Molecular therapy.
`y. 17, no. 8 (Aug. 2009)
`General Collection
`
`W1MO1975M
`4009-08-27 13:39:16
`
`A V A.
`
`=— —— << ~iieee”
`
`» |
`
`|
`J a T
`
`official journal ofthe
`American Societyof
`Gene ¢> Cell Therapy
`
`vol. 17 no. 8 august 2009
`
`www.moleculartherapy.org
`
`ees
`‘
`coe et hae
`ad i on G a
`a Ay et
`me et
`ee
`3
`
`Po
`
`Z—IKNS|
`Ry
`
` .
`
`PROPERTY OF THE
`NATIONAL
`LIBRARY OF
`MEDICINE
`
`Sonic hedgehog modulates
`the HSC niche
`
`Integration-deficient lentiviral vectors
`Newinsights on adenovirus as
`vaccine vectors
`
`|
`
`AMERICAN
`SOCIETYof
`
`ww GENE
`
`mel THERAPY
`
`nature publishing group &
`
`

`

`editorial board
`
`Molecular
`‘Therapy
`
`
`
`EDITOR-IN-CHIEF
`David A Williams
`
`Boston, MA
`
`EDITOR
`Robert M Frederickson
`
`Seattle, WA
`
`
`
`ASSOCIATE EDITORS
`
`Adrian J Thrasher, London, UK
`John Bell, Ottawa, Canada
`Helen E Heslop, Houston, TX
`Katherine High, Philadelphia, PA
`Jacques Tremblay, Quebec, Canada
`IA
`Beverly Davidson, lowa City,
`Mark A Kay, Stanford, CA
`Ernst Wagner, Munich, Germany
`HildegundCJ Ertl, Philadelphia, PA
`James Wilson, Philadelphia, PA
`Darwin J Prockop, Temple, TX
`Loren J Field, Indianapolis, IN
`John J Rossi, Los Angeles, CA
`Seppo Yla-Herttuala, Kuopio, Finland
`Shiroh Futaki, Kyoto, Japan
`
`
`
`EDITORIAL BOARD
`
`Valder Arruda, Philadelphia, PA
`Alberto Auricchio, Naples, Italy
`Andrew Baker, Glasgow, UK
`Christopher Baum, Hannover, Germany
`Mark Behlke, Coralville,
`[A
`Ben Berkhout, Amsterdam, Netherlands
`Mick Bhatia, Hamilton, Canada
`David Bodine, Bethesda, MWD
`Xandra O Breakefield, Charlestown, MA
`Malcolm Brenner, Blacksburg, VA
`Bruce A Bunnell, New Orleans, LA
`Peter A Campochiaro, Baltimore, MD
`Barrie J Carter, Seattle, WA
`Toni Cathomen, Berlin, Germany
`Sunil Chada, Blacksburg, VA
`Jeffrey Chamberlain, Seattle, WA
`Seng H Cheng, Framingham, MA
`E Antonio Chiocca, Columbus, OH
`John A Chiorini, Bethesda, MD
`Odile Cohen-Haguenauer, Paris, France
`Ken Cornetta, Indianapolis, IN
`Tim Cripe, Cincinnati, OH
`Ronald G Crystal, New York, NY
`Albert Deisseroth, San Diego, CA
`Ruxandra Draghia-Akli, Houston, TX
`Dong-Sheng Duan, Columbia, MO
`Cynthia E Dunbar, Bethesda, MD
`Paul B Fisher, Richmond, VA
`
`FOUNDING EDITOR
`Inder M Verma,La Jolla, CA
`
`Svend O Freytag, Detroit, MI
`Theodore Friedmann, San Diego, CA
`Steven Craig Ghivizzani, Gainesville, FL
`Joseph C Glorioso, Pittsburgh, PA
`Perry B Hackett, St Paul, MN
`Hideyoshi Harashima, Sapporo, Japan
`Gunther Hartmann, Bonn, Germany
`Stephen D Hauschka, Seattle, WA
`Roland Herzog, Gainesville, FL
`Johnny Huard, Pittsburgh, PA
`Zoltan Ivics, Berlin, Germany
`Yasufumi Kaneda, Osaka, Japan
`George Karpati, Montreal, Canada
`Brian K Kaspar, Columbus, OH
`Dave Kirn, San Francisco, CA
`Stephan Kochanek, Ulm, Germany
`Don B Kohn,Los Angeles, CA
`Robert Langer, Cambridge, MA
`Kam Leong, Durham, NC
`Andre Lieber, Seattle, WA
`Dexi Liu, Pittsburgh, PA
`Pedro Lowenstein, Los Angeles, CA
`lan MacLachlan, Burnaby, Canada
`Ron Mandel, Gainesville, FL
`R Scott Mclvor, Minneapolis, MN
`Phillipe Menasché, Paris, France
`Phillipe Moullier, Nantes, France
`John D Mountz, Birmingham, AL
`Nicholas Muzyczka, Gainesville, FL
`
`
`Glen R Nemerow,La Jolla, CA
`Philip Ng, Houston, TX
`James Norris, Charleston, SC
`Robin Parks, Ottawa, Canada
`Derek A Persons, Memphis, TN
`Glenn Pierce, Berkeley, CA
`Katherine Ponder, St Louis, MO
`Jesus Prieto, Pamplona, Spain
`Samuel D Rabkin, Charlestown, MA
`Dave Russell, Seattle, WA
`Stephen J Russell, Rochester, MN
`Michel Sadelain, New York, NY
`Richard J Samulski, Chapel Hill, NC
`David Schaffer, Berkeley, CA
`Dmitry Shayakhmetoy, Seattle, WA
`Lonnie D Shea, Evanston, IL
`Evan Snyder, La Jolla, CA
`Cy Stein, New York, NY
`Francis Szoka,Jr, San Francisco, CA
`Kenazaburo Tani, Fukuoka, Japan
`Thierry Van den Driessche, Leuven, Belgium
`Richard Vile, Rochester, MN
`Daniel Weiss, Burlington, VT
`Matthew D Weitzman, La Jolla, CA
`Dominic ] Wells, London, UK
`John H Wolfe, Philadelphia, PA
`Jon Wolff, Madison, WI
`Savio LC Woo, New York, NY
`
`STATISTICS CONSULTANT
`Mi-Ok Kim, Cincinnati, OH
`
`

`

`Molecular
`‘Therap www.moleculartherapy.org
`
`Molecular Therapy is published by Nature Publishing Group, a
`division of Macmillan Publishers Ltd on behalf of the American
`Society of Gene Therapy.
`
`SCOPE
`
`Molecular Therapy is the monthly publication of the American Society of Gene
`& Cell Therapy (ASGCT). The journal publishes original scientific papers in the
`areas of gene transfer, gene regulation, gene discovery, cell therapy, experi-
`mental models, correction of genetic and acquired diseases, andclinicaltrials.
`Manuscripts describing new methodological advanceswill also be considered
`for publication. In addition, Molecular Therapy publishes timely reviews, com-
`mentaries, and scientific correspondence. Although Molecular Therapyis the
`official journal of ASGCT,it is international in scope and publication. The major
`criteria for acceptance and publication of a manuscript are originality, high
`quality, scientific rigor, and interest to a wide audience of readers.
`This journal is covered by AIDS Abstracts, BIOBASE, Biotechnology Citation In-
`dex, Chemical Abstracts, Current Contents, Excerpta Medica, Abstract Journals,
`Inpharma Weekly, Index Medicus/MEDLINE, Pharmacogenomics and Outcomes
`News, Reactions Weekly, EMBase, EMBiology, and Scopus.
`
`EDITORIAL
`
`All correspondence should be addressed to: Robert Frederickson PhD, Editor for
`Molecular Therapy, 214 Summit AvenueEast, Suite 305, Seattle, WA 98102-
`5640. Tel/Fax: 206-724-7760. Email: editor@molther.org. All manuscripts
`should be submitted online at: https://www.editorialmanager.com/mthe/.
`
`PUBLISHER
`
`All business correspondence and inquiries should be addressed to: Molecular
`Therapy, Nature Publishing Group,25 First Street, Suite 104, Cambridge, MA
`02141. Tel: +1 617 475 9221, Fax: +1 617 494 4960.
`
`Publishing Manager: Elizabeth Durzy
`Publishing Assistant: Caitlin Stier
`
`Senior Production Editor: Anthony Dunlap
`
`SOCIETY
`
`AUSTRALIA AND NEW ZEALAND:Tel: +61 3 9825 1160. Fax: +61 3 9825 1010,
`E-mail: nature@macmillan.com.au
`INDIA: Tel: +91 124 288 1054. Fax: +91 124 288 1053.
`E-mail: npgindia@nature.com
`THE REST OF THE WORLD: Tel: +44 (0) 20 7843 4759. Fax: +44 (0) 20 7843
`4998. E-mail: institutions@nature.com
`
`print subscriptions (including single issue purchases)
`ALL CUSTOMERS(excluding Japan, Korea and China): Customer Service
`Department, Nature Publishing Group, Houndmills, Basingstoke, Hants, RG21
`6XS, UK. Tel: +44 (0) 1256 329 242. Fax: +44 (0) 1256 812 358.
`E-mail: subscriptions@nature.com
`
`JAPAN, KOREA AND CHINA: Nature Publishing Group, Nature Japan, Chiyoda
`Building 2-37, Ichigayatamachi, Shinjuku-ku, Tokyo 162-0843, Japan. Tel: +81 3
`3267 8751. Fax: +81 3 3267 8746. E-mail: institutions@natureasia.com
`
`Prices are applicable in the following regions: US dollars ($) for North, Central,
`South America and Canada; Euros (€)
`for all European countries (excluding the
`UK); Sterling (£) for UK and rest of world; Yen (¥)for Japan. Please ensure you
`use the appropriate currency. All prices, specifications and details are subject
`to change without prior notification. Single issues of Molecular Therapy are
`available.
`
`ADVERTISING:Inquiries concerning print and web advertisements should be
`addressed to: Ben Harkinson, Advertising Sales Executive. Tel: +1 617 475 9222.
`Fax: +1 617 494 4960. E-mail: b.harkinson@boston.nature.com
`
`SUPPLEMENTS: Inquiries concerning supplements should be addressed to:
`Michelle Libby, Commercial Projects Executive. Tel: +1 617 475 9230.
`Fax: +1 617 494 4960. E-mail: m.libby@boston.nature.com
`REPRINTS AND PERMISSIONS: For reprints of any article in this journal,
`please contact: Tel: +1 617-494-4900. Fax: +1 617-494-4960.
`E-mail: reprints@boston.nature.com.
`For reproduction rights: ajpermissions@nature.com.
`
`Copyright © 2009 American Society of Gene & Cell Therapy, Inc.
`ISSN 1525-0016
`EISSN 1525-0024
`
`All rights of reproduction are reserved in respectofall papers, articles, illustra-
`tions, etc. published in this journal in all countries of the world.
`
`For information, contact the American Society of Gene & Cell Therapy at:
`Tel: +1 414 278 1341. Fax +1 414 276 3349; E-mail: info@asgt.org;
`Web: www.asgt.org.
`
`2009 SUBSCRIPTIONS
`
`All material published in this journal is protected by copyright, which covers
`exclusive rights to reproduce and distribute the material. No material published
`in this journal may be reproduced or stored on microfilm or in electronic, optical
`or magnetic form withoutthe written authorization of the publisher.
`Authorization to photocopy material for internal or personal use, or internal
`institutional subscriptions
`or personal use ofspecific clients, is granted by Nature Publishing Group to
`libraries and others registered with the Copyright Clearance Center (CCC)
`NEW INSTITUTIONAL POLICY: NPG has movedtoasite license policy for
`Transactional Reporting Service, provided the relevant copyright fee is paid di-
`institutional online access, using prices based on Full-Time Equivalents (FTE)
`rect to CCC, 222 Rosewood Drive, Danvers, MA 01923, US.Identification code
`or Research and Development (R&D)staff. Institutions may also purchase a
`for Molecular Therapy: 1525-0016/07
`separate print subscription.
`SUBSCRIBING TO A SITE LICENSE: Contact your local sales representative for a
`tailored price quote for your institution. You will be required to complete a NPG
`site license agreement. More information, contact details and FTE/R&D defini-
`tions are available at the http://nature.com/libraries.
`INSTITUTIONALPRINT SUBSCRIPTIONS: Orders can be placed with your
`regular subscription agent or through NPG—eitheronline or by contacting our
`customerservice department. Prices are as follows: The Americas $1,669.00,
`Europe €1,436.00,
`Japan ¥245,600.00, UK/Rest of World £927.00.
`PERSONALSUBSCRIPTIONS: Personal customers who pay by personal check
`or credit card can either purchase a combined print plus online subscription or
`an online only subscription. Prices are as follows: Combined (print plus online)
`The Americas $556.00, Europe €484.00,
`Japan ¥81,900.00, UK/Rest of World
`£369.00. Personal (online only) The Americas $501.00, Europe €437.00,
`Japan
`¥73,700.00, UK/Rest of World £278.00.
`
`Apart from any fair dealing for the purposes of research or private study, or
`criticism or review, as permitted under the Copyright, Designs and Patent Act
`1988, this publication may be reproduced,stored or transmitted, in any form
`or by any means, only with the prior permission in writing of the publisher,
`or in the case of reprographic reproduction, in accordance with the terms of
`licenses issued by the Copyright Licensing Agency.
`Printed on acid-free paper, effective with Volume 15, Issue 1, 2007
`Printed and bound in the US by The Sheridan Press, Hanover, PA, US.
`Molecular Therapy (ISSN: 1525-0016) is published monthly by Nature
`Publishing Group, 75 Varick Street, 9th floor, New York, NY 10013-1917.
`Periodicals Postage paid at New York NY and additional mailing offices.
`Postmaster send address changes to Molecular Therapy, Nature Publishing
`Group, Subscription Dept, 342 Broadway, PMB 301, New York, NY 10013-3910
`
`CONTACT INFORMATION
`
`site licenses
`
`THE AMERICAS: Tel: +1 800 221 2123. Fax: +1 212 689 9711.
`E-mail: institutions@natureny.com
`
`ASIA PACIFIC (excluding South Asia, Australia and New Zealand): Tel: +81 3
`3267 8769. Fax: +81 3 3267 8746. E-mail: institutions@natureasia.com
`
`While every effort is made by the publishers to see that no inaccurate or
`misleading data, opinion or statement appears in this journal, they and the
`American Society of Gene & Cell Therapy wish to makeit clear that the data
`and opinions appearing in the articles and advertisements herein are the
`responsibility of the contributor or advertiser concerned. Accordingly, the
`American Society of Gene & Cell Therapy, the publishers and the editors and their
`respective employees, officers and agents accept noliability whatsoever for the
`consequences of any such inaccurate or misleading data, opinion or statement.
`
`

`

`originalarticle
`
`Chimeric Receptors Containing CD137 Signal
`Transduction Domains Mediate Enhanced
`Survival of T Cells and Increased Antileukemic
`Efficacy In Vivo
`Michael C. Milone’2, Jonathan D. Fish?*, Carmine Carpenito', Richard G. Carroll’,
`Gwendolyn K. Binder', David Teachey**, Minu Samanta’, Mehdi Lakhal', Brian Gloss',
`Gwenn Danet-Desnoyers°, Dario Campana®’, JamesL. Riley’?, Stephan A. Grupp** and Carl H. June!?
`
`‘Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania USA; Department of Pathology and Laboratory
`Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania USA; *Departmentof Pediatrics, University of Pennsylvania
`School of Medicine, Philadelphia, Pennsylvania USA;‘Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA;
`‘Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA; ‘Departmentof Oncology, St Jude Children’s
`Research Hospital, Memphis, Tennessee, USA; ‘Departmentof Pathology, St Jude Children’s Research Hospital, Memphis, Tennessee, USA
`
`INTRODUCTION
`Persistence of T cells engineered with chimeric antigen
`With the advent of efficient gene transfer technologies, such
`receptors (CARs) has been a major barrier to use of
`as murine retroviral and HIV-derived lentiviral vectors,
`it has
`these cells for molecularly targeted adoptive immuno-
`becomefeasible to confer novel antigenic specificity to T cells by
`therapy. To address this issue, we created a series of
`transfer of chimeric antigen receptors (CARs) with stable, long-
`CARs that contain the T cell receptor-¢ (TCR-C) signal
`term expression. This technology has been used to generate T cells
`transduction domain with the CD28 and/or CD137
`specific for HIV and several human tumorantigens, and some of
`(4-1BB) intracellular domains in tandem. After short-
`these engineered Tcells have been tested in Phase I/II studies in
`term expansion, primary humanTcells were subjected
`humans demonstrating the feasibility and relative safety of this
`to lentiviral gene transfer, resulting in large numbers
`approach,'* One study has demonstrated antitumor activity in
`of cells with >85% CAR expression.
`In an immuno-
`patients with neuroblastoma given a single CARinfusion."
`deficient mouse xenograft model of primary human
`CARs combine the antigen recognition domain of antibody
`pre-B-cell acute lymphoblastic leukemia, human T cells
`with the intracellular domain of the T cell receptor-¢ (TCR-C)
`expressing anti-CD19 CARs containing CD137 exhib-
`chain or FcyRI protein into a single chimeric protein that are
`ited the greatest antileukemic efficacy and prolonged
`capable of triggering T-cell activation in a manner very similar to
`(>6 months) survival
`in vivo, and were significantly
`that of the endogenous TCR.** Several studies demonstrate that
`more effective than cells expressing CARs containing
`the addition of costimulatory domains, particularly the intrac-
`TCR-C alone or CD28-€
`signaling receptors. We uncov-
`ellular domain of CD28 can significantly augment the ability
`ered a previously unrecognized, antigen-independent
`of these receptors to stimulate cytokine secretion and enhance
`effect of CARs expressing the CD137 cytoplasmic
`antitumorefficacy in preclinical animal models using both solid
`domain that likely contributes to the enhanced anti-
`tumors and leukemia that lack the expression of the CD28 recep-
`leukemic efficacy and survival in tumor bearing mice.
`tor ligands CD80 and CD86.”* Inclusion of domains from recep-
`Furthermore, our studies revealed significant discrep-
`tors such as the tumor necrosis factor receptor family members,
`ancies between jin vitro and in vivo surrogate mea-
`CD134 (OX-40) and CD137 (4-1BB) into CARs has also been
`sures of CAR efficacy. Together these results suggest
`shown to augment CAR-mediated T-cell responses.'*'' Gene
`that incorporation of the CD137 signaling domain in
`transfer approaches using these engineered CARs may therefore
`CARs should improve the persistence of CARs in the
`provide significant improvements over current adoptive immu-
`hematologic malignancies and hence maximize their
`notherapystrategies that must rely on the endogenous TCRspec-
`antitumor activity.
`ificities, for which significant issues of TCR repertoire limitation
`and impaired tumor major histocompatibility complex class I
`expression mayexist.
`
`
`Received 1 January 2009; accepted 25 March 2009; published online
`21 April 2009. doi:10.1038/mt.2009.83
`
`Correspondence: Michael C. Milone, Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, University of
`Pennsylvania, BRB 2/3, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-5160, USA. E-mail: milone@mail.med.upenn.edu or Carl H. June,
`Department of Pathology and Laboratory Medicine, Abramson Family Cancer ResearchInstitute, University of Pennsylvania, BRB 2/3,
`421] Curie Boulevard, Philadelphia, Pennsylvania 19104-5160, USA. E-mail: cjlune@exchange.upenn.edu
`
`
`Molecular Therapy vol. 17 no. 8, 1453-1464 aug. 2009
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`1453
`
`

`

`Chimeric Antigen Receptors
`
`© The American Society of Gene & Cell Ther spy
`
`acpi9-ac [Rae
`
`acdi9-¢ |
`
`oCD19-BB-t ocD19-28-¢ [Iq
`
`oCD19-28-BB-C ff
`
`
`
`
`
`
`—e— EF-10(CD4) —=~ EF-10 (CD8)
`—*— UbIC (CD4) —— UbiC (CD8)
`—e- PGK (CD4)
`—«~— PGK (CD8)
`
`m CMV(CD4)
`
`-—= CMV (CD8)
`
`
`
`
`
`
`
`b
`
`10,000
`
`1,000
`
`100
`
`4
`
`=
`3
`EoOo
`oO
`
`/
`
`aa
`
`=
`
`
`
`
`oi
`
`
`
`
`eeeprayeee
`0
`2
`4
`6
`8
`10
`12
`14
`16
`«+18
`Days following transduction
`
`d
`100
`+
`
`CD8"Tcells == Mock
`
`
`—e oCD19-¢
`Mock
`:
`CD19-BB-¢
`
`—«- oCD19-BB-¢
`oCD19-C
`—e o0D19-28-¢
`aCD19-BB-C
`4[—= acb19-28-BB-¢
`aCD19-28-¢
`oCD19-28BB-t!|
`
`5
`@
`a
`S 10
`2
`
`
`
`10° =640'=10, «10 10°
`
`scFv surface expression
`scFv surface expression
`o
`4
`2
`3
`4
`5
`6
`7
`8
`Q
`Days
`
`
`
`Figure 1 Lentiviral gene transfer combined with o@CD3/aCD28 coated magnetic bead activation of T cells permits generation of large
`numbers of CD19-specific chimeric antigen receptor (CAR*) T cells. (a) A schematic diagram showing the CD19-specific CAR used in this study.
`(b) Comparison of green fluorescent protein (GFP) expression under the controlof different eukaryotic promotersin primary human CD4* and CD8*
`T cells over time. GFP fluorescence was comparedin the indicated T cell subset in cells that were stimulated with oCD3/aCD28 coated beadsfollowed
`bylentiviral transduction at an multiplicity of infection (MOI) of 0.2 on day 1 with vector expressing enhanced GFP underthe controlof the promoter
`indicated. Flow cytometric detection of GFP fluorescence was calibrated using Rainbow Calibration Particles (Spherotech, Lake Forest, IL) to correct for
`day-to-day variation. (¢) «CD19-specific CAR surface expression in primary human CD4* and CD8* T cells. Expression was examined6 daysfollowing
`transduction with the indicated CAR-encodinglentiviral vector at a MOIof ~8. (d) In vitro expansion of CD4* and CD8*Tcells following activation
`with c.CD3/a@CD28 coated magnetic beads and transduction of the indicated CAR on day 1. Data are representative of >3 independentexperiments.
`In this study, we have addressedtheissue oflimited in vivo persis-
`_in vivo studies indicate that CARs containing CD137 have superior
`tenceofCARsbydefiningtherelative contributions ofTCR-¢, CD137
`antileukemic efficacy and improvedpersistence in a primary human
`and CD28 signaling domains in mice engrafted with hematopoietic—_acute lymphoblastic leukemia xenograft model. Furthermore, we
`malignancies. We chose the human CD19 antigen as our initial
`also find that CARs expressing CD137 signaling domains can pro-
`target for several reasons: (i) CD19 displays a pattern of expres-
`_vide significantactivity that appears to be antigen independent and
`sion thatis highlyrestricted to B cells and B-cell progenitor cells,'*__
`maycontributeto the efficacy of CARsinvivo.
`(ii) CD19 does not appear to be expressed by hematopoietic stem
`RESULTS
`cells permitting the targeting of the B-cell lineage withoutaffecting
`
`other hematopoietic lineages,'* and (iii) CD19 is widely expressed Efficient generation of CAR*Tcells using
`by malignantcells that are derived from the B-cell lineage includ-
`artificial bead-based antigen-presenting cells and
`ing most lymphomas and lymphocytic leukemias.After optimiz-
`lentiviral gene transfer
`ing the generation of CARs with anefficient T-cell culture process,
`Lentiviral vectors can transfer genes into activated CD4* and
`in vitro studies indicate that incorporation of either CD28 or 4-1BB_=CD8* humanT cells with high efficiency but expression of the
`signaling domains enhancesactivity over TCR-, confirming previ-
`vector-encoded transgene depends onthe internal promoterthat
`ousstudies, In contrast, compared to CARsthat contain CD28, our_drivesits transcription. Therefore, successful CAR expression and
`
`1454
`
`www.moleculartherapy.org vol. 17 no. 8 aug. 2009
`
`

`

`©The American Society of Gene & Cell Therapy
`
`Chimeric Antigen Receptors
`
`be expressed with high expression in >85% primary humanTcells
`(Figure Ic), Western blotting under both reducing and nonreduc-
`ing conditions demonstrated that the CARs are present as both
`covalent dimers and monomers within T cells (Supplementary
`Figure $2). Using theartificial bead-based antigen-presenting cell
`system previously described by our laboratory,'* >50-fold expan-
`sion of CAR* T cells could be achieved over the course of trans-
`duction and growth in ~10 days (Figure 1d).
`
`
`
`_
`[== Mock (K19)
`" —S— 19-¢ (K19)
`| ee 19-BB-¢ (K19)
`~~ 19-28-C (K19)
`s+ 19-28-BB-t (K19)
`~ Mock (Kwt)
`$19-¢, (Kit)
`“19-BB-¢ (Kwt)
`+ 19-28-0 (Kwt)
`
`20
`15
`10
`Effector:target ratio
`
`25
`
`30
`
`aCD19-BB-¢
`
`%Lysis
`
`5
`
`25
`
`20
`
`
`
`%LysisofprimaryALL
`
` thon - 19-28-BB-¢ (Kwt) 0
`
`
`Functional characterization of anti-CD19
`CAR-expressing primary humanT cells
`To enhance the functionality of the immunoreceptor, we intro-
`duce the signal transduction domains of CD28 or CD137 in the
`TCR-¢ containing CAR (Figure 1a). Similar to data reported
`by other groups,’ the introduction of costimulatory domains
`into CARs does not improve the antigen-specific cytotoxicity
`triggered by these receptors (Figure 2b). Lytic activity of trans-
`duced T cells against K562 target cells expressing CD19 cor-
`related with the transduction efficiency of the T cells (data not
`shown). CAR-triggered cytotoxicity is antigen-specific with only
`negligible lysis of wild-type K562 cells that lack expression of
`the CD19 antigen (Figure 2a). CAR*T cells are also able to effi-
`ciently kill primary pre-B acute lymphoblastic leukemia (ALL)
`cells that express physiologic levels of CD19 (Figure 2b). Ofnote,
`these primary ALLcells lack expression of endogenous CD80 or
`CD86 (Figure $3).
`Following CARactivation with CD19* K562cells, CD4* T cells
`expressing CARs produced abundant quantities of interleukin-2
`(IL-2) and interferon-y (Figure 3) comparableto cells stimulated
`via the endogenous TCR and CD28receptors (data not shown). T
`cells expressing CD28 and CD137 domain-containing CARspro-
`duced greater quantities ofIL-2 when comparedwith cells express-
`ing the aCD19-( receptor (Figure 3). The production of the type 2
`cytokines, IL-4 and IL-10, by CD4* Tcells wasalso stimulated by
`all of the CARstested; however, thelevels of these cytokines were
`muchlower, consistent with the Th1-like phenotypeofT cells gen-
`erated by anti-CD3 and CD28stimulatory beads."* It was notable
`that the incorporation of the CD137 domaininto CARsdecreased
`the production of these type 2 cytokines, consistent with previ-
`gene therapies with CAR-expressing Tcells rely on the ability of
`ous reportsof the 4-1 BB signaling pathwayin naturalT cells.’* All
`T cells to maintain adequate receptor expression over long peri-
`CARsstimulated interferon-y production by CD8*Tcells. These
`ods of time. We tested several promoters to identify the one with
`findings confirm thatthe addition of costimulatory domainsinto
`the highest stable expression in both primary CD4* and CD8* T
`CARs modulates cytokine secretion in a mannerthat is dependent
`cells. Transduction was performed at limiting dilution to ensure
`on the type of costimulatory domain.”*'”"” However,it is less well
`that the cells have a single integrated vector per cell (data not
`appreciated that the pattern of cytokine expression is altered by
`shown). Although the cytomegalovirus (CMV) promoter exhib-
`incorporation of different signal transduction domains into the
`ited high levels of expression ofa green fluorescent protein (GFP)
`CARs. These differences may have important consequences for
`transgeneearly after transduction, expression decreased to <25%
`the functionality of T cells engineered to express CARs.
`of the initial expressionafter 10 days of culture (Figure 1b). The
`distribution of CMV-driven GFP expression wasalso quite vari-
`able compared with the other promoters tested (Supplementary
`Figure $1). In contrast, the elongation factor-1a (EF-1a) promoter
`not only induced the highest level of GFP expression butalso opti-
`mally maintained it in both CD4cells and CD8cells (Figure 1b).
`These findings confirmed and extended other studies in primary
`humanT cells.!° The EF-1a promoterwasthereforeselectedforall
`future studies using CARs. By using lentiviral vectors and trans-
`ductionsata multiplicityofinfection of 5,the different CARs could
`
`aCD19-AC
`
`
`
`0
`
`2
`
`6
`4
`Effector:targetratio
`
`8
`
`10
`
`Figure 2 CD19-specific CAR* T cells dernonstrate antigen-specific
`killing of CD19* tumor cells. (a) CAR* T cell cytotoxic activity towards
`K562 cells that are engineered to express human CD19 (K19) or tar-
`get antigen negative wild-type K562 cells (K wild type). Following >10
`days expansion, CAR*T cells (Effector cells) were mixed with the K562
`cells (Target cells) at the indicated ratios. Results represent the mean
`percentof target cell lysis as described in materials. Results are repre-
`sentative of three-independent experiments. (bb) Cytotoxic activity of T
`cells expressing either the aCD19-BB-C or the aCD19-AC control recep-
`tor towards primary human ALL target cells using the same method
`described in (a). Error bars represent the standard error of the mean
`for three replicates.
`
`The effects of costimulatory domains on
`CAR-driven T cell proliferation
`The generation of a robust and sustained antitumor immune
`response requires not only triggering of cytotoxicity and cytokine
`production but also stimulation of T cell proliferation. To assess
`the relative contribution of different costimulatory domains to
`proliferative signals delivered by CARs, we engineered primary
`humanT cells to express CARs in conjunction with GFP to permit
`
`Molecular Therapy vol. 17 no. 8 aug. 2009
`
`1455
`
`

`

`Chimeric Antigen Receptors
`
`+s
`T
`G TheA erican Society of Gene & Cell
`G The American society of Gene & Cell TI erapy
`
`IFN-y
`
`CD8"T cells
`
`aCD19-28-BB-¢
`
`fF
`
`eCD19-28-¢
`aCb19-BB-¢
`F
`
`F
`
`ucDia~g
`Mock
`
`oCD19-28-BB-¢
`
`a@CD19-28-¢
`
`oCD19-BB-¢ —
`o@CD19-¢
`
`Mock
`
`2,000
`
`4,000
`
`6,000
`
`oCD19-28-BB-C
`
`oCD19-28-C
`
`aCD19-BB-¢
`
`aSD19-C
`Mock
`
`oCD19-28-BB-¢
`
`aCD19-28-C
`
`oCD19-BB-¢
`aCD19-¢
`Mock
`
`o
`
`100
`
`200
`
`300
`
`400
`
`500
`
`BLO
`
`IL-to
`
`o
`
`400
`
`800
`
`1,200
`
`1,600
`
`pg/ml
`
`0
`
`CD4' T cells
`
`aCD19-28-BB-C
`
`a@CD19-28-C
`aCD19-BB-C F
`
`oCD19-C
`Mock
`
`a@CD19-28-BB-¢ S
`
`aCD19-28-¢ SS
`oCD19-BB-t
`oCD19-t SSM
`Mock i
`
`
`
`2,000
`
`4,000
`
`6,000
`
`8,000
`
`Mock
`oCb19-28-88-¢ & ocb19-2¢-¢ SSSR
`
`oCD19-28-BB-¢ |
`
`aCD19-28-¢
`
`oCD19-BB-C
`
`SAAN
`aCD19-¢ ee
`
`0
`
`100
`
`200
`
`300
`
`400
`
`500
`
`oCD19-BB-¢
`aCD19-¢
`Mock
`
`0
`
`400
`
`800
`pg/ml
`
`1,200
`
`1,600
`
`Figure 3 The cytokines produced by CD19-specific CAR* T cells are
`dependent on the presence of costimulatory domains within the CAR.
`Following +10 days of expansion, 1 x 10° CARtTcells, as indicated, were stimulated with K562 cells expressing either human CD19 (hatchedbars) or
`antigen negative wild-type K562cells (open bars). Supernatant was harvested after 24 hours of incubation, and the indicated cytokines were measured by
`tive of two-independent experiments; BLQ, below thelimit of quantification.
`cytokine bead array (BD Biosciences, San Jose, CA). Results are representa
`
`This increased proliferation was observed in both CD4* and
`evaluation of both CARt and CAR™ Tcells in the same culture.
`CD8*Tcells (data not shown), and it was associated with a pro-
`Following T-cell restimulation with CD19* K562 (K562-CD19
`cells), T cells expressing the aCD19-28-C receptor exhibited pro-
`longed blast phaseafter the initial stimulation and transduction,
`as revealed by a longer maintenance of an elevated mean cel-
`liferation comparable to that obtained with full stimulationof the
`lular volume (Figure 4c), a parameter that correlates well with
`endogenous TCR complex with K562 cells loaded with anti-CD3
`log phase proliferation of T cells.'° These findings suggest that
`and CD28 antibodies, a condition shown previously to support
`long-term expansion of primary human T cells (KT32-BBL)°
`incorporation of the CD137 intracellular domain mediates anti-
`gen-independentactivity that is similar to that provided by the
`(Figure 4a[v]). The aCD19-28-BB-( triple receptor also stimu-
`natural 4-1BB receptor in T cells following ligation.” As a result
`lated CD19 driven proliferation (Figure 4a[iv]), but to a lesser
`of the enhanced proliferation observed following the initial acti-
`extent than that observed with the aCD19-28-C double costimu-
`vation of T cells via aCD3/aCD28-coated beads used to enhance
`latory receptor. No significant proliferation was observed when
`T-cell transduction”(Figure 4b), CARt T cells expressing the
`these same T cells were stimulated with wild-type K562 cells lack-
`aCD19-BB-¢ receptorhadrelatively low CAR-driven proliferation
`ing the CD19 antigen (K562 wild type). As previously shown by
`other investigators,'”'""!” T cells expressing the aCD19-C¢ recep-
`(Figure 4a[iii]).
`tor showedlittle proliferation on exposure to the surrogate CD19
`antigen (Figure 4a[ii]), demonstrating the dependence of CAR-
`driven proliferation on costimulatorysignals.
`Unexpectedly, T cells containing the aCD19-BB-¢ double
`costimulatory domain CARhadsignificantly increased prolifera-
`tive capacity during in vitro expansion independently of recep-
`tor ligation with the surrogate CD19 antigen (Figure 4a[iii],b).
`
`Evaluating antitumor responses of CAR* human
`primary T cells in vivo
`Other than the antigen-independent proliferation of the 4-1BB
`containing CAR,the abovein vitro findings, inaggregate, suggested
`that the aCD19-28-¢ CAR would be the mosteffective receptor for
`generating a sustained antileukemic T cell response in vivo. We
`
`1456
`
`www.moleculartherapy.org vol. 17 no. 8 aug. 2009
`
`

`

`© The American Society of Gene & Cell Therapy
`
`Chimeric Antigen Receptors
`
`a
`
`2
`3B
`s
`8
`3
`os
`3
`a
`&
`
`S
`=
`
`2 2
`
`5
`2
`3S
`2
`2
`ni
`
`b
`
`2
`=
`3S
`3
`mo}
`Je
`S
`3
`s&
`
`3 S
`
`c
`40
`
`30
`
`=
`2
`o 20
`o
`=
`&
`
`—cCD19-€
`—«oCD19-BB-¢
`>
`== CD19-28-0
`—oCD19-28-BB-C
`
`Days in culture
`
`0
`100
`
`600
`
`Vad\s
`1,100
`Cell volume (fl)
`
`eh
`1,600
`
`2,100
`
`Figure 4 CD28 and 4-1BB costimulatory domains enhance «CD19 CAR-inducedT cell proliferation in vitro with both antigen-dependent and
`antigen-independent effects. (a) In vitro expansion of CAR*Tcells following antigen stimulation. T cells were stimulated with wCD3/aCD28 coated
`magnetic beads on day 0, and transduced with the indicated CAR on day 1 using a bicistronic lentiviral vector expressing CAR along with eGFP using
`the 2A ribosomal skipping sequence as described in Materials. Cultures were restimulated (arrow) with either CD19* K562 cells (K562-CD19), wild-
`type K562 cells (KS62 wild type) or K562 cells expressing hCD32 and 4-1BBL in the presence of aCD3 and aCD28 antibody (K562-BBL-3/28) following
`washing. Exogenous IL-2 was added to the cultures every other day at 1001U/ml. GFPTT cells were enumerated by flow cytometry using bead-based
`counting. Results are reported as the number of population doublings, and they are representative of four-independent experiments. (b) Sustained
`CAR* T cell expansion in the absence ofrestimulation. CD4* and CD8*Tcells were engineered with the indicated CAR, expanded and enumerated as
`in panel (a) In the absence of any K562 stimulator cells. Results are representative of at least three-independent experiments. (¢) Histogram of mean
`T cell volume (fl) on day 8 of culture measured using a Coulter MultisizerIII particle counter following stimulation with «CD3/aCD28 coated magnetic
`beads on day 0, and transduction with the indicated CAR on day 1. Results are representative of at least three-independent experiments.
`
`2 weeks after establishing leukemia in the mice (Figure 5f). The
`evaluated the in vivo efficacy of aCD19 CARs using an in vive
`treatmenteffect was significant for the aCD19-¢ CAR (P < 0.05)
`model ofALL (Figure 5a) in which primary human pre-B ALL cells
`and for CARsthat expressed costimulatory d