11SOU HUMVEE OLE
`
`Human gene therapy.
`23, no. 5 (2012
`RB155
`
`May)Hl ,
`
`Cl
`
`
`VI OEee
`www.liebertpub.com/hum
`
` i
`
`
`
`Miltenyi Ex. 1026 Page 1
`
`

`

`
`
`Human Gene Therapy
`
`European Society of Gene and Cell Therapy * British Society for Gene Therapy * French Society of Cell and Gene Therapy
`Irish Society for Gene and Cell Therapy * The Israeli Society of Gene & Cell Tharapy * German Society for Gene Therapy
`Spanish Society of Gene and Cell Therapy * Netherlands Society of Gene and Cell Therapy * Finnish Society of Gene Therapy
`Human Gene Therapyis the premier, multidisciplinary journal covering all aspects of gene therapy. The
`Journal publishes in-depth coverage of DNA, RNA,andcell therapies by delivering the latest break-
`throughsin research and technologies. Human Gene Therapyis a central forum forscientific and clinical
`information, including ethical, legal, regulatory, social, and commercial issues, which enables the ad-
`vancementandprogressof therapeutic procedures leading to improvedpatient outcomes,andultimately,
`to curing diseases.
`Information for Manuscript Submission: www.liebertpub.com/hum
`Submit manuscripts online: http://mc.manuscriptcentral.com/hum
`All papers, news, comments, opinions, findings, conclusions, or recommendations in Human Gene
`Therapy are those of the authors and do not constitute those of the Journal, its Publisher,its editorial staff,
`orits affiliated societies.
`Reprints: For permission to photocopy 99 copies or fewer of an article for internal purposes please
`request permission andpay the appropriate fee by contacting the Copyright Clearance Center,Inc.: (978)
`750-8400. If the number of copies is 100 or greater please contact the Publisher at (914) 740-2100 or
`reprints@liebertpub.com
`Advertising inquiries, with the exception of those coming from Europe and Australia, should be ad-
`dressed to the Publisher: (914) 740-2100; E-mail: advertising@liebertpub.com For advertising inquiries
`from Europe and Australia: +44-0-1875-825700; E-mail: impress@impressmedia.com All advertise-
`ments are subject to approval by the Publisher. The acceptance of advertisements doesnot constitute an
`endorsement of the product or service advertised.
`Indexed in: MEDLINE; Current Contents”/Life Sciences; Science Citation Index Expanded; Science
`Citation Index”; Biotechnology Citation Index”; Biological Abstracts; BIOSIS Previews; Journal Ci-
`tation Reports/Science Edition; Derwent Drug File; EMBASE/Excerpta Medica, EMBiology; Scopus;
`and Chemical Abstracts.
`Subscriptions should be addressed to the Publisher and are payable in advance, Bulk subscriptions are
`available upon request from the Publisher. No refunds may be issued after publication of a volume’sfirst
`issue. Visit our website (www.liebertpub.com/hum) or contact our Customer Service Department for
`current subscription rates and pricing information.
`Human Gene Therapy (ISSN: 1043-0342)is owned and published 12 times a year by Mary Ann Liebert,
`Inc. Periodicals postage paid at New Rochelle, NY, andat additional mailing offices. Mailed in Canada
`under CPC CPM #40026674. Copyright © 2012. Printed in the United States of America. Postmaster:
`Send address changes to Human Gene Therapy, c/o Subscription Department, Mary Ann Liebert, Inc.,
`140 HuguenotStreet, New Rochelle, NY 10801-5215.
`Mary Ann Liebert, Inc.
`140 HuguenotStreet
`New Rochelle, NY 10801-5215
`Telephone: (914) 740-2100
`E-mail: info@liebertpub.com
`
`MaryArs Liebert, ineepi
`
`www.liebertpub.com/hum
`
`Miltenyi Ex. 1026 Page 2
`
`Miltenyi Ex. 1026 Page 2
`
`

`

`Human Gene Therapy
`
`The Official Journal of the
`Furopean Society of Gene and Cell Therapy ° British Society for Gene Therapy * French Society of Cell and Gene Therapy
`irish Society for Gene and Cell Therapy »* The Israeli Society of Gene & Cell Therapy * German Society for Gene Therapy
`Spanish Society of Gene and Gell Therapy « Netherlands Society of Gene and Cell Therapy * Finnish Society of Gene Therapy
`Akseli Hemminki, M.D.
`Izumu Saito, M.D., Ph.D.
`University of Helsinki
`University of Tokyo
`
`Ulrich R, Hengge, M.D.
`Heinrich-Heine-University
`
`Roland W. Herzog, Ph.D,
`University of Florida
`Steven Howe, Ph.D.
`UCL Institute of Child Health
`
`Wenlin Huang, Ph.D, M.D.
`Sun¥ar-sen University
`
`Carl H, June, M.D,
`University of Pennsylvania Perelman
`School af Medicine
`
`Chang-Yuil Kang, Ph.D.
`Seoul National University
`Hans-Peter Kiem, M.D.
`University of Washington School ofMedicine
`David Klatzmann, M.D., Ph.D.
`INSERM
`
`Robert M. Kotin, Ph.D.
`National Heart, Lung, and Blood Institute
`
`Francois M. Lemoine, M.D., Ph.D.
`Université Pierre and Marie Curie
`
`You Lu, M,D,
`Sichuan University
`
`Patrick Midoux, Ph.D.
`INSERM-CNRS
`
`Eugenio Montini, Ph.D.
`San Raffuele Telethon Institute
`for Gene Therapy
`
`Philippe Moullier, M.D., Ph.D.
`INSERM-Genethon
`
`Rita Mulherkar, Ph.D,
`Tata Memorial Centre
`
`Nicholas Muzyezka, Ph,D,
`University of Florida
`
`Hiroyuki Nakai, M.D., Ph.D.
`Health & Science University Schaal ofMedicine
`
`Timothy O'Brien, M.D., Ph.D,
`National University of Ireland, Galway
`Richard Peluso, Ph.D.
`Merck & Co., Ine.
`
`Katherine Ponder, M.D.
`Washington University School of Medicine
`
`ZhiYong Qian, M.D.
`Sichuan University
`
`John E.J. Rasko, M.B.B.S., Ph.D.
`CenfenaryInstitute and Royal Prince
`Alfred Hospital
`
`Paul D. Robbins, Ph.D.
`University of Pittshurgh School
`of Medicine
`John J. Rossi, Ph.D.
`City of Hope
`
`Debi P. Sarkar, Ph.D.
`University of Delhi South Campus
`
`Ton Schumacher, Ph.D.
`The Netherlands Cancer Institute
`
`Arun Srivastava, Ph.D.
`Universiry of Florida College of Medicine
`
`George Stamatoyannopoulos, M.D,, D.Sc.
`University af Washington
`
`Mark Tangney, Ph.D.
`Cork Cancer Research Center
`
`Victor W. van Beusechem, Ph.D.
`VU University Medical Cenier
`
`Christof von Kalle, M.D., Ph.D.
`National Centerfor Tumor
`Diseases (NCT)
`
`Simon N, Waddington, Ph.D.
`Jmperial College London
`
`Gerard Wagemaker, Ph.D,
`Erasmus University Medical Center
`
`Matthew D, Weitzman, Ph.D.
`The Children's Hospital of Philadelphia
`
`John EL. Wolfe, V.M.D., Ph.D.
`University of Pennsylvania
`Jon A, Wolff, M.D.
`Roche Madison Inc.
`
`Joseph C. Wu, M.D,, Ph.D.
`Stanford University School of Medicine
`
`Xiao Xiao, Ph.D.
`University of North Carolina
`
`Rafael J. Vaiiez-Munoz, Ph.D.
`Royal Holloway, University of Landon
`
`Yiping Yang, M.D., Ph.D.
`Duke University Medical Center
`
`Seppo Yla-Herttuala, M.D,, Ph.D.
`University af Kuopia
`
`Yoshi Yonemiisu, M.D, Ph.D.
`Kyushu University
`
`Gideon Zamir, M.D.
`Halayseli-Hebrew University
`Medical Center
`
`Xia Zhao, M.D,
`Sichuan University
`
`Editorial Office
`Scientific Editors
`Sadik H. Kassim, Ph.D,
`Rebeca M. Tenney
`E-wail; AGThewswire@lieberipub,com
`
`Martha Turnitsa
`Editorial Manager
`Human Gene Therapy
`University of Pennsylvania
`125 §. Fist St, Ste. 2000 TRL
`Philadelphia, PA 19104
`E-mail: ngi@mail.med.wpenn.edu
`
`Founding Editor
`W. French Anderson, M.D.
`
`Editor-in-Chief
`James M. Wilson, M.D., Ph.D.
`Univ. of Fennsylvania Perelman School
`af Medione
`Gene Thenipy Program
`Dept of Pathalogy and Laboratory Medicine
`sy 2000, Translational Research
`Laboratories
`125 §. 34st Street
`Philadelphia, PA 19104-3403
`E-mail? wilsonjm@ mail, med.upenn.edu
`
`Deputy Editor
`Professor George Dickson
`Justitite of Biomedical & Life Sciences
`Shoal of Biological Sciences
`Roval Holloway - University of London
`Egium, Surrey, TW20 OEX, UK
`E-mail: g.dickson@rhul.ac.uk
`
`
`Methods Editor
`Thierry VandenDriessche, Ph.D.
`Head-Division of Gene Therapy
`& Regenerative Medicine
`Faculty of Medicine & Pharmacy
`Free University of Brussels (VUB)
`Laarbeeklaan 103
`Building A
`B-J090 Brussels
`Belgium
`E-mail: thierry.vandendniessche@ yub.ac.be
`
`Associate Editors
`Chiara Bonini, M.D.
`Experimental Hematology Unit
`Division af Regenerative Medicine,
`Gene Therapy and Stem Cells
`Son Raffaele Scientific Instinite
`Via Olgettina 58
`20132, Milano, laly
`E-niail: bommi.chiara@hsr.it
`
`Scientific Editorial Board
`Kari Airenne, Ph.D.
`University af Easter Finland
`
`Ramon Alemany, Ph.D.
`Institut Catald d'Oncologia-IDIBELL
`Tan E. Alexander, M.B.B.S,, Ph.D.
`University of Sydney
`
`Daniel G, Anderson, Ph.D.
`Massachusetts Institute of Technology
`
`Christopher Baum, M.D., Ph.D.
`Hannover Medical School
`
`Alessundra Biff, M.D.
`San Raffaele Scientific Institute
`
`Attilio Bondanza, M.D., Ph.D.
`San Raffaele Hospital
`Xandra O. Breakefield, Ph.D.
`Massachusetts General Hospital
`Juan A. Bueren, Ph.D.
`CIEMAT
`
`Hildegard Biining, Ph.D.
`Uniklinik Kain
`
`Barrie J. Carter, Ph.D.
`Carter BioConsulting
`
`Nathalie Cartier-Lacave, M.D.
`INSERM
`
`Toni Cathomen, Ph.D.
`Hanmaver Medical School
`
`Saswati Chatierjee, Ph.D.
`City of Hope Natl. Medical Center
`
`June-Key Chung, M.D,, Ph.D.
`Seayl National University
`
`Mary Collins, Ph.D.
`Royal Free and Universiry Callege
`Medical School
`
`Terence R, Flotte, M.D.
`Gene Therapy Center
`Depariments of Pediatrics and Microbiology
`& Physiologic Systems University
`of Massachusetts Medical School
`55 Lake Avenue North, Suite $1-340
`Worcester, MA O0/655
`E-mail: terry.flotte@umassmed.edu
`
`Kenneth Cornetta, M.D.
`Indiana University School of Medicine
`
`Frangois-Loic Cosset, Ph.D.
`Ecole Normale Supérieure de Lyon
`
`Gay M. Crooks, M.B,, B.S.
`University of California, Lox Angeles
`
`Ronald G. Crystal, M.D,
`Weill Comell Medical College
`
`Anja Ehrhardt, Ph.D.
`University of Munich
`
`John F. Engelhardt, Ph.D.
`University of Jowa
`
`Erik Falck-Pedersen, Ph.D,
`Weill Comel! Medical College
`
`Sarah Ferber, Ph.D.
`Sheba Medical Center
`
`Anne Galy, Ph.D.
`GENETHON
`
`Mark A, Kay, M.D., Ph.D.
`Departments of Pediatrics and Genetics
`Stanford University School of Medicine
`269 Campus Drive #2105
`Stanford, CA 94305
`E-mail: markay@stanford.edu
`
`Adrian J, Thrasher, M.D., Ph.D.
`Centre for Immunodeficiency
`Molecular Jmmunalogy Unit
`UCL Instinite of Child Health
`40) Guilford Street
`London WCIN LEH, UK
`E-mail: A.Thrasher@ich.ucl.ac.uk
`
`Yu-quan Wei, M.D., Ph.D.
`National Key Laboratory of Biotherapy and
`Cancer Center
`West China Hospital, West China Medical
`School, Sichuan University
`Guopeng Street, Keyuan Street Road 4, No. |
`Chengdu, Sichuan 61004)
`PR. China
`E-mail: yuquanwei@scu.edu,cn
`
`Guangping Gao, Ph.D.
`University of Massachusetts Medical School
`
`David W- Russell, M.D,, Ph.D.
`University of Washingron
`
`Manuel Grez, Ph.D.
`University of Frankfurt
`
`Stephen J. Russell, M.D., Ph.D.
`Maya Clinic College of Medicine
`
`Michel Sadelain, M.D., Ph.D.
`Yajun Guo, M.D., Ph.D.
`Shanghai Second Miltary Medical University Memorial Sloan-Kettering Cancer Cenier
`
`
`r *
`
`Marg Ams Liebert, Ine.
`
`poblishers
`
`Miltenyi Ex. 1026 Page 3
`
`Miltenyi Ex. 1026 Page 3
`
`

`

`Human Gene Therapy
`
`
`
` Volume 23 Number 5 May 2012
`
`
`
`EDITORIAL
`
`A New Open Access Partner
`JM. Wilson
`
`COMMENTARY
`
`Double-Blinded, Placebo-Controlled, Randomized Gene Therapy Using Surgery
`for Vector Delivery
`R.G. Crystal, 8.M. Kaminsky, N.R. Hackett, and T.K. Rosengart
`
`GENE THERAPY BRIEFS
`A. Philippidis
`
`RESEARCH ARTICLES
`
`Infusing CD19-Directed T Cells to Augment Disease Control in Patients
`Undergoing Autologous Hematopoietic Stem-Cell Transplantation for Advanced
`B-Lymphoid Malignancies
`P. Kebriaei, H. Huls, B. Jena, M. Munsell, R. Jackson, D.A. Lee, P.B. Hackett, G. Rondon,
`E. Shpall, R.E. Champlin,-and L.J.N. Cooper
`
`Kebriaei and colleagues report on their phase 1 protocol to examine the safety and feasibility of administering
`genetically modified autologous T cells expressing a CD19-specific chimeric antigen receptor (CAR)to patients
`with high-risk B-lymphoid malignancies undergoing autologous hematopoietic stem cell transplantation, CAR
`was introduced to T cells by nonviral gene transfer, using the Sleeping Beauty system and signals through
`chimeric CD28 and CD3-t.
`
`AAVrh.10-Mediated Expression of an Anti-Cocaine Antibody Mediates Persistent
`Passive Immunization That Suppresses Cocaine-Induced Behavior
`J.B. Rosenberg, M.J, Hicks, B.P. De, O. Pagovich, E. Frenk, K.D. Janda, S. Wee, G.F. Koob,
`N.R. Hackett, S.M. Kaminsky, S. Worgall, N. Tignor, J.G. Mezey, and R.G. Crystal
`
`Rosenberg and colleagues construct an AAV serotype rh.10 vector that encodes the heavy andlight chains
`of the high-affinity anti-cocaine monoclonal antibody GNC92H2. They demonstrate that intravenous administration
`of this vector leads to high serum levels of cocaine-specific antibodies that block administered cocaine from access
`to ihe brain, prevent cocaine-induced hyperlocomotoractivity, and lead to anticocaine passive immunity in mice.
`
`Immunodominant Liver-Specific Expression Suppresses Transgene-Directed
`Immune Responsesin Murine Pompe Disease
`P. Zhang, B. Sun, T. Osada, R. Rodtiguiz, X.Y. Yang, X. Luo, A.R. Kemper, T.M. Clay,
`and D.D. Koeberl
`
`Zhang and colleagues investigate the mechanism for immunomodulatory gene therapy by evaluating two AAV
`vectors encoding human acid a-glucosidase (hGAA), including both a tolerogenic vector containing theliver-specific
`promoter (LSP) and an immunogenic vector containing the ubiquitously active cytomegalovirus enhancer and chicken
`B-actin promoter (CB) regulatory cassette. The authors find that simultaneous administration of tolerogenic
`and immunogenic vectors induces immunetolerance in mice and leads to higher levels ofliver transduction,
`in comparison with either vector alone,
`
`Characterization of an HIV-Targeted Transcriptional Gene-Silencing RNA
`in Primary Cells
`A.-M.W. Turner, A.M. Ackley, M.A. Matrone, and K.V. Morris
`
`437
`
`438
`
`442
`
`444
`
`451
`
`460
`
`473
`
`Turner and colleagues demonstrate that transcriptional gene silencing (TGS) can reduce viral transcription
`in primary human CD4*Tcells, although increasing viral burden resulted in the loss of this antiviral effect.
`Studies into off-target effects additionally identified a novel potential interaction between the small nucleolar
`RNA pathway and the TGS-based antisense RNA, resulting in activation of p53.
`
`(continued)
`
`Miltenyi Ex. 1026 Page 4
`
`Miltenyi Ex. 1026 Page 4
`
`

`

`re
`
`*
`=
`
`ie
`A
`a
`
`byl
`
`
`
`
`Retargeting Vesicular Stomatitis Virus Using Measles Virus Envelope Glycoproteins
`C. Ayala-Breton, G.N. Barber, S.J. Russell, and K.-W. Peng
`Ayala-Breton and colleaques use the measles virus (MV) hemagglutinin (H) and fusion (F) envelope glycoproteins
`ta redirect vesicular stomatitis virus (VSV) entry and infection specifically to tumor-associated receptors
`such as epidermal growth factor receptor,folate receptor, and prostate membrane-specific antigen. /n vivo
`expression ofthe all retargeted VSV was restricted to receptor-positive human tumor xenograits.
`
`Development and Validation of Novel AAV2 Random Libraries
`Displaying Peptides of Diverse Lengths and at Diverse Capsid Positions
`M. Naumer, Y. Ying, S. Michelfelder, A. Reuter, M. Trepel, O.J. Muller, and J.A. Kleinschmidt
`Naumer and colleagues expand the combinatorial AAV2 display system with a panelof novel AAVlibraries displaying
`peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or 7-mer peptides at position 453.
`Characterization of vector selectivity by transduction of nontarget cells and comparative genetransduction analysis
`using a panel of 44 human tumorcell lines revealed that insertion of different-length peptides allows targeting
`of distinct cellular receptors for cell entry with similar efficiency, but different selectivity.
`
`484
`
`A492
`
`Silencing of miR20a Is Crucial for Ngn1-Mediated Neuroprotectionin Injured
`Spinal Cord
`M.K. Jee, J.S. Jung, Y.B. Im, S.J. Jung, and S.K. Kang
`Jee and colleagues demonstrate that small interfering RNA (siRNA)-mediatedinhibition of microRNA 20a (miR20a)
`leads to increased expression of a key neuronaltranscription factor, neurogenin-1 (Ngn1). They show that siRNA-
`mediated knockdown of miR20a enhances neural cell survival and neurogenesis and leads to recovery of motor
`function in a mouse model of traumatic spinal cord injury.
`Asymmetric siRNA: New Strategy to Improve Specificity and Reduce Off-Target
`Gene Expression
`Z, Yuan, X. Wu, C. Liu, G. Xu, and Z. Wu
`
`508
`
`521
`
`In this study, Yuan and colleagues demonstrate that asymmetrically shortened siRNAs cansilence their targets
`as efficiently as their traditional 19+ 2 counterpartsin vitro. Moreover, the authors suggestthat shortening siRNAs
`from their 3‘ end improves the loading specificity of the RNA-induced silencing complex and results in feweroff-target
`gene expression effects.
`
`BRIEF REPORT
`
`Hepatic Gene Transfer in Neonatal Mice by Adeno-Associated Virus
`Serotype 8 Vector
`L, Wang, H. Wang, P. Bell, D. McMenamin, and J.M, Wilson
`Wang and colleagues conducta series of preclinical animal studies examining the kinetics of AAV gene transfer.
`They demonstrate that self-complementary AAV8results in robust andefficient hepatic genetranster in neonatal
`mice. Yet, this transduction quickly decreases over a few weeks because ofvectordilution caused by rapid cell
`proliferation in the liver of growing young mice.
`
`
`533
`
`British Society for Gene Therapy 2012
`March 9, 2012
`UCL Institute of Child Health, London
`
`pp A1-A22
`ABSTRACTS AVAILABLE ONLINE ONLY
`
`
`Instructions for Authors can be found on our website at www.liebertpub.com/hum
`
`Cover: Adeno-associated virus type 2 (AAV2) capsid protein structures showing two peptide insertion
`sites at the top of the spikes around the threefold axes, enabling the development of AAV2 random
`libraries displaying peptides at two different positions. Insertion sites are marked together with two
`neighboring amino acids in purple (arginine 588) and cyan (glycine 453) in the side and top view of a
`capsidprotein trimer. For further details, see the article by Naumeref a/. on page 492.
`
`MaryArn Liebert, ee
`
`www.liebertpub.com/hum
`
`Miltenyi Ex. 1026 Page 5
`
`Miltenyi Ex. 1026 Page 5
`
`

`

`
`
`HUMAN GENE THERAPY 23:444-450 (May 2012)
`© Mary Ann Liebert, Inc.
`DOI: 10.1089/hum.2011 .167
`
`ResearchArticles
`
`Infusing CD19-Directed T Cells to Augment Disease Control
`in Patients Undergoing Autologous Hematopoietic Stem-Cell
`Transplantation for Advanced B-Lymphoid Malignancies
`
`Partow Kebriaei’ Helen Huls2 Bipulendu Jena? Mark Munsell! Rineka Jackson? Dean A. Lee??
`Perry B, Hackett? Gabriela Rondon! Elizabeth Shpall! Richard E. Champlin! and Laurence J.N. Cooper?*
`
`Abstract
`Limited curative treatment options exist for patients with advanced B-lymphoid malignancies, and new ther-
`apeutic approaches are needed to augmenttheefficacy of hematopoietic stem-cell transplantation (HSCT).
`Cellular therapies, such as adoptive transferofT cells that are being evaluated to target malignant disease, use
`mechanisms independent of chemo- and radiotherapy with nonoverlapping toxicities. Gene therapy is em-
`ployed to generate tumor-specific T cells, as specificity can be redirected through enforced expression of a
`chimeric antigen receptor (CAR) to achieve antigen recognition based on the specificity of a monoclonal anti-
`body. By combining cell and gene therapies, we have opened a new Phase | protocolat the MD Anderson Cancer
`Center (Houston, TX) to examine the safety and feasibility of administering autologous genetically modified T
`cells expressing a CD19-specific CAR (capable of signaling through chimeric CD28 and CD3-() into patients with
`high-risk B-lymphoid malignancies undergoing autologous HSCT. The T cells are genetically modified by
`nonviral gene transfer of the Sleeping Beauty system and CAR™Tcells selectively propagated in a CAR-de-
`pendent manneron designerartificial antigen-presenting cells. The results of this study will lay the foundation
`for future protocols including CAR” T-cell infusions derived from allogeneic sources.
`
`Introduction
`
`for patients with
`oe TREATMENT OPTIONS exist
`B-lymphoid malignancies whorelapse after autologous or
`allogeneic hematopoietic stem-cell transplantation (HSCT). Re-
`garding non-Hodgkin lymphoma (NHL), 50% of patients who
`relapse after conventional chemotherapy may be salvaged by
`autologous HSCT. Amongthose whoare transplanted,residual
`disease at the time oftransplantation predicts an increased rate
`of relapse (Mills et al., 1995; Caballero et al., 2003), Allogeneic
`HSCT, which may deliver a potent graft-versus-tumor (GVT)
`effect mediated by donor-derived immunocompetentcells, can
`be potentially curative in 30-50%of patients (Ratanatharathorn
`et al, 1994; Verdonck, 1999). However, because of the high
`treatment-related mortality (TRM) associated with allogeneic
`HSCT,the GVT effect is not always correlated with improved
`overall survival (Peniket et al., 2003). Thus, novel therapeutic
`approaches are urgently needed for these patients.
`
`T-cell therapy can specifically target disease, employing
`anti-tumor mechanisms that are nonoverlapping with the
`conditioning regimen used for HSCT. Therefore, investiga-
`tors have developed adoptive immunotherapy as a strat-
`egy to augment
`the GVT effect. To overcome immune
`tolerance to tumor-associated antigens (TAAs)investigators
`have developed and implemented immunotherapies infus-
`ing T cells genetically modified to express a chimeric antigen
`receptor (CAR)(Paulos etal., 2008;Jenaet al., 2010). CARs are
`typically engineered to provide the genetically modified T
`cell with the specificity of a murine monoclonal antibody
`and, on binding a cell surface TAA, can specifically activate
`the T cell for proliferation, cytokine production, and cytolysis
`(Eshharef al., 1993; Brentjenset al., 2003; Cooper et al., 2003;
`Park et al,, 2007; Brenner and Heslop, 2010; Porter et al.,
`2011). CARs are “universal” in that they bind TAAs inde-
`pendent of major histocompatibility complex (MHC), and
`thus one receptor construct can be used to treat a population
`
`
`Ipivision of Cancer Medicine, M.D. Anderson Cancer Center, Houston, TX 77005.
`2Division of Pediatrics, M.D. Anderson Cancer Center, Houston, TX 77005.
`3Graduate Schoolof Biomedical Sciences, University of Texas, Houston, TX 77005.
`“Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455.
`
`444
`
`Miltenyi Ex. 1026 Page 6
`
`Miltenyi Ex. 1026 Page 6
`
`

`

`DISEASE CONTROL AUGMENTED WITH CD19-DIRECTED T CELLS
`
`445
`
`lymphoma (SLL), chronic lymphocytic leukemia (CLL), fol-
`licular lymphoma, or mantle cell lymphoma;that are beyond
`first relapse or primary refractory to conventional treatment.
`Additionaleligibility criteria include adequate organ func-
`tion, a Zubrod performancestatus of 0 or 1, no evidence of
`uncontrolled infection, and negative serology for hepatitis B
`(HBV), hepatitis C (HCV), and HIV. Patients with active
`CNSdisease are excluded. In addition, patients with known
`allergy to bovine or murine products will be excluded, Fur-
`thermore, patients must not be taking systemic corticoste-
`roids within 3 days before T-cell infusion or experiencing any
`new significant toxicity within 24hours before T-cell infu-
`sion. Last, to be eligible for [L-2 injections after administra-
`tions of T cells, patients must not experience any new
`significant adverse events (AEs) probably or definitely at-
`tributed ta the adoptively transferred T cells.
`
`T cell manufacture and release
`
`of patients with the same TAA* tumors. Multiple CAR de-
`signs have emerged to dock with multiple TAAs and to
`provide a fully competentactivation signal in desired T-cell
`subsets (Berry et al., 2009; June etal., 2009; Jena et al., 2010).
`CAR* T cells have been tested in multiple trials at multiple
`centers, as we have reviewed (Jena etal., 2010).
`Here we describe a new PhaseI cell and gene immuno-
`therapy trial administering autologous ex vivo-expanded
`genetically modified T cells expressing a CD19-specific CAR
`into patients with high-risk B-lymphoid malignancies un-
`dergoing autologous HSCT.Therationale for redirecting the
`specificity of T cells for CD19, using a CAR,is based on the
`following: (1) CD19 is a B-lineage cell surface TAA expressed
`on lymphoid malignancies; (2) CD19 is not expressed on
`hematopoietic stem cells (HSC) and (3) soluble CD19 is ap-
`parently not shed into the circulation (to act as a competitor
`for binding CAR to CD19 on tumorcells). Preliminary
`studies demonstrated the safety, feasibility, and efficacy of
`infusing CD19-specific CAR therapy for lymphoid malig-
`The two SB DNAplasmids (manufactured by Waisman
`nancies (Kochenderferet al., 2010). Our results on infusing a
`Biomanufacturing, Madison, WI), encoding the CAR (des-
`first-generation CD19-specific CAR into patients with re-
`ignated CD19RCD28) transposon (Cooper, 2007; Singhetal.,
`current follicular lymphoma after a preparative regimen
`2008; Jena et al., 2010) and SB11 transposase (Singh et al.,
`consisting of rituximab and fludarabine demonstrated limited
`2008; Manuri et al., 2009), will be simultaneously electro-
`persistence due to mcomplete signaling through CD3-{ and an
`transferred into T cells with Nucleofector device (Lonza
`immune response to immunogenic transgenes coexpressed
`Group, Basel, Switzerland). The genetically modified T cells
`with CAR inTcells (Jensen, 2007). Therefore, we developed a
`will be selectively propagated in a CAR-dependent manner
`new platform for the genetic modification of T cells based on
`on y-irradiated K562 cells that have been genetically modi-
`(1) a second-generation CAR that signals through both chi-
`fied by transduction with lentivirus (in collaboration with C.
`meric CD28 and CD3-{ (Singh, 2008), (2) electrotransfer of the
`June at the University of Pennsylvania, Philadelphia, PA)
`Sleeping Beauty (SB) system to improve integration efficiency
`and cloned by limiting dilution to function as CD19* aAPC.
`of CAR transgene, and (3) outgrowth of CAR* T cells on
`The CD19RCD28 transgene is a second-generation CAR that
`designerartificial antigen-presenting cells (aAPC)to select for
`activates T cells via chimeric CD28 and CD3-f. The aAPC
`T cells with proven proliferative potential.
`(clone #4) were manufactured as a Master Cell Bank by
`Production Assistance for Cellular Therapies (PACT) under
`the auspices of the National Heart, Lung, and Blood Institute
`(NHLBI, Bethesda, MD). Subsequently, a Working Cell Bank
`of clone #4 was derived at MDACC and used to support
`numeric expansion of CAR* T cells as cultured in the pres-
`ence of soluble recombinant human IL-2 and IL-21. The
`validation studies describing phenotype and function of the
`CAR* T cells manufactured in compliance with current good
`manufacturing practice (CGMP) are being published (Singh
`et al., 2012).
`In the event
`there is an overgrowth of
`CD3"°®CD56* lymphocytes early in the culturing process,
`these cells will be removed using CD56-specific monoclonal
`antibody and paramagnetic selection. The electroporation
`and propagation ofclinical-grade T cells will occur at the
`MDACC in compliance with cGMP for Phase I/II trials.
`The release criteria, undertaken in compliance with Clinical
`Laboratory Improvement Amendments (CLIA),
`for
`the
`manufactured T cells are (1) sterility (bacteria, fungi, myco-
`plasma, endotoxin),
`(2) chain of custody (low-resolution
`MHCclass I typing), (3) phenotype (presence of T cells [CD3
`expression], presence of transgene [CAR expression], ab-
`sence of aAPC [CD32 expression], absence of B cells [CD19
`expression]), (4) safety (absence of autonomous cell growth),
`and (5) viability.
`
`Clinical Trial Design
`
`Regulatory approvals
`
`The clinical trial is open only at the MD Anderson Cancer
`Center (MDACC, Houston, TX) as IRB #2007-0635 (IBC
`#RM0508-070). Approval was obtained from the NIH Office
`of Biotechnology Activities (OBA, #0804-922) and U.S. Food
`and Drug Administration (FDA) (IND #14193). Thetrial is
`listed at ClinicalTrials.gov (identifier: NCT00968760).
`
`Objectives
`
`The primary objective is to assess the safety, feasibility,
`and persistence of autologous ex vivo-expanded genetically
`modified CD19-specific CAR®* T cells intravenously admin-
`istered to patients with persistent or relapsed CD19* lym-
`phoid malignancies, The secondary objectives will include
`(1) screening for the developmentof host immune responses
`against the CD19-specific CAR, (2) describing the ability of
`the infused T cells to hometo sites of disease, such as bone
`marrow and lymph nodes, (3) assessing the impact of in-
`terleukin (IL)-2 on T-cell persistence, and (4) assessing dis-
`ease response.
`
`Patient eligibility
`
`Patients are eligible if they are between 18 and 60 years of
`age with advanced CD19* lymphoid malignancies, includ-
`ing non-Hodgkin's lymphoma (NHL), small
`lymphocytic
`
`Peripheral blood stem cell mobilization and collection
`
`The methods of peripheral blood stem cell (PBSC) mobi-
`lization, collection, storage, and infusion have been described
`
`Miltenyi Ex. 1026 Page 7
`
`Miltenyi Ex. 1026 Page 7
`
`

`

`446
`
`KEBRIAEI ET AL.
`
`TABLE 1, RESEARCH PARTICIPANT ASSIGNMENT
`to T Ceti Dosinc CoHoRTS
`
`
`
` Dose cohort Single T cell dose* IL-2
`
`
`
`No
`>10’/m? but <5x10’/m?
`Dose level X
`No
`>5x107/m?* but <5 10°/m?
`Dose level A
`No
`>5x108/m? but <5x10°/m?
`Dose level B
`Yes
`>5x107/m? but <5 10°/m?
`Dose level C
`
`
`>5x10°/m? but <5 10°/m?Dose level D Yes
`
`*Tcell dosing, starting at Dose level A, is calculated on the basis of
`prefteeze counts, and infused over a 2-day split.
`
`istered as a single daily injection at 0.3 x 10° [U/m*beginning
`within 2 days of the last split of the T-cell infusion and
`continuing for up to 14 daily doses. Patients may be pre-
`medicated with acetaminophen and diphenhydramine be-
`fore each injection. The [L-2 dose will be reduced by 50%if
`the patient develops a new adverse event of grade >2 (CTC,
`version 4) involving cardiopulmonary, hepatic (excluding al-
`bumin), gastrointestinal, neurological, or renal toxicity prob-
`ably or definitely attributed to IL-2 administration.IL-2 will be
`stopped in patients with persistent grade >2 (CTC,version 4)
`toxicity despite IL-2 dose reduction. Patients taking IL-2 with
`a grade 4 adverse event will stop IL-2. If the adverse event
`does not improve to grade <3 within 36 hr of stopping IL-2,
`then corticosteroids will be started. Isolated injection site skin
`toxicity attributable to IL-2 administration may be an indica-
`tion to discontinue IL-2, but not to ablate T cells.
`
`Supportive care
`Institutional HSCT guidelines for antimicrobial, antifun-
`gal, and antiviral prophylaxis will be followed. Patients will
`not receive filgrastim to enhance neutrophil recovery unless
`there is concern for serious infection. Packed red bloodcells
`will be administered to maintain hemoglobin levels 2>8 g/dl.
`Platelet transfusions will be administered to keep platelet
`counts 210x10°/liter. All blood products are filtered and
`irradiated, All research participants who are able to have
`children mustpractice effective birth control while on study.
`
`Study assessments
`
`Miltenyi Ex. 1026 Page 8
`
`Patients will have an initial assessment to include HLA
`typing, serology for cytomegalovirus (CMV), HIV, human T-
`lymphotropic virus (HTLV)-1, HCV, and HBV, as well as
`peripheral blood (PB) collected before manufacture of CAR*
`T cells and analyzed for protein expression and genetic
`profiling. Before initiating the conditioning regimen for
`HSCT,
`the patients will undergo institutional standard-of
`care evaluations to validate adequate organ functions and
`standard restaging studies, including bone marrow biopsy
`and positron emission tomography/computed tomography
`(PET/CT)scans, as clinically indicated. Immediately after the
`T-cell infusion (last split dose) and then weekly for 2 weeks,
`and then at 1, 3, 6, and 12 months, the research participants
`will receive physical and laboratory evaluations, such as PB
`for protein expression and genetic profiling, including PCR
`After we have established safety and feasibility of the
`analyses for the presence ofinfusedTcells. Skewing ofthe T-
`T-cell dosing in the first two cohorts of the study (Table 1),
`cell receptor repertoire may indicate that someinfusedTcells
`we will assess whether low-dose IL-2 can support the sur-
`can preferentially survive and thus the PB will be serially
`vival and/or numeric expansion of CAR* T cells in the last
`analyzed for emergenceofoligoclonal or clonal population(s)
`two cohorts ofthe trial. IL-2 will be subcutaneously admin-
`
`(Hosing et al., 2006). Patients will receive nonpurged autol-
`ogous HSC collected after mobilization with filgrastim and
`chemotherapy. The target progenitor cell dose is 410°
`CD34" cells/kg with a minimal acceptable dose of 2x 10°
`CD34* celis/kg. Patients failing to reach that target cell
`number can undergo bone marrow harvestat the discretion
`of the treating physician. Bone marrow will be obtained by
`multiple aspirations from the right and left iliac crest under
`general anesthesia, with a target total nucleated cell dose of
`3x 108 cells/kg. All products are cryopreserved according to
`standardinstitutional techniques.
`
`Conditioning regimen for HSCT
`
`We will use our standard-of-care conditioning regimen
`as a means to lympho-deplete the patients in an effort to
`improve the persistence of CAR* T cells. The conditioning
`regimen consists of intravenous carmustine on day —6 ata
`(300 mg/m?) infused over 2hours, fo