly provided by Drs.
`Phillip Greenberg and Stanley Riddell (Fred Hutchinson Cancer Research
`Center, Seattle, WA). These lines were cultured as previously described (8).
`Daudi cells were electroporated with the linearized plasmid hRLuc:Zeocin-
`pceDNA3.1 as previously described (27). Three days after electroporation,
`zeocin (InvivoGen) was addedto theculture at a cytocidal concentration of
`0.4 mg/mL.
`Flow cytometry. F(ab), fragment of FITC-conjugated goat anti-human
`Fey (Jackson ImmunoResearch, West Grove, PA) was usedata 1:20 dilution to
`detect cell surface expression of CD19R and CD19RCD28. Theother FITC-,
`phycoerythrin-, and CyChrome-conjugated reagents were obtained from BD
`Biosciences (San Jose, CA). Data acquisition was done on a FACScan and
`FACSCalibur (BD Biosciences). The median fluorescentintensity, coefficient
`ofvariation (CV), and percentageofcells in a region ofinterest was calculated
`using CellQuest version 3.3 (BD Biosciences) and FCS Express version 3
`(De Novo Software, Thornhill, Ontario, Canada). Fluorescence-activated
`cell sorting was done on an Epics Altra (Beckman Coulter, Fullerton, CA).
`IL-2 production. Secretion of IL-2 was assessed following stimulation
`with Daudi cells
`(10° per well) of a CD19-specific T-cell clone or
`CD28 modified CD19-specific T-cell clone (2 x 10° per well). CTLA-4 Ig
`(10 pg/mL), kindly provided by Dr. George Georges (Fred Hutchinson
`Cancer Research Center), was added to selected wells; supernatants were
`harvested after 36 hours; and IL-2 production was analyzed by ELISA (R&D
`Systems, Minneapolis, MN).
`Chromium release assay. A 4-hour chromium release assay (CRA)
`was used to determine thecytolytic activity of T cells. The assay was carried
`out and analyzed as previously described using effector to target (£/T) cell
`ratios of 50:1, 25:1, 5:1, and 1:1 (8). Data are reported as mean + 1 SD.
`
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`Ul
`CAR to Enhance T-Cell Persistence and Efficacy—_-
`$MM—_——>cwwoq_“weuwe——-rr—_—Ee
`
`then subjected to 45 rounds of amplification for 10 seconds at 95°C and
`was restored in transduced CD8*CD19R*CD28* T cells, and IL-2
`30 seconds at 60°C with the Bio-Rad iCycler iQ Multiple-Color Real-time
`secretion could be partially blocked with CTLA-4 Ig (Fig. 1C). This
`PCR Detection System (Hercules, CA) using real-time PCR tested primer
`endogenous production of IL-2 led to the CD19-dependent
`pairs for IL-2, IFN-y, Bel-X,, and glyceraldehyde-3-phosphate dehydroge-
`proliferation of CD8*CD19R*CD28* T cells in the absence of
`nase (GAPDH; Biosource, Camarillo, CA). To confirm accuracy and
`exogenousrhIL-2, which could mostly be blocked by the addition of
`reproducibility, the real-time PCR was carried out in duplicates for each
`CTLA-4 Ig (Fig. 1D). The CD8*CD19R*CD28~ T-cell clone did not
`sample within one iCycler run. Amplification of the GAPDH mRNA was
`proliferate in absence of rhiL-2, but proliferation resulted when
`done on all tested samples as a control for variations in amounts of
`rhIL-2 was provided. Addition of exogenous rhIL-2 further
`RNA. The quantitative PCR results were analyzed using the comparative
`enhanced [*H]thymidine uptake by CD8*CD19R*CD28* T cells.
`C, method (29).
`This was not significantly blocked by CTLA-4 Ig, which is
`Proliferation assay. CD19R* or CD19RCD28" T cells were cocultured for
`72 to 90 hours at a 1:1 ratio with mitomycin C-treated (10% mitomycin C
`consistent with T-cell activation for proliferation through the IL-2
`for 90 minutes) Daudi, LCL, SUP-B15, or K562 cells. CTLA-4 Ig (10 g/mL)
`receptor beyond activation achieved after binding of introduced
`and/or rhIL-2 (10 units/mL) was added to selected wells as indicated.
`CD28 to CD80/CD86 (Fig. 1D). As previously shown for CD8*
`During the last 18 hours, the cultures were pulsed with 1 wCi/well (0.037
`cytomegalovirus-specific T cells, our results suggest that CD8*
`MBq/well) methyl-[*H]thymidine (ICN, Costa Mesa, CA). Cell-associated
`CD19-specific effector T cells do not have an intrinsic block to
`radioactivity was measured byscintillation counting (TopCount, Perkin-
`IL-2 production; rather, the lack of costimulation is due toafailure
`Elmer, Shelton, CT). Data are reported as mean + 1 SD.
`of endogenous CD28 expression (28).
`Xenograft tumor model. On day 0, 24 to 48 hours after irradiation
`Generation of CD19RCD28* T cells. Having shown that
`(y-irradiated to 2.5 Gy using an external '’Cs-source (JL Shepherd Mark I
`enforced expression of full-length CD28 could reestablish fully
`Irradiator, San Fernando, CA), 6- to 10-week-old female NOD/scid (NOD/
`LtSz-Prkdc*/}) mice (Jackson Laboratory, Bar Harbor, ME) wereinjected
`competent CD19-dependent signaling, we investigated whether
`in the peritoneum with 10° hRluc* Daudicells. Beginning on day 3, tumor
`the CD19-specific CAR itself could be modified to provide CD28-
`engraftment was evaluated by serial biophotonic imaging (see below). Mice
`mediated signaling. This is desirable because some CD19*
`with progressively growing tumors were segregated into treatment groups
`malignant
`targets that
`lack CD80/CD86 expression will not
`bearing comparable tumorloads. The mice were subsequently injected in
`provide