`
`
`
`Mary Arn Lichert, (ne.«publishers
`
`
`
` TTTTETETeee
`
`VOLUME 10, NUMBER 2, January 20, 1999
`
`ISSN: 1043-0342
`
`TIN)
`
`Gen
`Therapy
`
`Editor-in-Chief:
`
`W. French Anderson
`
`Associate Editors:
`
`Malcolm K. Brenner
`
`James M. Wilson
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`European Editors:
`Claudio Bordignon
`Jean Michel Heard
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`Asian Pacific Associate Editor:
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`Full Text Online: http://www.catchword.com/titles/10430342.htm
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`Miltenyi Ex. 1021 Page 1
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`2
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`General Information
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`Human Gene Therapy answers the need for a central forum dealing with all aspects of gene transferin
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`Miltenyi Ex. 1021 Page 2
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`

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`
`Human Gene Therapy
`
`www.humangenetherapy.com
`
`ee
`
`oh
`
`+inthe
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`
`
`Editor-in-Chief
`W.French Anderson, M.D.
`Journal Office
`University ofSouthern California
`School ofMedicine
`1975 Zonal Avenue, KAM 300
`Los Angeles, CA 90033
`(323) 442-GENE (4363)
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`Xandra Breakfield, Ph.D.
`Massachusetts General Hospital
`Charlestown
`Barrie J. Carter, Ph.D.
`Targeted Genetics Corporation
`Seattle
`C. Thomas Caskey, M.D.
`Merck Research Laboratory
`West Point, PA
`Francis S. Collins, M.D., Ph.D.
`NIH
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`Associate Editors
`Malcolm Brenner, M.D., Ph.D.
`F.R.C.P., M.R.C.Path.
`Baylor College ofMedicine
`Cell and Gene Therapy Program
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`Houston, TX 77030
`(713) 770-4663
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`James M.Wilson, M.D., Ph.D.
`Director, Institutefor Human Gene Therapy
`Wistar Institute
`University ofPenn Medical Center
`3601 Spruce Street
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`European Editor
`Clavdio Bordignon, M.D.
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`Laboratoire Rétrovirus et Génétique
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`Asian Pacific Associate Editor
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`First Department ofInternal Medicine
`Kanazawa University
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`Kanazawa 920 Japan
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`Staff
`Tris E. Samson
`Dipika Patel
`FlorencePaillard, Ph.D.
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`Scientific Editorial Board
`ArthurL. Beaudet, M.D.
`Baylor College ofMedicine
`Houston
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`R. Michael Blaese, M.D.
`NIH
`Thomas Blankenstein, M.D.
`Max Delbriick Centrumfiir
`Molekulare Medizin, Germany
`Richard C. Boucher, M.D.
`University ofNorth Carolina at Chapel Hill
`
`Kenneth Cornetta, M.D.
`Indiana University SchoolofMedicine
`Ronald G. Crystal, M.D.
`New York Hospital-Cornell Medical Center
`David T. Curiel, M.D.
`University ofAlabama at Birmingham
`Albert B. Deisseroth, M.D., Ph.D.
`Yale University
`New Haven, CT
`David Dichek, M.D.
`Gladstone Institute ofCardiovascular Disease
`San Francisco
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`Theodore Friedmann, M.D.
`University of California, San Diego
`La Jolla
`Eli Gilboa, Ph.D.
`Duke University Medical Center
`Michael M. Gottesman, M.D.
`Laboratory of Cell Biology, NIH
`Philip D. Greenberg, M.D.
`University of Washington
`Seattle
`Katherine High, M.D.
`University ofPennsylvania
`Keith Humphries, M.D., Ph.D.
`Terry Fox Laboratories
`Vancouver, BC, Canada
`Douglas Jolly, Ph.D.
`Chiron Technologies
`San Diego
`MarkA.Kay, M.D., Ph.D.
`Stanford University
`Stanford, CA
`_ William Kelley, M.D.
`University ofPennsylvania Medical
`Center
`Donald B. Kohn, M.D.
`University ofSouthern California
`Los Angeles
`Robert Kotin, Ph.D.
`NIA
`
`Jeffrey M. Leiden, M.D., Ph.D.
`University of Chicago
`.
`R. Scott McIvor, Ph.D.
`University ofMinnesota, Minneapolis
`A. Dusty Miller, Ph.D.
`Fred Hutchinson Cancer Research Center
`Seattle, WA
`Richard Morgan, Ph.D.
`Clinical Gene Therapy, NIH
`James Moulé, Ph.D.
`University ofMichigan Medical Center
`Ann Arbor
`.
`Richard C. Mulligan, Ph.D.
`Whitehead Institute for Biomedical Research
`Cambridge
`Gary J. Nabel, M.D., Ph.D.
`University ofMichigan Medical Center
`Ann Arbor
`Arthur Nienhuis, M.D.
`St Jude Children’s Research Hospital
`Memphis
`Philip Noguchi, M.D.
`Food and Drug Administration
`Rockville, MD
`
`William Osborne, Ph.D.
`University of Washington School ofMedicine
`Seattle
`Robertson Parkman, M.D.
`Children’s Hospital ofLos Angeles
`Amy Patterson, M.D.
`
`Michel Perricaudet, M.D.
`Institut Gustave Roussy
`Villejuif, France
`Steven J. Russell, M.D., Ph.D.
`Mayo Foundation
`Rochester, MN
`R. Jude Samulski, Ph.D.
`University ofNorth Carolina at ChapelHill
`Brian Sorrentino, M.D.
`St. Jude Children’s Research Hospital
`Memphis
`Dinko Valerio, Ph.D.
`University ofLeiden
`Rijswijk, The Netherlands
`Inder Verma,Ph.D.
`Salk Institute
`San Diego
`Jeffrey Weber, M.D.
`USC-Norris Cancer Center
`Los Angeles
`Jeffrey A. Whitsett, M.D.
`Children’s Hospital Medical Center
`Cincinnati, OH
`John Wolfe, V.M.D., Ph.D.
`University ofPennsylvania
`Jon A. Wolff, M.D.
`Waisman Center
`Madison, WI
`Savio L.C. Woo, Ph.D.
`MountSinai Medical Center
`New York
`
`
`
`é
`
`Ethics/Legal/Regulatory
`Editorial Board
`Arthur Caplan, Ph.D.
`University ofPennsylvania
`Alexander M.Capron, LL.B.
`University ofSouthern California
`James F. Childress, Ph.D.
`University of Virginia
`Robert Cook-Deegan, M.D.
`National Academy ofSciences
`John ¢.Fletcher, Ph.D.
`University of Virginia
`Schoal ofMedicine
`Eric Juengst, Ph.D.
`Centerfor Human Genome Research
`Debra Knorr
`NIA
`
`Sheldon Krimsky, Ph.D.
`Tufts University
`Charles McCarthy, Ph.D.
`Kennedy Institute ofEthics
`Gary McGarrity, Ph.D.
`Genetic Therapy, Inc.
`Henry I. Miller, M.D.
`Hoover Institution
`
`Thomas H. Murray, Ph.D.
`Case Western Reserve SchoolofMedicine
`Robert Nelson, Ph.D.
`Institute ofReligion
`at the Texas Medical Center Houston
`LeRoy Walters, Ph.D.
`Kennedy Institute ofEthics
`Nelson Wivel, M.D.
`University ofPennsylvania Medical Center
`Miltenyi Ex. 1021 Page 3
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`
`Miltenyi Ex. 1021 Page 3
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`

`

`
`
`Human Gene Therapy
`
`JANUARY 20, 1999.
`NUMBER2
`VOLUME 10
`
`
`COMMENTARY
`
`Immunotherapy with T Cells Bearing Chimeric Antitumor Receptors
`
`,
`
`151
`
`SCIENTIFIC ARTICLES
`
`Hexokinase Type II: A Novel Tumor-Specific Promoter for Gene-Targeted Therapy
`Differentially Expressed and Regulated in Human CancerCells
`M.M. Katabi, H.L.B. Chan, S.E. Karp, and G. Batist
`
`.
`
`155
`
`*Anti-Tumor Activity of Human T Cells Expressing the CC49-¢ Chimeric
`Immune Receptor
`R.P. McGuinness, Y. Ge, §.D. Patel, S.V.S. Kashmiri, H.-S. Lee, P.H. Hand,
`J. Schlom, M.H. Finer, and J.G. McArthur
`
`High Level of Retrovirus-Mediated Gene Transfer into Dendritic Cells Derived from
`Cord Blood and Mobilized Peripheral Blood CD34*+ Cells
`M. Movassagh, C. Baillou, F.L. Cosset, D. Klatzmann, M. Guigon, and F.M. Lemoine
`
`Functional Characterization of Adenoviral/Retroviral Chimeric Vectors and Their
`Use for Efficient Screening of Retroviral Producer Cell Lines
`G. Duisit, A. Salvetti, P. Moullier, and F.-L. Cosset
`
`Adeno-Associated Virus-Mediated Gene Transfer to the Brain: Duration and Modulation
`of Expression
`W.D. Lo, G. Qu, T.J. Sferra, R. Clark, R. Chen, and P.R. Johnson
`
`.
`
`:
`
`Upregulation of Fibrinolysis by Adenovirus-Mediated Transfer of Urokinase-Type
`Plasminogen Activator Genes to Lung Cells in Vitro and in Vivo
`N. Hattori, T.H. Sisson, Y. Xu, and R.H. Simon
`
`Contribution of Plasmid DNA to Inflammation in the Lung after Administration of
`Cationic Lipid:pDNA Complexes
`N.S. Yew, K.X. Wang, M. Przybylska, R.G. Bagley, M. Stedman, J. Marshall, R.K. Scheule,
`and S.H. Cheng
`
`Toward an Enzyme/Prodrug Strategy for Cancer Gene Therapy: Endogenous
`Activation of Carboxypeptidase A Mutants by the PACE/Furin Family of Propeptidases-
`D.A. Hamstra and A. Rehemtulla
`
`In Vivo Hepatocyte Retrovirus-Mediated Gene Transfer through the Rat Biliary Tract
`J.L. De Godoy, R. Malafosse, M. Fabre, M. Mehtali, D. Houssin, and O. Soubrane
`
`Fas-Fas Ligand Interactions Play a Major Role in Effector Functions of Cytotoxic
`T Lymphocytes after Adenovirus Vector-Mediated Gene Transfer
`N. Chirmule, A.D. Moscioni, Y. Qian, R. Qian, Y. Chen, and J.M. Wilson
`
`Toward Autologous ex Vivo Gene Therapy for the Central Nervous System with Human
`Adult Astrocytes
`J.-L. Ridet, O. Corti, P. Pencalet, N. Hanoun, M. Hamon, J. Philippon, and J. Mallet
`
`165
`
`175
`
`189
`
`201
`
`215
`
`223
`
`235
`
`249
`
`259
`
`271
`
`(continued)
`
`Miltenyi Ex. 1021 Page 4
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`Miltenyi Ex. 1021 Page 4
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`e
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`!e
`
`281
`
`291
`
`301
`
`311
`
`319
`
`333
`335
`
`Intraarterial Delivery of Adenovirus Vectors and Liposome-DNA Complexes to
`Experimental Brain Neoplasms
`N.G. Rainov, K. Ikeda, N.H. Qureshi, S. Grover, U. Herrlinger, P. Pechan, EA. Chiocca,
`X.O. Breakefield, and F.H. Barnett
`Branched Cationic Peptides for Gene Delivery: Role of Type and Numberof Cationic
`Residues in Formation and in Vitro Activity of DNA Polyplexes
`C. Plank, M.X. Tang, A.R. Wolfe, and F.C. Szoka, Jr.
`
` Expression of Biologically Active Atrial Natriuretic Factor Following Intrahepatic Injection
`
`of a Replication-Defective Adenoviral Vector in Dogs
`V. Chetboul, M. Adam, I. Deprez, A. Ambriovic, D. Rosenberg, F. Crespeau, M. Saana,
`L Pham, M. Eloit, 8. Adnot, and J.-L. Pouchelon
`
`DNA Injection into Single Cells of Intact Mice
`J.K. Utvik, A. Njd, and K. Gundersen
`Recombinant Adeno-Associated Virus-Mediated Expression of 0°-Alkylguanine-
`DNA-alkyltransferase Protects Human Epithelial and Hematopoietic Cells against
`Chloroethylating Agent Toxicity
`S.J. Longhurst, J.A. Rafferty, J.R. Arrand, N. Cortez, C. Giraud, KI. Berns,
`and L.J. Fairbairn
`
`NEWS AND COMMENTS
`
`Regulatory Issues
`Future Meetings of the NIH Recombinant DNA Advisory Committee (RAC) and Gene
`Therapy Policy Conferences (GTPC)
`.
`Corrigendum
`*-
`‘
`7
`
`Instructions for Authors can be found at the back of the issue.
`
`*Commentary on this article appears in this issue.
`
`MaryAmn Liebert, tnebpled
`
`www.liebertpub.com
`
`Miltenyi Ex. 1021 Page 5
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`Miltenyi Ex. 1021 Page 5
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`

`

`
`
`American Society of
`Gene Therapy
`
`
`
`
`June 9 — 13, 1999
`
`Washington, DC
`28 ANGELES
`
`BiOMEDICA.
`
`.iGRARY
`
`FEB 26 1999
`
`UNIVEPS TY OF CALIFORNIA
`
`Marriott Wardman Park Hotel
`
`(Abstract Deadline: March 2)
`
`For information contact:
`
` 2" Annual Meeting
`
`
`
`|
`
`
`
`ASGT Registration Manager
`6900 Grove Rd.
`Thorofare, NJ 08086
`Phone 609-848-1000
`Fax 609-848-5274
`Email: ASGT@slackinc.com
`
`Website : http//www.asgt.org
`
`
` oe
`Miltenyi Ex. 1021 Page 64
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`Miltenyi Ex. 1021 Page 6
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`

` cancer.
`
`HUMANGENE THERAPY 10:165-173 (January 20, 1999)
`Mary Ann Liebert, Inc.
`
`Anti-Tumor Activity of Human T Cells Expressing the
`CC49-¢ Chimeric Immune Receptor
`
`RYAN P. McGUINNESS,! YING GE,! SALIL D. PATEL,! SYED V.S. KASHMIRI,? HYUN-SIL LEE,?
`PATRICIA HORAN HAND,? JEFFREY SCHLOM,? MITCHELL H. FINER,! and JAMES G. McARTHUR!
`
`Miltenyi Ex. 1021 Page 7
`
`
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`
`
`
`A chimeric immunereceptor consisting of an extracellular antigen-binding domain derived from the CC49
`humanized single-chain antibody,linked to the CD3¢ signaling domain of the T cell receptor, was generated
`(CC49-2). This receptor binds to TAG-72, a mucin antigen expressed by most human adenocarcinomas. CC49-
`¢ was expressed in CD4* and CD8*Tcells and induced cytokine production on stimulation. HumanTcells
`expressing CC49-¢ recognized and killed tumorcell lines and primary tumorcells expressing TAG-72. CC49-
`{T cells did not mediate bystanderkilling of TAG-72-negative cells. In addition, CC49-¢T cells not only killed
`FasL-positive tumor cells in vitro and in vivo, but also survived in their presence, and were immunoprotec-
`tive in intraperitoneal and subcutaneous murine tumor xenograft models with TAG-72-positive human tumor
`cells. Finally, receptor-positive T cells were still effective in killing TAG-72-positive targets in the presence of
`physiological levels of soluble TAG-72, and did not induce killing of TAG-72-negative cells under the same
`conditions. This approach is being currently beingutilized in a phaseI clinicaltrial for the treatment of colon
`
`ABSTRACT
`
`OVERVIEW SUMMARY
`
`Tumor-specific T cells are generated in cancer patients, but
`often fail to eradicate the tumor. Ex vivo expansion of these
`tumor-infiltrating lymphocytes (TILs) followed by reinfu-
`sion into the patient*has shown some efficacy, but fails to
`provide significant long-term responses. This may be a re-
`sult of the low frequency of high-affinity tumor-specific T
`cells. To circumventthese issues, we have generated T cells
`bearing a chimeric immune receptor composed of an ex-
`tracellular, TAG-72 tumor antigen-specific scFv domain
`and an intracellular CD3{ domain (CC49-. Genetically
`modified human T cells expressing CC49-¢ lyse TAG-72-
`positive tumorcells and are immunoprotective in xenograft
`tumor models with TAG-72-positive human tumorcells.
`
`2 (IL-2) was usedto drive the ex vivo expansion of lymphokine-
`activated killer (LAK)cells or tumor-infiltrating lymphocytes
`(TILs). LAKcell- and TIL-based therapies for most tumor types
`did not demonstrate significant long-term clinical responses,
`perhaps owingto the low frequency of high-affinity tumor anti-
`gen-specific T cells (Herbermanetal., 1987; Blasse, 1995; Yan-
`nelli et al., 1996). Ex vivo antigen-driven expansion of syn-
`geneic T cells can produce large numbers of antigen-specific T
`cells (Riddell and Greenberg, 1995; Rooneyet al., 1995; Rid-
`dell et al., 1996). Even though impressive clinical efficacy has
`been observed in the treatment of cytomegalovirus (CMV)in-
`fections and Epstein-Barr virus (EBV) lymphoproliferative dis-
`ease with this approach, the use of antigen-specific T cells is
`not without difficulties, including patient-to-patient variability
`in cytotoxic T lymphocyte (CTL) frequency, the prolongedcul-
`ture times required to generate therapeutic numbers of CTLs,
`and the low frequencyof antigen-specific T cells for non-virus-
`INTRODUCTION
`associated malignancies.
`The genetic modification of humanTcells to express high-
`UMOR-DIRECTED CYTOLYTIC CELLS have been observed in
`affinity,
`tumor antigen-specific chimeric immune receptors
`patients that have later succumbed to their disease. To in-
`(CIRs) offers an alternative method to produce large numbers
`crease the numbersof these nascentcytolytic cells, interleukin
`of tumor-specific T cells. These receptors are created through
`
`Department of Preclinical Biology and Immunology and Department of Vector Biology, Cell Genesys, Inc., Foster City, CA 94404.
`Laboratory of Tumor Immunologyand Biology, National Cancer Institute, Bethesda, MD 20892.
`
`165
`
`Miltenyi Ex. 1021 Page 7
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`

`166
`
`McGUINNESSETAL.
`
`ture to CC49-2, with the CC49 scFv replaced with the 447D
`scFv (Patel et al., 1999).
`Virus was generated by transient CaPO,-mediated trans-
`fection of 293 cells as described previously (Roberts et al.,
`1994), with the following modification; three different plas-
`mids, the kat retroviral plasmid encoding CC49-¢ and two
`plasmids expressing the retroviral Gag—Pol and Envproteins,
`necessary for packaging functions, were used to decrease the
`likelihood of generating replication-competent
`retrovirus
`through recombination between the CC49-é-viral and pack-
`aging vectors.
`
`Tumorcells
`
`lines
`The LS174T, KLE-B, CCRF-CEM,and MIP-1 cell
`were obtained from J. Schlom (National Cancer Institute,
`Bethesda, MD). LS180, Snu-1, Jurkat, NCI H716, and NCI
`H508cell lines were obtained from the American Type Culture
`Collection (ATCC, Rockville, MD). Human colon carcinoma
`liver metastases were a generousgift of R. Warren (University
`of California, San Francisco). Tumor metastases were disasso-
`ciated in Dulbecco’s modified Eagle’s medium (DMEM) with
`collagenase (1 mg/ml) and 0.002% DNase in MEM at 37°C
`with mechanical agitation. The tumor cell suspensions were
`washedextensively and cultured for 4-18 hr at 37°C in 5% CO,
`in MEM supplemented with 10% fetal calf serum (FCS), 2 mM
`sodium pyruvate, penicillin-streptomycin, and L-glutamine.
`
`the use of tumor- or virus-specific single-chain antibodies
`(scFvs) or ligands fused to the intracellular signaling domains
`of the FcRor the T cell receptor (TCR) CD3¢chains. T cells
`expressing a variety of scFv-based receptors have been shown
`to lyse tumor- or virus-infected targets in vitro (Stancoviski et
`al., 1993; Moritz et al., 1994; Roberts er al., 1994; Hwu et al.,
`1995). In somecases, the chimeric immunereceptor-bearing T
`cells extended the survival of tumor-bearing mice.
`T cell immunotherapy of cancer patients will likely entail
`repeat administrations of the engineered T cells. All scFv-
`based receptors constructed previously utilized murine anti-
`bodies for ligand binding. Multiple administrations of T cells
`expressing these receptors will likely be prohibited owing to
`generation of a humananti-mouse antibody (HAMA)response
`and subsequenteliminationof the genetically modifiedT cells.
`For clinical development, chimeric immune receptors con-
`structed from human ligands or humanized scFvs will be nec-
`essary. We describe here a CIR specific for the panadenocar-
`cinoma surface antigen, TAG-72, constructed from the
`humanized scFv of the CC49 antibody (Shuef al., 1993; Kash-
`miri et al., 1995). The murine CC49 antibody (Molinolo et
`al., 1990) has been usedin clinical trials in more than 500 pa-
`tients as either a radiotherapeutic agent or radiodiagnostic tool
`for colon, ovarian, prostate, and breast cancers, and has shown
`no significant secondary organ toxicity (Greineret al., 1992;
`Meredithet al., 1994; Murray et al., 1994). Primary human T
`cells were genetically modified with high efficiency using a
`retrovirus encoding the CC49-¢ CIR (hereafter referred to as
`Culture and transduction of humanTcells
`CC49-£). The CC49-¢ T cells demonstrated antigen-specific
`lysis of all TAG-72-positive tumor cell lines and primary cells
`from patient isolates tested, produced immunostimulatory cy-
`tokines on receptor stimulation, and were immunoprotective
`in murine xenograft tumor models. In addition, CC49-¢ T cell
`activity was retained in the presence of Fas ligand (FasL)-ex-
`pressing tumortargets and in the presence of physiologic lev-
`els of soluble TAG-72.
`
`cells
`blood mononuclear
`peripheral
`Ficoll-purified
`(PBMCs), depleted of adherent cells, were resuspended at 3 X
`10° cells/ml in T cell medium (TCM) (AIM-V [Gibco, NY] and
`RPMI [1:1] with HEPES, sodium pyruvate, glutamine, peni-
`cillin-streptomycin, and 10% human serum) and stimulated
`with anti-CD3 and anti-CD28 antibody-coated Dynal (Lake
`Success, NY) beads at a density of three beads per cell in TCM
`for 48 hr at 37°C in 5% COp. On day 2, the beads were re-
`moved by magnetic separation. The PBMCs were then resus-
`pended at 1 X 106 cells/ml in TCM with IL-2 (100 IU/ml). One
`daylater, the T cells were resuspended at 10® cells/ml] in TCM
`with IL-2 and mixed with an equal volume of supernatant con-
`taining the CC49-£ retrovirus and supplemented with IL-2 (100
`TU/ml) and Polybrene (4 g/ml). This step was repeated twice:
`24 and 48 hr later. On day 6 the cells were removed from the
`transduction mix and resuspended in TCM with IL-2 at 10°
`cells/ml. The culture was then split every 1-2 days to 2 X 10°
`cells/ml for an additional 4-10 days.
`
`
`
`MATERIALS AND METHODS
`
`Generation of CC49-€
`
`receptor retrovirus
`
`The CC49-£ TAG-72 antigen recognition domain consists of
`an scFv of the Vy and Vy, regions from the humanized CC49
`antibody (Shu etal., 1993; Kashmiri et al., 1995) joined through
`a (Gly,-Ser)3 linker. A 788-bp fragment was obtained by di-
`gestion with Ncol
`and Rsrll
`from plasmid pTAHU-
`CC49SCIgDCH1] andligated to the [y], hinge and CH3 domain
`of human IgG,
`(residues 221-230 and 341-444 of im-
`munoglobulin [Ig] )), using an oligonucleotide linker flanked
`CC49-¢ or nontransduced T cells (5 X 105) were incubated
`by RsrlII and NspIsites. The extracellular portion of CC49-2 is
`attached to the human CD3¢ intracellular region (residues
`with 100 ng of a CC49 anti-idiotypic antibody, AI49-3, in the
`31-142 of CD3) through the human membrane-associated IgG
`presence of 3 mg of normal mouse IgG at room temperature
`for 20 min, and then incubated with 1 mg of goat anti-mouse
`M1 transmembrane-spanning domain and human CD4trans-
`membrane-spanning region (residues 372-395 of CD4). The
`IgG-PE(phycoerythrin) in the presence of 3 mg of normal goat
`IgG in 100 ml of phosphate-buffered saline (PBS)—2% FCSat
`CIR backbone, hinge--yi(CH3)-yM1-CD4 TM-CD34, was gen-
`room temperature for 20 min. For dual staining for CD4* and
`erated at Cell Genesys (Foster City, CA). CC49- was cloned
`into the retroviral rkat43.2 vector backbone, which has been
`CD8tTcells, the cells were then stained with 1 mg ofanti-
`previously described (Finer et al., 1994). The anti-HIVeny
`CD8-FITC (fluorescein isothiocyanate) or anti-CD4—FITC an-
`tibodies.
`gp120, 447D-£ chimeric immune receptor is similar in struc-
`
`FACSanalysis
`
`
`
`Miltenyi Ex. 1021 Page 8
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`

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`167
`
`ANTI-TUMOR ACTIVITY OF CC49-¢ T CELLS
`
`*ICr release assays
`
`cc49 scFv
`
`CD3zeta IC
`
`at
`51Cy-Jabeled target cells (5000) were mixed with various
`numbers of T cell effectors in a total volume of 200 ml in a
`cc49 V,,
`cc49 Vv.
`round-bottom 96-wellplate. In bystanderkilling analysis, TAG-
`
`72-negative cells were labeled and mixed with nonlabeled
`TAG-72 cells. After a 4-hr incubation at 37°C and 5% COb,
`
`the cells were pelleted at 1200 rpm for 4 min and 100 ml of
`the supernatant removed for counting of the released >!Cr.
`(G4°S)3
`yl
`The percent specific release is calculated as follows: per-
`Hinge|CD4 TM
`cent
`specific release = [(cpmyeteased — CPMspontaneous
`release)!
`(CpMmaximum released — CPMspontaneous release)] X 100.
`
`Soluble TAG-72 inhibition assays
`
`CC49-£ and nontransduced T cells were incubated with sol-
`uble TAG-72 (sTAG-72) from bovine submaxillary mucin
`(Sigma Chemicals, St. Louis, MO) or with sTAG-72-positive
`patient serum for 1 hr at 37°C and then mixed with an equal
`volume of medium containing >!Cr-labeled target cells and used
`in a 4-hr >!Cr release assay.
`
`Animal tumor models
`
`Mice were maintained in the Cell Genesys animal facility.
`Four- to 6-week-old SCID-NOD mice were injected intraperi-
`toneally with 10° KLE-B cells of subcutaneously with 10°
`LS174T or KLE-B cells in 200 ml of PBS. Prior to injection,
`tumor cells were trypsinized and washed three times in PBS.
`Treated mice received 3 X 10° CC49-¢-transduced or normal
`humanT cells by the same route. The T cells were mixed with
`the tumorcells immediately prior to injection into the animals.
`Mice were monitored daily for the development of subcuta-
`neous tumors in the subcutaneous tumor model or sacrificed at
`6 or 12 weeks and examined for the presence of tumors in the
`intraperitoneal tumor model.
`
`RESULTS
`
`Generation and growth of CC49-¢ T cells
`
`Human T cells were stimulated to proliferate (see below for
`conditions) and then transduced with retrovirus encoding CC49-
`¢ (Fig. 1). After a short expansion period, the T cells were
`stained with anti-CC49 idiotype and anti-CD8 antibodies and
`the cells analyzed by fluorescence-activated cell
`sorting
`(FACS). The average T cell transduction efficiency was 31%,
`with the range being 21 to 95% (Fig. 2A), without selection of
`receptor-positive cells. Both CD4* and CD8* T cells were
`transduced to express CC49-¢ (54 and 56% CC49-£ positive,
`respectively, in this representative example) (Fig. 2B). Recep-
`tor expression was stable; in one experiment CC49--positive
`T cells were maintained for 35days of continuousculture with-
`out any observable loss of receptor expression (Fig. 2C).
`Since the state of activated T cells is known to affect T cell
`function and survival, we tested the effect of culture conditions
`on T cell responsiveness andfunction mediated by CC49-¢. T
`cells that were stimulated with an anti-CD3 antibody became
`unresponsive to IL-2 following CC49-¢ stimulation mediated
`by interaction with TAG-72-expressing target cells (Fig. 3A).
`
`y1 CH3
`
`FIG. 1. The CC49-¢ chimeric immunereceptor. A CIR con-
`sisting of an extracellular antigen-binding domain derived from
`the CC49 humanized single-chain antibody, linked to the CD3£
`signaling domain of the T cell receptor, was generated. scFv,
`Single-chain variable fragment; IC, intracellular domain; Vy
`and V,, variable heavy and light chains, respectively; (G4-S)3,
`(Gly4-Ser)3 peptide linker; yl, immunoglobulin +; TM,trans-
`membrane region.
`.
`
`The responsiveness to IL-2 is measured as proliferation. In con-
`trast, T cells costimulated by anti-CD3 and anti-CD28 treat-
`ment were responsive to IL-2-induced proliferation (Fig. 3B).
`Thus, all further studies were conducted using T cells grown
`under these conditions.
`
`Antigen-specific lysis of tumor cells by CC49-{ T cells
`
`The lytic activity and specificity of CC49-¢-positive T cells
`weretested against several TAG-72-positive and -negative tumor
`cell lines. CC49-¢ T cells killed all TAG-72-positive targets tested,
`including LS174T and KLE-B cells (Fig. 4A and B) and the de-
`gree of lysis correlated with the level of TAG-72 expression on
`the targets (Table 1). CC49-é-positive T cells did not kill TAG-
`72-negative targets, including NCI H716 (Fig. 4C), MIP-1, and
`Snu-1. Lysis of these TAG-72-negative targets did not increase in
`the presence of TAG-72-positive tumorcells, indicating that the
`lytic activity of the CC49-¢ T cells was specific and did notresult
`in any bystander lysis of TAG-72-negative cells (Fig. 4D and E).
`CC49-€ T cells kill patient-derived tumorcells
`The sensitivity of tumor-derived cell lines and primary tu-
`morcells to T cell-mediated lysis can be vastly different. How-
`ever, CC49-¢-transduced T cells efficiently lysed primary tu-
`morcells from liver metastasis explants from two patients with
`advanced colon carcinoma. Even though the colon carcinoma
`cells expressed low, heterogeneouslevels of TAG-72 (Fig. 5A),
`CC49-£ T cells generated from two independent donors lysed
`the tumor cells, wile nontransduced T cells from the same
`donors did not kill the colon carcinomacells (Fig. 5B and C).
`
`CC49-f-mediated function is similar to
`TCR-mediated activity
`
`Weanalyzed theability of CC49-¢ to mediate target killing
`and to generate the production of cytokines following stimula-
`
`Miltenyi Ex. 1021 Page 9
`
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`

`

`168
`
`McGUINNESSETAL.
`
`QO
`
`nN °
`
` o o%cc49-CPositiveTCells
`
`
`
`o o a
`
`-_ Oo
`
`0
`
`40
`30
`20
`10
`Days Post-Transduction
`
`
`
`anti-cc49id
`
`—s¥%¥a‘.
`6’t
`
`anti-cc49 id
`
`
`
`anti-CD8
`
`FIG. 2. T cell expression of CC49-¢. (A) Four days after transduction CD8* T cells were stained for expression of CC49-£
`with an anti-idiotype antibody (solid line) or an isotype-matched control antibody (dashed line). (B) Ten days after transduction
`PBMCswerestained for expression of the CC49-¢ chimeric immune receptor and CD8. (C) Overthe course of 35 days of con-
`tinuous culture following transduction, T cells were stained for expression of the CC49-¢ chimeric immune receptor.
`
`tion. T cells that were stimulated through the TCR or the CIR
`(with anti-CD3or anti-CC49 idiotype antibodies) shared a sim-
`ilar increasein activity (Fig. 6).
`In addition to killing TAG-72-positive cells, CC49-¢-posi-
`tive T cells secreted granulocyte-macrophage colony-stimulat-
`ing factor (GM-CSF; 150 to 600 pg/ml/10° T cells/24 hr), in-
`terferon -y (IFN-y; 20 to 8500 pg/ml/10® T cells/24 hr), and
`tumornecrosis factor w (TNF-a; 60 to 90 pg/ml/10® T cells/24
`hr) following stimulation with tumortargets. IL-2 and IL-4 were
`not detected. No cytokines were detected in the cultures con-
`taining CC49-£ T cells in medium alone. This pattern of cy-
`tokine expression wassimilar to the cytokine profile of cytolytic
`T cells stimulated through the T cell receptor (data not shown).
`Thus,
`the CC49-¢-mediated functional effects are similar to
`those generated on T cell receptor engagement.
`
`Soluble TAG-72 does not affect the function of
`CC49-¢ T cells
`
`A potential inhibitor of CC49-£-positive T cell activity is the
`presence of soluble TAG-72 (sTAG-72), which can be detected
`in the serum of patients with advanced disease in concentra-
`tions up to 600 U/ml (Molinolo etal., 1990). However, sTAG-
`72 did not significantly inhibit T cell lysis of TAG-72-positive
`targets when CC49-{ T cells were preincubated with sTAG-72
`concentrations up to 2000 U/ml (Fig. 7). After longer incuba-
`tions of 2 days in the presence of sTAG-72 (100 U/ml), 60%
`of the CC49-¢T cell lytic activity was retained (data not shown).
`These results suggest that sSTAG-72 does not block sufficient
`numbers of the T cell CC49-£ receptors to abrogate CC49-¢ T
`cell-mediated lysis of tumortargets.
`
`
`
`TCellProliferation
`
`(Tcellsx108)
`
`>_>noOo
`
`=
`
`oo
`
`Days
`
`FIG. 3. T cell activation and growth of CC49-{ T cells (A and B) Culture conditions impact T cell responsiveness. Long-term
`culture of T cells activated by anti-CD3 treatment induces T cell unresponsiveness to IL-2 on signaling through the CC49-{ re-
`ceptor from TAG-72-expressing targets (A, circles). However, short-term culture of T cells activated by anti-CD3 and anti-CD28
`costimulation maintains T cell responsiveness to IL-2 following CC49-¢ receptor stimulation from TAG-72-expressing targets
`(B, circles). Nontransduced T cells proliferate in response to IL-2 irrespective of growth conditions, as they cannot respond to
`TAG-72-expressing targets (A and B, squares).
`
`Miltenyi Ex. 1021 Page 10
`
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`Miltenyi Ex. 1021 Page 10
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`

`

`
`
`laterssagempraaeapemntedsj:nerettnnenentA
`
`
`weenRORI
`
`ANTI-TUMOR ACTIVITY OF CC49-¢ T CELLS
`
`169
`
`A
`
`B
`
`aonoeo8
`nN2>
`
`o
`
`%SpecificLysis
`
`non-transduced
`
`50:1
`
`30:1
`
`10:1
`
`3:1
`
`non-transduced
`“ o8S
`Lysis
`%SpecificLysis
`%Specific
`
`MIP-1/Jurkat Targets
`
`H716/Jurkat Targets
`
`FIG. 4. CC49-¢ T cell antigen-specific lysis of TAG-72-positive and -negative tumor cells. The specificity of CC49-¢ T cells
`(A-C,circles) was tested with numerous TAG-72-positive targets including the colon carcinoma-derived LS174Tcell line (A)
`and the KLE-B cell line (B). The TAG-72-negativetargets included the colon carcinoma-derived NCI H716 (C). Targetcell ly-
`sis with nontransduced T cells is indicated (A—C, squares). CC49-T cells did not induce bystanderkilling of TAG-72-negative
`cells such as MIP-1 and H716. CC49-¢ T cells lysed radiolabeled TAG-72-positive Jurkat cells (D and E, circles) but not radi-
`olabeled TAG-72-negative MIP-1 (D, squares) and H716 (E,squares). Bystander lysis was not observed with radiolabeled TAG-
`72-negative MIP-1 (D, triangles) and H716 (E,triangles) were mixed with G-72-positive Jurkat cells.
`
`In addition, we investigated the ability of sSTAG-72 to induce
`killing of TAG-72-negative cells, possibly by adhering to those
`c