EXHIBIT C1
`EXHIBIT Cl
`
`ALVOGEN, Exh. 1055, p. 0119
`
`ALVOGEN, Exh. 1055, p. 0119
`
`

`

`myeloma
`
`Ornencirn for
`lapsed lymphoma
`
`URNAL OF
`AMERICAN
`
`MATOLOGY
`
`Boca le
`
`induced
`
`lymphocyte
`
`apoptosis
`
`(tone UE haels
`|
`CLL
`
`SUCee
`:
`band 3
`
`q
`
`| Thalidomide
`
`DE)
`BSEEile
`
`ALVOGEN, Exh. 1055, p. 0120
`
`

`

`blood
`
`JOURNAL OF
`
`1 NOVEMBER 2000 - VOLUME 96, NUMBER 9
`
`CONTENTS
`
`COVER FIGURE
`
`The tetramerstructure of cytoplasmic erythrocyte membrane band 3.Sec the
`related article by Zhanget al, beginning on page 2925,
`
`THE AMERICAN
`2917=CD40-ligand (CD154) gene therapy for chronic lymphocytic leukemia
`SOCIETY OF
`W. G. Wierda, M. J. Cantwell, S. J. Woods, L. Z. Rassenti, C. E. Prussak, and T. J, Kipps
`HEMATOLOGY
`
`PLENARY PAPERS
`
`2925 Crystallographic structure and functional interpretation of the cytoplasmic domain
`of erythrocyte membrane band 3
`D. Zhang, A. Kiyatkin,J. T. Bolin, and P. S. Low
`
`2934
`
`A phaseI/Htrial of iodine-131-tositumoemab (anti-CD20), etoposide,
`cyclophosphamide, and autologous stemcell transplantation for
`relapsed B-cell lymphomas
`O. W. Press, J. FE. Eary, T. Gooley, A. K. Gopal, S. Liu, J. G. Rajendran, D. G. Maloney,
`S. Petersdorf, S. A. Bush, L. D. Durack, P. J, Martin, D. R, Fisher, B. Wood, J. W. Borrow,
`B. Porter, J. P. Smith, D. C, Matthews, F. R. Appelbaum,and I. D. Bernstein
`
`2943
`
`Thalidomide and its analogs overcome dru
`g resistance of human multiple myeloma
`cells to conventional therapy
`T. Hideshima,D. Chauhan, Y. Shima, N. Raje, F. E, Davies, Y.-T. Tai, S. P. Treon, B. Lin,
`R. L. Schlossman, P. Richardson, G, Muller, D.I. Stirling, and K. C. Anderson
`
`REVIEW ARTICLE
`
`2951 Mechanisms of HIV-associated lymphocyte apoptosis
`A. D. Badley, A. A. Pilon, A. Landay, and D. H. Lynch
`
`CHEMOKINES
`
`:
`.
`2965 Down-regulationof neutrophil functions by the ELR*+ CXC chemokineplatelet
`basic protein
`J. E, Ehlert, A. Ludwig, T. A. Grimm,B. Lindner, H.-D. Flad, and E. Brandt
`
`‘T-lymphocyte production of macrophage inflammatory protein-La is critical to the
`2973
`recruitment of CD8*Tcells to the liver, lung, and spleen during
`graft-versus-host disease
`J. S. Serody, S. E. Burkett, A. Panoskaltsis-Mortari, J. Ng-Cashin, E. McMahon,
`G. K. Matsushima, S. A. Lira, D. N. Cook, and B. R. Blazar
`
`ALVOGEN, Exh. 1055, p. 0121
`
`ALVOGEN, Exh. 1055, p. 0121
`
`

`

`CLINICAL
`OBSERVATIONS,
`
`INTERVENTIONS, AND
`THERAPEUTIC TRIALS
`
`eee
`
`2981
`
`Long-term follow-upof emnsionduration, mortality, and second malignancies in
`hairy cell leukemia patients treated with pentostatin
`I. W. Flinn, K. J. Kopecky, M. K. Foucar, D, Head, J. M. Bennett, R. Hutchison, W. Corbett,
`P. Cassileth, T. Habermann, H. Golomb,K.Rai, E. Eisenhauer, F. Appelbaum,
`B. Cheson, and M. R. Grever
`
`2987~~Diagnostic value of dominantT-cell clones in peripheral bloodin 363 patients
`presenting consecutively with a clinical suspicion of cutaneous lymphoma
`M.-H.Delfau-Larue, L. Laroche, J. Wechsler, E. Lepage, C. Lahet, M. Asso-Bonnet,
`M. Bagot, and J.-P. Farcet
`
`2993
`
`Prognostic significance of CD56 expression for ALK-positive and ALK-negative
`anaplastic large-cell lymphomaof T/null cell phenotype
`R. Suzuki, Y. Kagami, K. Takeuchi, M. Kami, M. Okamoto,R. Ichinchasama, N. Mori,
`M. Kojima, T. Yoshino, H. Yamabe, M. Shiota,S. Mori, M. Ogura, N. Hamajima,
`M.Seto, T. Suchi, Y. Morishima, and S. Nakamura
`
`3001
`
`Ex vivo expanded peripheral bloodprogenitorcells provide rapid neutrophil
`recovery after high-dose chemotherapy in patients with breast cancer
`I. MeNicce,R. Jones, S. I. Bearman, P. Cagnoni, Y. Nieto, W. Franklin, J. Ryder, A. Steele,
`J. Stoltz, P. Russell, J. McDermitt, C. Hogan, J. Murphy, and E. J. Shpall
`
`GENE THERAPY —————
`3008
`Induction of B-cell tolerance by retroviral gene therapy
`J. L, Bracy and J. lacomini
`
`HEMATOPOIESIS
`
`rT
`3016 Murine hematopoietic stem cell characterization andits regulation in BM
`transplantation
`Y. Zhao,Y. Lin, Y. Zhan, G. Yang, J. Louie, D, E. Harrison, and W. F. Anderson
`
`3023=Imprintingof insulin-like growth factor 2 ismodulated during hematopoiesis
`I. M. Morison, M. R. Eccles, and A. E. Reeve
`
`3029 Generation of murine dendritic cells from fit3-ligand-supplemented bone
`marrowcultures
`K. Brasel, T. De Smedt, J. L. Smith, and C. R. Maliszewski
`
`3040
`
`A minimal c-fes cassette directs mycloid-specific expression in transgenic mice
`A, Heydemann, S. Warming, C. Clendenin,K. Sigrist, J. P. Hjorth, and M. C. Simon
`
`ALVOGEN, Exh. 1055, p. 0122
`
`ALVOGEN, Exh. 1055, p. 0122
`
`

`

`HEMOSTASIS,
`THROMBOSIS, AND
`VASCULAR BIOLOGY
`
`IMMUNOBIOLOGY
`
`3049
`
`3056
`
`3064
`
`3070
`
`3078
`
`3086
`
`3094
`
`3102
`
`3109
`
`3118
`
`Characterizationof the protein Z-dependentprotease inhibitor
`X. Han,R. Fiehler, and G. J. Broze Jr
`
`Smooth muscle cell surface tissue factor pathwayactivation by oxidized low-density
`lipoprotein requires cellular lipid peroxidation
`M.S. Penn, M.-Z. Cui, A, L, Winokur,J. Bethea, T. A. Hamilton,
`P. E. DiCorleto, and G. M. Chisolm
`
`Platelet release of trimolecular complex components MT1-MMP/TIMP2/MMP2:
`involvement in MMP2activation and platelet aggregation
`I. Kaves, I. Elalamy, J.-D. Sraer, M, Hatmi, and G. Nguyen
`
`Dimerizationof P-selectin in platelets and endothelial cells
`FJ. Barkalow, K.L, Barkalow, and T. N. Mayadas
`
`Role of SCL/Tal-1, GATA,and Ets transeription factor bindingsites for the
`regulation of F/k-J expression during murine yascular development
`A. Kappel, T. M. Schlaeger, I. Flamme, S. H. Orkin, W. Risau, and G. Breier
`
`The humanantimicrobial and chemotactic peptides L1-37 and «-defensins are
`expressed by specific lymphocyte and monocyte populations
`B. Agerberth, J. Charo, J. Werr, B. Olsson, F Idali, L. Lindbom, R. Kiessling, H. Jérnvall,
`H. Wigzell, and G. H. Gudmundsson
`
`Impaired functionofcirculating HIV-specific CD8* T cells in chronic human
`immunodeficiency virus infection
`P. Shankar, M. Russo, B. Harnisch, M. Patterson,P. Skolnik, and J. Lieberman
`
`Induction of cytotoxic T-lymphocyte responsesin vivo after vaccinations with
`peptide-pulsed dendritic cells
`P. Brossart, S. Wirths, G. Stuhler, V. L. Reichardt, L. Kanz, and W, Brugger
`
`Interferon y and interleukin 6 modulate the susceptibility of macrophages to human
`immunodeficiencyvirus type 1 infection
`M. Zaitseva, S. Lee, C. Lapham,R.Taffs, L. King, T. Romantseva,
`J. Manischewitz, and H. Golding
`
`Correlation of mutations of the SH2D1A gene and Epstein-Barrvirus infection with
`clinical phenotype and outcomein X-linked lymphoproliferative disease
`J. Sumegi, D. Huang, A. Lanyi, J. D, Davis, T. A, Seemayer, A. Maeda, G, Klein, M. Seri,
`H. Wakiguchi, D. T. Purtilo, and T. G. Gross
`
`ALVOGEN, Exh. 1055, p. 0123
`
`ALVOGEN, Exh. 1055, p. 0123
`
`

`

`CONTENTS(continued)
`
`3126
`
`UTYgene codes for an HLA-B60-restricted human male-specific minor
`histocompatibility antigen involved in stemcell graft rejection: characterization
`of the critical polymorphic aminoacid residuesfor T-cell recognition
`M. FE. J. Vogt, E. Goulmy, I. M. Kloosterboer, E. Blokland, R. A, de Paus,
`R. Willemze, and J. H. FE. Falkenburg
`
`NEOPLASIA
`
`3133
`
` Molecularsingle-cell analysis of the clonal relationship of small Epstein-Barr
`virus-infected cells and Epstein-Barr virus-harboring Hodgkin and
`Reed/Sternberg cells in Hodgkindisease
`T. Spicker, J. Kurth, R. Kiippers, K. Rajewsky, A. Briuninger, and M.-L. Hansmann
`
`3139
`
`3147
`
`3154
`
`Highlevels ofsoluble syndecan-1 in mycloma-derived bone marrow: modulation of
`hepatocyte growthfactor activity
`C.Seidel, M. Berset, O. Hjertner, D. Cao, N, Abildgaard, H. Hjorth-Hansen, R. D. Sanderson,
`A. Waage, and A. Sundan
`
`Elevated soluble MUCT levels and decreased anticMUCI antibody levels in patients
`with multiple myeloma
`S. P. Treon, P. Maimonis, D. Bua, G. Young, N. Raje, J. Mollick, D. Chauhan,Y.-T. Tai,
`T. Hideshima, Y. Shima, J. Hilgers, 8. von Mensdorff-Pouilly, A. R. Belch,
`L. M, Pilarski, and K. C, Anderson
`
`Mutations of the AMLI genein myelodysplastic syndrome and their functional
`implications in leakemogenesis
`Y. Imai, M. Kurokawa, K. Izutsu, A. Hangaishi, K. Takeuchi, K. Maki, 8, Ogawa, S. Chiba,
`K. Mitani, and H, Hirai
`
`3161
`
`3168
`
`3175
`
`3181
`
`3188
`
`3195
`
`Involvement of CD44-hyaluronaninteraction jn malignantcell homing and
`fibronectin synthesis in hairy cell Jeakemia
`K. A. Aziz, K. J. Till, M. Zuzel, and J. C. Cawley
`
`Chronic lymphocytic leukemia Bcells inhibit spontancous Ig production by
`autologous bone marrowcells: role of CD95-CD95L interaction
`A, Sampalo, G. Navas, F. Medina, C. Segundo, C. Camara, and J. A. Brieva
`
`Heterogeneity in therapeutic response of genetically altered myelomacell lines to
`interleukin 6, dexamethasone, doxorubicin, and melphalan
`M. Rowley, P. Liu, and B. Van Ness
`
`In vitro and in vivo productionof vascular endothelial growth factor by chronic
`lymphocytic leukemia cells
`H. Chen, A. T. Treweeke, D. C. West, K. J. Till, J.C. Cawley, M. Zuzcl, and C. HL. Toh
`
`Kaposi sarcomais a therapeutic target for vitamin D, receptor agonist
`R. Masood, S. Nagpal, T. Zheng, J. Cai, A. Tulpule, D. L. Smith, and P. S. Gill
`
`Efficacy of STI571, an Abl tyrosine kinase inhibitor, in conjunction with other
`antileukemic agents against Ber-A bl-positive cells
`J.T. Thiesing, S. Ohno-Jones, K. S. Kolibaba, and B. J. Druker
`This material was copied:
`atthe NUMtand may be
`Subject US Copyright Laws
`
`ALVOGEN, Exh. 1055, p. 0124
`
`ALVOGEN, Exh. 1055, p. 0124
`
`

`

`3200 Altered ligand binding and transcriptional regulation by mutations in the
`PML/RAR@ligand-binding domainarising in retinoic-acid—resistant
`patients with acute promyelocytic leukemia
`S. Cété, D. Zhou, A. Bianchini, C. Nervi, R. E. Gallagher, and W. H. Miller Jr
`
`3209 Anovel gene, MELI, mapped to 1p36.3 is highly homologous to the MDSI/EVII
`gene andis transcriptionally activated in 1(1;3)(p36;q21)-positive leukemia cells
`N. Mochizuki, S. Shimizu, T. Nagasawa, H. Tanaka, M. Taniwaki, J. Yokota, and K. Morishita
`
`PHAGOCYTES
`
`
`3215
`Identification and characterization of CKLiK, a novel granulocyte
`Cat*t/calmodulin-dependent kinase
`S. Verploegen, J.-W. J. Lammers, L. Koenderman, and P. J. Coffer
`
`RED CELLS
`
`
`3224
`The Fanconi anemia proteins FANCA and FANCGstabilize each other and promote
`the nuclear accumulation of the Fanconi anemia complex
`I, Garcia-Higuera, Y. Kuang, J. Denham, and A. D. D’Andrea
`
`3231
`
`Nonopsonic monocyte/macrophage phagocytosis of Plasmodium
`falciparum—parasitized erythrocytes: a role for CD36 in malarial clearance
`I. D. McGilvray, L. Serghides, A. Kapus, O, D. Rotstein, and K. C. Kain
`
`3241
`
`Regulation of hemoglobin synthesis andproliferation of differentiating erythroid
`cells by heme-regulated elF-2« kinase
`J. S. Crosby, P. J. Chefalo, I. Yeh, S. Ying, 1. M. London, P. Leboulch, and J.-J. Chen
`
`3249 Apoptosis in megaloblastic anemia occurs during DNA synthesis by a
`p53-independent, nucleoside-reversible mechanism
`M. J. Koury, J. O. Price, and G, G. Hicks
`
`3256 Human ABC7 transporter: gene structure and mutation causing X-linked
`sideroblastic anemia with ataxia with disruptionof cytosolic iron-sulfur
`protein maturation
`S. Bekri, G. Kispal, H. Lange,E. Fitzsimons, J. Tolmie, R. Lill, and D. F. Bishop
`
`3265
`
`Retinoic acid stimulates erythropoietin gene transcription in embryonal carcinoma
`cells through the direct repeat of a steroid/thyroid hormone receptor response
`element half-site in the hypexia-response enhancer
`T. Kambe, J. Tada-Kambe, Y. Kuge, Y. Yamaguchi-Iwai, M. Nagao, and R. Sasaki
`
`BRIEF REPORTS
`
`
`3272 Assessment of bone marrowstemcell reserve and function and stromal cell function
`in patients with autoimmunecytopenias
`H. A. Papadaki, F. M. Gibson, S. Rizzo, E. C. Gordon-Smith, and J, C. W. Marsh
`
`This material was copied
`at the NiMtand may be
`Subject USCopyright Laws
`
`ALVOGEN, Exh. 1055, p. 0125
`
`ALVOGEN, Exh. 1055, p. 0125
`
`

`

`CONTENTS(continued)
`
`3276
`
`Secretory phospholipaseA, predicts impending acute chest syndromein sickle
`
`cell disease
`L. A,Styles, A. J. Aarsman,E. P. Vichinsky, and F. A. Kuypers
`
`3279 Molecular evidence of organ-related transmission of Kaposi sarcoma~associated
`herpesvirus or humanherpesvirus-8 in transplant patients
`M.Luppi, P. Barozzi, G. Santagostino, R. Trovato, T. E Schulz, R. Marasca, D. Bottalico,
`L. Bignardi, and G. Torelli
`
`3282 Adhesion to 90K (Mac-2 BP) as a mechanism for lymphomadrugresistance in vivo
`B. Fornarini, C. D’Ambrosio, C. Natoli, N, Tinari, V. Silingardi, and S. Iacobelli
`
`3286 Drug-resistant human cytomegalovirus infection in children after allogeneic stem
`cell transplantation may havedifferent clinical outcomes
`T. Eckle, L. Prix, G. Jahn, T. Klingebiel, R. Handgretinger, B. Selle, and K, Hamprecht
`
`3290
`
`Low B-cell and monocytecounts on day 80 are associated with high infection rates
`between days 100 and 365 after allogeneic marrow transplantation
`J. Storek, G. Espino, M. A. Dawson,B.Storer, M. E. D. Flowers, and D. G. Maloney
`
`CORRESPONDENCE
`
`3294
`
`The ABLkinaseinhibitor STI571 does not affect survival of hematopoietic cells
`after ionizing radiation
`N. Uemuraand J. D. Griffin
`
`3295 Hyperprothrombinemia mayresult in acquiredactivated protein C resistance
`A. Tripodi, V. Chantarangkul, and P. Mannucci
`
`SPECIAL DEPARTMENTS
`
`xix Author guide
`
`xxx
`
`Futurearticles
`
`. 3297. Announcements
`
`Manuscripts to be considered for publication in Bloodshould be sentto our newaddress:
`Kenneth Kaushansky, MD
`Editor-in-Chief
`Blood, Journalof the
`AmericanSociety of Hematology
`1900 M Street, NW, Suite 200
`Washington, DC 20036
`
`Forother information, call (202) 776-0548,All manuscripts submitted to Bloodand
`subsequently acceptedfor publication will be assessed a $50 charge per published page.
`This chargeis necessary to offset the increasing costof publication.
`
`ALVOGEN, Exh. 1055, p. 0126
`
`ALVOGEN, Exh. 1055, p. 0126
`
`

`

`blood
`
`JOURNAL OF
`
`THE AMERICAN
`
`SOCIETY OF
`
`HEMATOLOGY
`
`Blood, Journal of The American Sectety ef Hentatology
`(ISSN 0006-4971), is published 25 times (in 2 volumes)
`per year by The American Society of Hematology
`(ASH). 1900 MStreet. NW, Suite 200, Washington, DC
`20036. Dates ofissue are the Ist and the [5th of each
`month, except
`in November, when 3 issues are pub-
`lished. Printed in the United States of America. Periodi-
`cals postage paid at Washington, DC, and additional
`mailing offices.
`Postmaster: Send changes of address to Blood,
`Journal ofThe American Society afHematology, 1900 M
`Street, NW, Suite 200, Washington, DC20036,
`
`Manuscript submissions
`Consult the Author Guide printed in each issue of Blood!
`fand
`posted
`on
`the web
`site
`at) www.blood
`Journal.org) before submitting 3 hard copies of the
`Manuscript,
`| diskette, and 3 complete sets offigures to:
`Kenneth Kaushansky, MD
`Editor-in-Chief, Blood
`1900 M Street, NW, Suite 200
`Washington, DC 20086
`
`Authorproofs
`Blood will be copyedited by the Blood Production staff
`in’
`the Washington, DC, Publishing Office. Authors
`should return their Page proofs and all related material
`within 48 hours of receipt
`to the Blood Production
`Office, 1900 MStreet, NW, Suite 200, Wsishington, DC
`20036,Tel: 202-299-0027,
`
`2000 subscription rates
`United States and Possessions:individual
`. B5IF2> insti-
`tution, $748. AW other Countries:
`individual, $747:
`lastituion, $897. There is no additional charge for
`surface delivery to any area outside the United States and
`possessions. Morairmail delivery, add $144 to your subscrip-
`ion price. Blood offers a reduced rate for students,
`interns, and residents whe
`are in approved hematology
`or oncology programs: United St
`ates and possessions,
`$244: afl other countries, $667,
`‘To qualify, supply your
`name, dates ofstudy, andthe
`signature of your academic
`advisor on institutional Lette
`rhead. Orders will be billed
`atthe individual rate until proofofstatusis received, All
`subscriptions are accepted on a calendar-year basis,
`The price forsingle/back issues is $38, Singleissues,
`both current and back, exist in limited qu
`antities and are
`offeredforsale subject to
`availability, Back issues sold
`iN conjunction with
`a subscription are prorated.
`Subscription information, claims,
`or changesof address
`CustomerService for Subscribers: 1-800-654-2452 (US
`& Canada); 1-407-345-4000 (outside US & Canada).
`Correspondence regarding Chonmemiber} subscriptions
`or change of address should be addressed to Blood
`Journal Subscriptions (e/o W. B. Saunders), PO Box
`628239, Orlando, FL 32862-8239, Checks should be
`made payable to Blood Subscriptions,
`ASH Members should send their change-of-address
`information to the ASH Membership Department, L906
`MStreet, NW, Suite 200, Wi
`ashington, DC 20036,
`All chanve-of-add
`fess notices should be sent at least
`6 weeks priorto the d
`ale ofissue, with both old and new
`addresses given,
`
`The American Society of Hematology
`1900 M Street, NW,Suite 200
`Washington, DC 20036
`
`“a one
`Internet
`Blood HomePage: www.bloodjourna org
`ASH HomePage: www. hematology.org.
`
`society of Hematol-
`;
`cannes
`Copyright © 2000 by The American s e
`blication my
`‘
`=
`.
`.
`rey. All rights reserved. No part of this Oi
`any means
`ORY.
`BH res
`tar
`“py dby
`.
`oe
`.
`any
`101
`.
`ced
`or
`transmitied in any forn
`y
`: ae
`bevepraducedor transmaitte
`«or mechanical. includ
`now or hereafter known, siertean y informationst
`wage
`ing photocopying, recording, of any P sain writing
`from
`and retrieval syster, without nan mealies
`the Publisher, The American Society ° ion to reprin
`t all
`Correspondence regarding Seonavail should
`| be
`or part of any article published in mt 9u0 M Street.
`addressed tor Blood Publishing eee
`NW, Suite 200, Washington, DC 200 - art
`y
`at
`the
`peal
`oeeet this journalr
`The appearance ol
`dete th
`Ba
`ave
`of an aruicie
`bottom of
`the first page of
`t that copies °
`indicates the copyright owner s vomns . oo
`L use, oF
`the article may be made for paca one Revie.
`for
`for the personal or internal use a veiearance Center.
`those registered with the Copyright MA 01923: (978)
`Inc. (222 Rosewood Drive. pane manson’ is givel
`750-8400: www.copyright.com). me » crated
`per-copy
`on the condigien that the copter payt ete Clearanee
`fee for that article through the ay iene by
`Center,
`Inc.,
`for copying beyond =
`Law.
`‘This
`Sections 107 or LOS of the US reeopyille such
`op Kine
`vee
`or
`consent does not extend to other ki
`soe aefyertisthye
`+
`phased
`yp adver
`/
`:
`foo
`gracing
`stribution, f
`estes
`as copying for general dist
`work
`weollective
`for ereating ne
`‘clon
`ses
`.
`promotional purposes, for creating
`sermisslel
`i
`:
`poses. f
`or for resale. For those kinds of pur]
`1)
`,
`isher (see above
`must be sought from the Publisher (
`
`Advertising representation
`atieds:
`‘or
`product advertise!
`nents, classifies
`spates.
`4
`To place orders for product advert
`ringhamAs le:
`or commercial reprints, contact¢ unt
`NI 07079: vit
`180 Old Tappan Road, Old a RUNS:
`e-mail:
`phone: 201-767-4170;
`fax: 201-76
`cunnasso @cybermex. net,
`;
`Jo nel
`2
`Lin Blood
`The ideas and opinions SEEcit
`1 Society ©an
`necessarily reflect
`those of The —
`ation oF
`Hematology or the Editors of Blood.3
`. Yaoi S
`pould
`sation
`in Blo"
`the
`advertisement or other produc
`re product ol
`:
`2
`oor
`‘? mention
`—
`arsemnent
`oftl
`not be construcd as an endorsenie ne aesoei®
`{te cole
`manufacturer's claims. Readers are ¢H u :
`vans 20%
`it
`the
`
`
`
`.
`The
`tact
`the manufacturer with any ve
`ad.
`imitati
`:
`s PTO
`ucts mention aassume HDar
`features or limitations of
`the pr
`1
`24 nol
`i
`Society
`smatolagy does
`apsans ©
`American
`2
`and/or damage t
`via
`‘rican
`Society of Hema
`st)
`-
`pers
`responsibility
`camel
`sibility for any injury
`ee
`re
`he mate
`“
`{0
`propertyarising fromorrelated
`{vised
`:
`fer is ae
`‘al. The
`.
`ee
`gypert
`.
`red
`contained in this periodic
`ane
`ae
`{the product
`literature
`‘
`sdical
`check the appropriate medical ae oan
`PootyTel
`sanufact
`.
`pitt
`rhe
`.
`eeyectede®
`yy
`u
`information currently provided bs
`ify the dosage
`; or contraindi
`‘
`each drug to be administered|
`“Hei
`inistcred fo verus
`:
`-administratior
`thet
`method and duration of administ
`roll
`Par
`‘the treating
`shysichin ©
`a
`tions, It is the responsibility of the eating |
`~
`her ine
`4
`fepe
`is
`or herd
`—
`ve on Dis
`nt,
`to dete!
`health care professional. rely
`of the patie
`atient
`i
`SOW
`
`the patie
`dent experience and knowle
`for
`treatment fe
`asf
`mine drug dosages and the best trea
`.
`Indexing & abstracting
`Medici
`Cut
`Index
`Blood is
`indexed and abstracted oe
`Science
`e@ cle
`x,
`Excerpta Medica, Current Cenieih nce Cit
`jon Indes
`rent Contents/Clinical Medicine. Scien Avil Alert.
`(sll
`SCISEARCH, Automatic Subject Ot
`BIOMED, and BIOSIS.
`
`
`
`ALVOGEN, Exh. 1055, p. 0127
`
`

`

`rienary paper
`
`'his material may be protected py Lopyrignt law (Iitle 17 U.S. Lode)
`
`
`Thalidomide andits analogs overcome drug resi
`myelomacells to conventional therapy
`Teru Hideshima, Dharminder Chauhan, Yoshihito Shima, Noopur Raj
`Robert L. Schlossman, Paul Richardson, George Muller, David |. Stirl
`
`stance of human multiple
`
`é, Faith E. Davies, Yu-Tzu Tai, Steven P. Treon, Boris Lin,
`ing, and Kenneth C. Anderson
`
`ini-
`Although thalidomide (Thal) was.
`tially used to treat multiple myeloma
`(MM) becauseof its known antiangio-
`genic effects,
`the mechanism of
`its
`anti-MM activity is unclear. These stud-
`ies demonstrate clinical activity of Thal
`against MMthatis refractory to conven-
`tional
`therapy and delineate mecha-
`nismsof anti-tumor activity of Thal and
`its potent analogs (immunomodulatory
`drugs [IMiDs]). Importantly, these agents
`
`Introduction
`
`act directly, by inducing apoptosis or
`G1 growtharrest, in MM cell lines and in
`patient MM celis that are resistant to
`melphalan, doxorubicin, and dexameth-
`asone (Dex). Moreover, Thal and the
`IMiDs enhance the anti-MM activity of
`Dex and, conversely, are inhibited by
`interleukin 6. As for Dex, apoptotic sig-
`naling triggered by Thal and the IMiDsis
`associated with activation of related
`adhesion focal tyrosine kinase. These
`
`Studies establish the framework for the
`developmentandtesting of Thal and the
`IMiDs in a new treatment paradigm to
`target both the tumor cell and the micro-
`environment, overcomeclassical drug
`resistance, and achieve improved out-
`come in this presently incurable dis-
`ease. (Blood. 2000;96:2943-2950)
`
`‘9 2000 by The American Society of Hematology
`
`Thalidomide (Thal) was originally usedin Europeforthe treatment
`of morning sickness in the 1950s but was withdrawn from the
`market
`in the 1960s because of reports of teratogenicity and
`phocomelia associated with its use. The renewed interest
`in Thal
`stems from its broad spectrumof pharmacologic and immunologic
`effects.£ Because of its immunomodulatory and antiangiogenic
`effects,
`it has been used to effectively treat erythema nodosum
`leprosum, an inflammatory manifestation of leprosy.” Potential
`therapeutic applications span a wide spectrum of diseases. melud
`ing cancer and related conditions, infectious diseases, autoimmune
`diseases, dermatologic diseases, and other disorders such as
`sarcoidosis, macular degeneration, and diabetic retinopathy. ' Re
`cent
`reports of increased bone marrow (BM) angiogenesis in
`multiple myeloma (MM)."° coupled with the known antianyio
`gene properties of Thal.® provided the rationale for its use to treat
`MM./ importantly, Thal induced clinical responses in 326 of MM
`patients whose disease was refractory to conventional and high
`dose therapy,’ suggesting that
`it can overcome drug resistance
`because of its alternative mechanisms of anticM acliVity, Besides
`alkylating agents and corticosteroids, Thal how, therefore, repre
`semis
`the third distinct chiss of agents useful
`in the trestment
`ol MM
`Given its broad spectrim: of acuvities, Thal may be aching
`apainst MM in several ways.” First. Thal may have a direct effect
`on the MMcell and/or BM stromal cell to inhibit their growth and
`survival
`Por example,
`tree radical-mediated oxidative DNA
`damage may play a rote in the teratogenicity of Thal? and may also
`have anti-tumor effects. Second, adhesion of MM cells to BM
`stromal cells both triggers secretion of cytokines that augment MM
`
`cell growth and survival!’ and confers drug resistance: Thal
`modulates adhesive interactions! and,
`thereby, may alter tumor
`cell growth, survival, and drug resistance. Third, cytokines secreted
`into the BM microenvironment by MMand/or BM stromalcells.
`suchas interleukin (1L)-6, IL-1, IL- 10, and tumor necrosis factor
`(ENP }-c, may augment MMcell growth and survival.and Thal
`mayalter their secretion and bioactivity.’ Fourth, vascular endothe-
`Hal growth factor (VEGE) and basic fibroblast growth factor 2
`(bEGE-2) are seereted by MMand/or BMstromal cells and may
`play a role both in tumor cell growth and survival. as well as BM
`dngiogenesis.!" Given its known antiangiogenic activity. Thal
`may inhibit activity of VEGbrGP-2. and/or angiogenesis jn
`MM. However, Singhal et al” observed no correlation of BM
`angiogenesis with response to Thal, suggesting that it may not
`be mediating antiiMM activity by its antiangiogenic effects.
`Finally, Thal may be acting against MMbyits immunomodula-
`fory effects. such as induction of a Tht T-cell response with
`secretion of interferon gamma (IPN-y) and TL-2.!7 Already 2
`classes of Thal analogs have been reported, including phospho-
`diesterase 4 inhibitors that
`inhibit TNF-a but do not enhance
`T-cell activation (selected cytokine inhibitory drugs [ScICIDs})
`and others that are not phosphodiesterase 4 inhibitors bur
`markedly stimulate T-cell proliferation as well as IL-2 and
`IPN~y production immunomodulatory drugs {IMiDs]}).!5
`In this study, we have begunto characterize the mechanisms of
`acuivily of Thal and these analogs against human MM_ cells.
`Delineation oftheir mechanisms ofaction, as well as mechanisms
`of resistance to these agents, will both enhance understanding of
`MMdisease pathogenesis and derive novel treatment strategies.
`
`
`
`From the Department of Adult Oncology, Dana-Farber CancerInstitute, and
`Reprints: Kenneth C. Anderson, Dana-Farber CancerInstitute, 44 Binney St.,
`Department of Medicine, Harvard Medical School, Boston, MA: and Celgene
`Boston, MA 02115; e-mail: kenneth anderson@dici.harvard.edu.
`Corporation, Warren, NJ.
`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 U.S.C. section 1734.
`© 2000 by The American Society of Hematology
`
`Submilted March 8, 2000; accepted June 28, 2000.
`Supported by National Institutes of Health grant PO1 78378 andthe Doris Duke
`Distinguished Clinical Research Scientist Award (K.C.A,).
`
`BLOOD, 1 NOVEMBER2000 « VOLUME 96, NUMBER9
`
`This material was copied
`atthe NLM and may be
`Subject US Copyright Laws
`
`2943
`
`ALVOGEN, Exh. 1055, p. 0128
`
`ALVOGEN, Exh. 1055, p. 0128
`
`

`

`2944
`
` HIDESHIMA etal
`
`BLOOD, 1 NOVEMBER 2000 - VOLUME 96, NUMBER 9
`
`Materials and methods
`
`MM-derivedcell lines and patient cells
`
`(MM.IR)
`Dexamethasone (Dex )-sensitive (MM.1S) and Dex-resistant
`human MMcell Jines werekindly provided by Dr Steven Rosen (Northwest-
`ern University, Chicago, IL). Doxorubicin (Dox)-. mitoxantrone (Mib-, and
`melphalan (Mel)-sensitive and -resistant RPMI-8226 human MM_ cells
`were kindly provided by Dr William Dalton (Moffitt Cancer Center, Tampa,
`PL), RPMI-8226cells resistant to Dox, Mit, and Mel included Dox 6 and
`Dox 40 cells. MR20cells. and LRS cells, respectively. Hs Sultan human
`MMcells were obtained from the American Type Culture Collection
`Rockville, MD). All MMcell lines were cultured in RPMI-[640 media
`(Sigma Chemical, St Louis, MO) that contained 10% fetal bovine serum, 2
`minol/L L-glutamine (GIBCO, Grand Island, NY), 100 U/mL penicillin,
`and 100 pg/mL, streptomycin (GIBCO), Drug-resistant cell
`lines were
`cultured with either Dox. Mit, Mel, or Dex to confirmtheir lack of drug
`sensitivity. MMpatient cells (96% CD38'CD4S5RA—) were purified from
`patient BM samples, as previously described. !*
`
`Thal and analogs
`
`Thal and analogs (Celgene, Warren, NJ) were dissolved in DMSO (Sigma)
`and stored at
`20°C until use. Drugs were diluted in culture medium
`(0.0001 to 100 2M) with < 0.1% DMSO immediately before use. The Thal
`analogs usedin this study were 4 SelCIDs (SelCIDs 1. 2. 3. and 4), which
`are phosphodiesterase 4 inhibitors that
`inhibit TNF-c production and
`increase [L-10 production from lipopolysaccharide UPS )-stimulated periph-
`eral blood mononuclear cells (PBMCs) but do not.
`stimulate ‘T-cell
`proliferation; and 3 IMiDs (IMiDL. IMiD2, and IMiD3), which do stimulate
`‘Teel profiferation, as well as IL-2 and IPN-y secretion, but are not
`phosphodiesterase 4 inhibitors. The IMiDs also inhibit TNE-c, IL-1 8. and
`IL-6 andgreatly increase H.-10 production by LPS-stimulated PBMCs,!5
`
`DNA synthesis
`
`DNA synthesis was measured as previously deseribed.!” MMcells 3% 104
`cells/well) were incubated in 96-well culture plates (Costar, Cambridge,
`MA)
`in the presence of media, Thal, SelCID1, SelCID2. SelCiD3,
`SelCID4, IMiD1, IMiD2, IMiD3. and/or recombinant
`IL-6 (50 ng/mL)
`(Genetics Institute, Cambridge, MA) for 48 hours at 37°C, DNA synthesis
`was measured by [‘H]-thymidine (H-TdR: NEN Products. Boston, MA)
`uptake. Cells were pulsed with “H-TdR (0.5 pCi/well) during the last 8
`hours of 48-hour cultures, harvested onto glass filters with an automatic cell
`harvester (Cambridge Technology, Cambridge, MA), and counted by using
`the LKB Betaplate scintillation counter (Wallac, Gaithersburg, MD), All
`experiments were performed tn triplicate.
`Colorimetric assays were also performed to assay drug activity. Cells
`from 48-hour cultures were pulsed with 10 ql of 5 mye/mL, 3-(4.5-
`dinethylthiavol-2-yl)-2.5-diphenyl tetrasodium bromide (MTT: Chemicon
`International Inc, Temecula, CA) to each well for 4 hours, followed by 100
`BL isopropanol
`that contained 0.0¢ HCI. Absorbance readings at
`a
`wavelength of 570 nm were taken on a spectrophotometer (Molecular
`Devices Corp, Sunnyvale, CA),
`
`Ceil cycle analysis
`
`in media (0.01% DMSO) or with 10 pmol/Lof Thal or | mol/L IMIDE.
`IMiD2, and IMiD3 at 37°C for 72 hours, with addition of drugs at
`24-hour intervals. Cells were then washed twice with ice-cold PBS and
`resuspended (1 * 10° cells/mL) in binding buffer (10 mmol/L HEPES ,
`pH 7.4. 140 mmol/L. NaCl. 2.5 mmol/L. CaCl). MMcells (1
`& 10°) were
`incubated with annexin V-PITC (5 pL: Pharmingen, San Diego, CA) and
`PI
`(S pg/mL) for 1S minutes at room temperature. Annexin V4 PI
`apoptotic cells were enumerated by using the Epics cell sorter (Coulter).
`
`immunoblotting
`
`MMcells were cultured with 10 pmol/L of Thal, IMiDT, IMiD2, or
`IMiD3: harvested: washed: and lysed using lysis buffer: 50 mmol/L.
`HEPES (pH 7.4), 150 mmol/L NaCl.
`1% Triton-X 100, 30 mmol/L.
`sodium pyrophosphate, 5 mmol/L EDTA, 2 mmol/L NasVO,. 5 mmol/L.
`NaF.
`| mmol/L. phenylmethy! sulfonyl
`fluoride (PMSF), 5 pe/mL.
`leupeptin, and 5 pg/mL aprotinin. For detection of p21, cell Lysates
`were subjected to SDS-PAGE, transferred to polyvinylidene difuoride
`(PYDF) membrane, and immunoblotted with anti-p2]
`antibody:
`(Ab: Santa Cruz Biotech, Santa Cruz, CA). The membrane was stripped
`and reprobed with anti-alpha tubulin Ab (Sigma) to ensure equivalent
`protein loading. For detection of p53. cell lysates were prepared from
`MMcells (2 * 107) with the useoflysis butfer. Lysates were incubated
`with anti-mutant (mt) or wild-type (wt) pS3 monoclonal Abs (Calbio~
`chem. San Diego, CA) and then immunoprecipitated overnight with
`protein A Sepharose (Sepharose CL-4B: Pharmacia, Uppsala. Sweden).
`Immune complexes were analyzed by immunoblotting with horseradish
`peroxidase-conjugated anti-p53 Ab reactive with both mt and wt ps3
`(Calbiochem).
`
`immunoblotting was also done
`To characterize growth signaling,
`with anti-phospho-specific MAPK Ab (New England Biolabs, Bey erly.
`MA) in the presence or absence of IL-6 (Genetics Lastitutes and/or the
`MEK f inhibitor PD98059 (NewEngland Biolabs), as in prior studies!
`Antigen-antibody complexes were detected by using enhanced chemilu-
`minescence (Amersham, Arlington Heights, IL). Blots were stripped and
`reprobed with anti-ERK2 Ab (Santa Cruz Biotech) to ensure equivalent
`protein loading.
`To characterize apoptotic signaling. MM cells were cultured with 100
`pmol/L of Thal, IMiD 1. IMiD2, or IMiD3: harvested: washed: and lysed in
`I mL oflysis buffer (S0 mmol/L Tris, pH 7.4. 150 mM NaCl, 14 NP-4Q, &
`mmol/L. EDTA, 2 mmol/L Na;VO;. 5 mmol/l. Nak | mmel/t. PMSE, S
`g/mL leupeptin, and 5 e/mL aprotinin}. as in prior studies. Lysates
`were incubated with anti-related adhesion focal tyrosine kinase (RAETK}
`Ab for | hour at 4°C and then for 45 minutes after the additien of protent
`G-agarose (Santa Cruz Biotech). Immune complexes were analyzed bs
`immunoblotting with anti-P-Tyr Ab (RC20: Transduction Laboratories.
`Lexington, KY) or anticRAPTK Abs. Proteins were separated by clectio-
`phoresis in 7.5% SDS-PAGEgels, transferred to nitrocellulose paper, and
`analyzed by immunoblotting. The antigen-antibody complexes were visual
`ized by chemiluminescence.
`
`Statistical analyses
`
`in driig-treated versus
`Statistical significance of differences observed.
`contol cultures was determined by u