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`[ Volume 18, Number 4
`April 2004
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`~,, Normal and Malignant Hemopoiesis
`lJ
`www.nature.com/leu
`Official Journal of the
`f Leukaemia Research Fund, UK
`•
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`nature publishing group
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`Miltenyi Ex. 1022 Page 1
`
`

`

`LEUKEMIA
`The Journal of Normal and Malignant Hemopoiesis
`
`EDITOR-IN-CHIEF
`C Nicole Muller-Berat Killmann MD
`SECTION EDITORS
`Apoptosis Editorial Network: E Solary, France; DE Johnson, USA;
`Molecular Targets forTherapy; Basic aspects and therapeutic
`JA McCubrey, USA
`implications: N Muller-Berat, France in conjunction with
`Biology of Gene Therapy: J Neita, USA
`JA Mccubrey, USA;
`Bone Marrow Transplantation and Myelodysplasias:
`DE Johnson, USA; JD Licht, USA; JP Marie, France.
`T de Witte, The Netherlands
`Myeloma: RM Kyle, USA
`Chronic Myeloid Leukemia: R Hehlmann, Germany
`New Cell Lines: H Drexler, Germany
`Clinical Studies (Adult Leukemias) J Rowe, Israel; X Thomas, France
`Normal Hemopoiesis and Stem Cells:
`CLL and B cell malignancies: M Albitar, USA
`RE Ploemacher, The Netherlands; P Quesenberry, USA
`lmmunophenotyping: MJ Borowitz, USA
`Pediatric Hemopathies: CH Pui, USA
`Leukemogenomics: J Radich, USA; JA McCubrey, USA;
`Pharmacodynamics and Pharmacogenomics: WK Plunkett, USA
`N Muller-Berat Killmann
`Sensitivity and Resistance to Therapy: JP Marie, France
`Lymphoma: H Tilly, France
`Signal Transduction and Cytokines: JA Mccubrey, USA
`Molecular Cytogenetics: R Berger, France; A Hagemeijer,
`Transcriptional Control and Deregulation: JD Licht, USA
`Belgium
`Virology: Z Berneman, Belgium
`
`Clinical Trial Editorial Research Group
`BM Camilla, USA; E Estey, USA; $J Forman, USA; W Hiddemann, Germany;
`R Larson, USA; R Ohno, Japan; CH Pui, USA; J Rowe, USA; M Tallman, USA
`
`T Abe,Japan
`BV Afanassiev, Russia
`M Baccarani, Italy
`MRBaer,USA
`KBallen,USA
`J Bartek, Denmark
`C Bastard, France
`OA Bernard, France
`F Bertrand, USA
`J-YCahn, France
`D Campana, USA
`Chang-an Deng, China
`Z Chen, China
`Z-Z Chen, China
`GCohen,UK
`NCross,UK
`A Cuneo, Italy
`W Dalton, USA
`Z Darzynkiewicz, USA
`L Degas, France
`H Dehner, Germany
`JR Downing, USA
`V Duronio, Canada
`WE Evans, USA
`
`Biotechnical Methods Section Leukemia
`Editors: JJM van Dongen, The Netherlands;
`D Grimwade, UK; A Hochhaus, Germany
`
`P Fenaux, France
`A Ferrando, USA
`R Franklin, USA
`R Furman, USA
`JA Gabert, France
`RCGallo,USA
`A Ganser, Germany
`JGil,Spain
`SGrant,USA
`A Gratwohl, Switzerland
`G Grosveld, USA
`U Gullberg, Sweden
`C Harrison, UK
`H Hasle, Denmark
`H Hirai, Japan
`0 Hrusak, Czech Republic
`U Jager, Austria
`C Jordan, USA
`WMKast,USA
`TJ Kipps, USA
`M Kneba, Germany
`S Knuutila, Finland
`M Lanette, France
`HJ Lawrence, USA
`
`EDITORIAL BOARD
`DC Linch, UK
`T Lion, Austria
`F Lo Coco, Italy
`E Macintyre, France
`JA Madrigal, UK
`F Mandelli, Italy
`E Matutes, UK
`EA McCulloch, Canada
`A Melnick, USA
`D Metcalf, Australia
`K Miyazawa, Japan
`SD Mundie, USA
`V Najfeld, USA
`T Nace, Japan
`KOhyashiki, Japan
`D Olive, France
`A Orfao, Spain
`N Panoskaltsis, UK
`J Pedersen-Bjergaard, Denmark
`S Pemrick, USA
`S Pileri, Italy
`Y Pommier, USA
`PPorcu,USA
`
`CONSULTANTS
`Statistics: S Suciu, Belgium
`Liaison editor with Autoimmunology: A Wiik, Denmark
`Liaison with Leukaemia Research Fund, UK, David Grant, Scientific Director
`
`J Bernard, France
`F Gavosto, Italy
`
`HONORARY MEMBERS
`R Zittoun, France
`D van Bekkum, The Netherlands
`GE Francis, UK
`
`J Freireich, USA
`C Rozman, Spain
`
`A Rapoport, USA
`S Raynaud, France
`MV Reiling, USA
`D Ribatti, Italy
`C Rosenfeld, USA
`JD Rowley, USA
`JE Rubnitz, USA
`P Ruvolo, USA
`D Scheinberg, USA
`B Schlegelberger, Germany
`M Schrappe, Germany
`AShimoni, Israel
`R Siebert, Germany
`P Sonneveld, The Netherlands
`F Marc Stewart, USA
`CA Stiller, UK
`T Szczepanski, Poland
`U Testa, Italy
`R Van Etten, USA
`CVerfaillie, USA
`D Viswanatha, USA
`D Weisdorf, USA
`C Willman, USA
`
`OFFICIAL JOURNAL OF THE LEUKAEMIA RESEARCH FUND U.K.
`published in alliance with Association Pour Les Maladies du Sang (AEMS), France
`
`Miltenyi Ex. 1022 Page 2
`
`

`

`LEUKEMIA
`www .nature.com/leu
`Leukemia is published by Nature Publishing Group, a division of
`Macmillan Publishers Ltd.
`
`Scope Leukemia aims to provide a vehicle for all disciplines which directly or
`indirectly contribute to our understanding and treatment of leukemia,
`lymphoma and allied diseases. Studies on normal hemopoiesis are as
`important as those on leukemia because of their comparative relevance.
`This journal is covered by Current Contents, SCIExpanded, Research
`Alert, Current Contents Life Sciences EMBASE/Excerpta Medica,
`Current Advances in Cell and Developmental Biology, Index Medicus/
`Medline, Cambridge Scientific Abstracts, BIOSIS, Reference Update,
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`Current Advances in Genetics and Molecular Biology and SIIC.
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`Editorial Manuscripts (plus two copies) and all editorial correspondence
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`C Nicole Muller-Berat Killmann, MD, Editor in Chief
`Leukemia, Hopital St Louis, I, Avenue Claude Vellefaux, 75475 Paris
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`Tel: + 33 I 40 03 67 68/69
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`Miltenyi Ex. 1022 Page 3
`
`

`

`• Leukemia (2004) 18, 676-684
`
`© 2004 Nature Publishing Group All rights reserved 0887-6924/04 $25.00
`www.nature.com/leu
`
`l
`
`j
`
`Chimeric receptors with 4-1 BB signaling capacity provoke potent cytotoxicity against
`acute lymphoblastic leukemia
`C Imai 1, K Mihara 1, M Andreansky1, IC Nicholson2, C-H Pui 1•3·4, TL Geiger3 and D Campana 1,3,4
`
`7 Department of Hematology-Oncology, St Jude Children's Research Hospital, Memphis, TN, USA; 2 Child Health Research
`Institute, Women's and Children's Hospital, Adelaide, South Australia; 3Department of Pathology, St Jude Children's Research
`Hospital, Memphis, TN, USA; and 4 Department of Pediatrics, University of Tennessee College of Medicine, Memphis, TN, USA
`
`To develop a therapy for drug-resistant B-lineage acute
`lymphoblastic leukemia (ALL), we transduced T lymphocytes
`with anti-CD19 chimeric receptors, consisting of an anti-CD19
`single-chain variable domain (reactive with most ALL cases),
`the hinge and transmembrane domains of CD8ix, and the
`signaling domain of CD3C, We compared the antileukemic
`activity mediated by a novel receptor ('anti-CD19-BB-C') con(cid:173)
`taining the signaling domain of 4-18B (CD137; a crucial
`molecule for T-cell antitumor activity) to that of a receptor
`lacking costimulatory molecules. Retroviral transduction pro(cid:173)
`duced efficient and durable receptor expression in human
`T cells. Lymphocytes expressing anti-CD19-BB-C receptors
`exerted powerful and specific cytotoxicity against ALL cells,
`which was superior to that of lymphocytes with receptors
`lacking 4-18B. Anti-CD19-BB·C lymphocytes were remarkably
`effective in cocultures with bone marrow mesenchymal cells,
`and against leukemic cells from patients with drug-resistant
`ALL: as few as 1% anti-CD19-BB-(-transduced T cells elimi(cid:173)
`nated most ALL cells within 5 days. These cells also expanded
`and produced interleukin-2 in response to ALL cells at much
`higher rates than those of lymphocytes expressing equivalent
`receptors lacking 4-1B8. We conclude that anti-CD19 chimeric
`receptors containing 4-18B are a powerful new tool for T-cell
`therapy of B-lineage ALL and other CD19 + B-lymphoid
`malignancies.
`Leukemia (2004) 18, 676-684. doi:1O.1O38/sj.leu.24O33O2
`Published online 12 February 2004
`Keywords: T-cell receptor; CD137; acute lymphoblastic leukemia;
`B-cell lymphoma
`
`Introduction
`
`In approximately 20% of children and 65% of adults with
`acute lymphoblastic leukemia (ALL), drug-resistant leukemic
`cells survive intensive chemotherapy and cause disease recu(cid:173)
`rrence.1 ·2 For patients with recurrent disease or with certain
`adverse disease features, such as B-lineage ALL with the
`t(9;22)(q34;q11 ), hypodiploidy < 45 chromosomes, or MLL
`gene rearrangements in infants, current chemotherapy regimens
`are mostly ineffective.3 Significant improvements in cure rates
`require the development of treatments that bypass cellular
`mechanisms of drug resistance and that have high therapeutic
`indexes.
`Clinical observations suggest that T lymphocytes can control
`the
`recurrence of chemotherapy-refractory
`leukemia. For
`example, T-cell-mediated graft-versus-host disease (GvHD) is
`associated with delay or suppression of leukemia relapse after
`transplantation.4-6 Infusions of donor
`allogeneic stem cell
`
`Correspondence: Dr D Campana, Department of Hematology(cid:173)
`Oncology, St Jude Children's Research Hospital, 332 North Lauder(cid:173)
`dale, Memphis TN 38105-2794, USA; Fax: +901-495 3749;
`E-mail: dario.campana@stjude.org
`Received 31 December 2003; accepted 5 January 2004; Published
`online 12 February 2004
`
`lymphocytes can have antileukemic effects,7-10 but they carry
`the risk of severe GvHD and their antileukemic effect is often
`inadequate in ALL.8·11 ·12
`T-lymphocyte specificity can be redirected by the transduc(cid:173)
`tion of artificial immune receptors, which typically consist of an
`extracellular antibody-derived single-chain variable domain
`(scFv) and an intracellular signal transduction molecule (eg,
`15 Allogeneic or autologous T lymphocytes expressing
`CD3(). 13
`-
`these receptors can be activated by cell surface epitopes
`targeted by the scFv and kill the epitope-presenting cells. In
`ALL, CD1 9 is an attractive target because it is expressed on
`virtually all leukemic cells in around 85% of cases (ie, B-lineage
`ALL), it is not expressed by normal nonhematopoietic tissues,
`and among hematopoietic cells, it is only expressed by B(cid:173)
`lineage lymphoid cells. 16- 19 However, CD3( signaling may not
`be sufficient to produce a durable immune response; without a
`second signal, or costimulus, T cells rapidly undergo apoptosis
`after stimulation. 19-22 This is a central issue for T-cell therapy of
`ALL because ALL cells generally lack the ligands of CD28,2 and
`of 4-1 BB (C Imai, D Campana, unpublished observations), the
`two major T-cell costimulatory molecules.
`In this study, we compared the function of human T cells
`expressing an anti-CD19-CD3( receptor to that of T cells
`expressing a novel chimeric receptor that contains the signal
`transduction domain of 4-1 BB (CD137) as well as anti-CD19
`scFv and CD3( (anti-CD19-BB-(). 4-1 BB, a tumor necrosis
`factor-receptor family member, was selected because it prevents
`activation-induced death of T cells,24-27 induces expansion of
`-CD8 + cells, 28 and enhances CD8 + T-cell responses during
`viral infection and allograft rejection.28-31 Most importantly,
`extensive experimental evidence with animal models of cancer
`points to a crucial role of 4-1 BB signaling for effective antitumor
`responses. 32-36 We found that anti-CD19-BB-(-transduced T
`cells have powerful antileukemic activity: they can destroy
`CD19+ ALL cell lines and primary leukemic cells at low
`effector: target (E:T) ratios and under conditions that approx(cid:173)
`imate the in vivo microenvironment where leukemic cells grow.
`
`Materials and methods
`
`Cells
`
`The human B-lineage ALL cell lines OP-1
`[t(9;22)(q34;q11 )/
`BCR-ABL], 37 and RS4;11 [t(4;11) (q21;q23)/MLL-AF4], 38 the T~
`cell lines Jurkat39 and CEM-C7,40 and the myeloid cell lines
`K562 41 and U-93742 were available in our laboratory. Cells
`were maintained in RPMl-164O (Gibco, Grand Island, NY, USA)
`with 10% fetal calf serum (FCS; BioWhittaker, Walkersville,
`MD, USA) and antibiotics. Human adenocarcinoma HeLa cells
`and embryonic kidney fibroblast 293T cells were maintained in
`DMEM (MediaTech, Herndon, VA, USA) supplemented with
`10% FCS and antibiotics.
`
`Miltenyi Ex. 1022 Page 4
`
`

`

`Primary leukemia cells were obtained from patients with
`newly diagnosed B-lineage ALL with the approval of the St Jude
`Children's Research Hospital Institutional Review Board and
`with appropriate informed consent. The diagnosis of B-lineage
`ALL was unequivocal; in each case, more than 95% of leukemic
`cells- were positive for CD19. Peripheral blood samples were
`obtained from healthy adult donors. Mononuclear cells were
`collected from the samples by centrifugation on a L ymphoprep
`density step (Nycomed, Oslo, Norway) and were washed two
`times in phosphate-buffered saline (PBS) and once in AIM-V
`medium (Gibco).
`
`Plasmids
`
`The plasmid encoding anti-CD19 scFv was previously re(cid:173)
`ported.43 The pMSCV-IRES-GFP, pEQPAM3(-E), and pRDF were
`obtained from the St Jude Vector Development and Production
`Shared Resource. Signal peptide, hinge and transmembrane
`domain of CD8cc, and intracellular domains of 4-1 BB, CD3(,
`and CD19 were subcloned by PCR using a human spleen cDNA
`library (from Dr G Neale, St Jude Children's Research Hospital)
`as a template (Figure 1 ). We used splicing by overlapping·
`to assemble several genetic
`extension by PCR (SOE-PCR)
`fragments. 44 The sequence of each genetic fragment was
`confirmed by direct sequencing. The expression cassettes were
`subcloned into fcoRI and Xhol sites of MSCV-IRES-GFP vector
`To transduce CD19-negative K562 cells with CD19, we
`constructed an MSCV-IRES-DsRed vector. The IRES and DsRed
`from MSCV-IRES-GFP and
`subcloned
`sequences were
`pDsRedN1 (Clontech, Palo Alto, CA, USA), respectively, and
`assembled by SOE-PCR. The IRES-DsRed cassette was digested
`and ligated into Xhol and Notl sites of MSCV-IRES-GFP. The
`expression cassette for CD19 was subsequently ligated into
`fcoRI and Xhol sites of MSCV-IRES-DsRed vector.
`
`Virus production and gene transduction
`
`To generate RD114-pseudotyped retrovirus, we used calcium
`phosphate DNA precipitation to transfect 3 x 106 293T cells,
`maintained in 10-cm tissue culture dishes (Falcon, Becton
`Dickinson, Franklin Lakes, NJ, USA) for 24 h, with 8 µg of one of
`the vectors, anti-CD19-(, anti-CD19-BB-(, or anti-CD19-trun(cid:173)
`cated, 8 µg of pEQ-PAM3(-E), and 4 µg of pRDF. After 24 h, the
`medium was replaced with RPMl-1640 with 10% FCS and
`antibiotics. Conditioned medium containing retrovirus was
`harvested 48 and 72 h after transfection, immediately frozen in
`dry ice, and stored at -80°C until use. HeLa cells were used to
`titrate virus concentration.
`Peripheral blood mononuclear cells were incubated in a
`tissue culture dish for 2 h to remove adherent cells. Nonadherent
`
`D CDBa signal peptide
`I Linker
`~ CDBa hinge & transmemb. domain
`~ 418B intracellular domain
`
`I
`VL
`CD19-truncated wl~A_..:.::..__,_--'-"-----"~==I
`VH
`I
`VH
`VL
`lccc:c.Lil -=---"---'-''---"'~="'---'-'·.:::.Ch=ainc......JI
`I
`VL
`VH
`l"":}'"--J -=---"---'-''---"'~==0.i£.<_---.-'.,-c.:::.h=•inc......J
`
`CD19-s
`
`CD19-BB·s
`
`anti•CD19 scFv
`
`signaling domain
`
`Schematic representation of the chimeric receptor
`Figure 1
`constructs used in this study.
`
`1 kb
`
`2 kb
`
`T-cell gene therapy for leukemia
`C Imai et al
`
`cells were collected and prestimulated for 48 h with 7 µg/ml
`PHA-M (Sigma, St Louis, MO, USA) and 200 IU/ml human IL-2
`(National Cancer Institute BRB Preclinical Repository, Rockville,
`in RPMl-1640 and 10% FCS. Cells were then
`MD, USA)
`transduced as fol lows. A 14-ml polypropylene centrifuge tube
`(Falcon) was coated with 0.5 ml of human fibronectin (Sigma)
`diluted to 100 µg!ml
`for 2 h at room temperature and then
`incubated with 2% bovine serum albumin (Sigma) for 30 min.
`Presti mu lated cells (2 x 105
`) were resuspended in the fibronec(cid:173)
`tin-coated tube in 2-3 ml of virus-conditioned medium with
`polybrene (4 µg!ml; Sigma) and centrifuged at 2400 g for 2 h.
`in each
`identical
`The multiplicity of infection (4-8) was
`experiment comparing the activity of different chimeric recep(cid:173)
`tors. After centrifugation, cells were left undisturbed for 24 h in a
`incubator at 37°C, 5% CO2 • The transduction
`humidified
`procedure was repeated on two successive days. Cells were
`then washed twice with RPMl-1640 and maintained in RPMl-
`1640, 10% FCS, and 200 IU/ml of IL-2 until use.
`A similar procedure was used to express chimeric receptors in
`Jurkat cells, except that cells were not prestimulated. K562 cells
`expressing CD19 were created by resuspending 2 x 105 K562
`cells in 3 ml of MSCV-CD19-IRES-DsRed virus medium with
`4 µglml polybrene in a fibronectin-coated tube; the tube was
`centrifuged at 2400 gfor 2 hand left undisturbed in an incubator
`for 24 h. Control cells were transduced with the vector only.
`These procedures were repeated on 3 successive days. After
`confirming CD19 and DsRed expression, cells were subjected to
`single-cell sorting with a fluorescence-activated cell sorter
`(MoFlo, Cytomation, Fort Collins, CO, USA). The clones that
`showed the highest expression of DsRed and CD1 9 and of
`DsRed alone were selected for further experiments.
`
`Detection of chimeric receptor expression
`
`Cells were stained with goat anti-mouse (Fabh polyclonal
`lmmunoresearch,
`antibody conjugated with biotin Uackson
`West Grove, PA, USA) followed by streptavidin conjugated to
`peridinin chlorophyll protein (PerCP; Becton Dickinson, San
`Jose, CA, USA). Anti-CD4 and anti-CD28 antibodies conjugated
`(from Becton
`to PerCP
`to PE and anti-CDS conjugated
`Dickinson, and Pharmingen, San Diego, CA, USA) were also
`used. Antibody staining was detected with a FACScan flow
`cytometer (Becton Dickinson).
`For Western blotting, 2 x 107 cells were lysed in 1 ml RIPA
`buffer (PBS, 1 % Triton-X 100, 0.5% sodium deoxycholate, 0.1 %
`SDS) containing 3 µg!ml of pepstatin, 3 µg!ml of leupeptin, 1 mM
`of PMSF, 2 mM of EDTA, and 5 µg!ml of aprotinin. Cell lysates
`were separated by SOS-PAGE on a 12% acrylamide gel (BioRad,
`Hercules, CA, USA). After transfer to a PVDF membrane, this
`was incubated with a mouse anti-human CD3( (clone 8D3;
`Pharmingen) and then with a goat anti-mouse lgG horseradish
`peroxidase-conjugated antibody. Antibody binding was re(cid:173)
`vealed by using the ECL kit (Pharmacia, Piscataway, NJ, USA).
`
`Expansion of receptor-transduced primary T cells
`and IL-2 production
`
`lymphocytes (3 x 105
`) were cocultured
`Receptor-transduced
`with 1.5 x 105 irradiated OP-1 cells in RPMl-1640 with 10%
`FCS with or without exogenous IL-2. Cells were pulsed weekly
`with irradiated target cells at an f:T ratio of 2:1. Viable cells
`were counted by Trypan-blue dye exclusion and by flow
`cytometry to confirm the presence of GFP-positive cells and
`
`• 677
`
`Leukemia
`
`Miltenyi Ex. 1022 Page 5
`
`

`

`T-cell gene therapy for leukemia
`C Imai et al
`
`the absence of CD19-positive cells. To prepare pure populations
`of CD8 + cells expressing chimeric receptors, we labeled cells
`with a PE-conjugated anti-COB antibody (Becton Dickinson)
`that had been previously dialyzed to remove preservatives and
`then sterile-filtered. CD8 + GFP + cells were isolated using a
`fluorescence-activated cell sorter (MoFlo).
`For IL-2 production, primary lymphocytes (2 x 105 in 200 µI)
`expressing chimeric receptors were stimulated with OP-1 cells
`at a 1 :1 f: T ratio for 24 h. Levels of IL-2 in culture supernatants
`were determined with a Bio-Plex assay (BioRad).
`
`Cytotoxicity assays
`
`The cytolytic activity of transductants was measured by assays of
`lactate dehydrogenase (LOH)
`release after 5 h using
`the
`Cytotoxicity Detection Kit (Roche,
`Indianapolis,
`IN, USA)
`according to the manufacturer's instructions. Percent-specific
`cytolysis was calculated by using the formula: (Test-effector
`control-low control/high control-low control) x 100,
`in
`which 'high control' is the value obtained from supernatant
`of target cells exposed to 1 % Triton-X 100, 'effector control' is
`the spontaneous LOH release value of lymphocytes alone, and
`'low control' is the spontaneous LOH release value of target
`cells alone; background control (the value obtained from
`medium alone) was subtracted from each value before the
`calculation.
`The anti leukemic activity of receptor-transduced lymphocytes
`was also assessed in 7-day cultures using lower E:T ratios. For
`this purpose, we used bone marrow-derived mesenchymal cells
`to support the viability of leukemic cells. 45
`8 Briefly, 2 x 104
`-4
`human mesenchymal cells immortalized by enforced expression
`of telomerase reverse transcriptase were plated on a 96-well
`tissue culture plate precoated with 1 % gelatin. After 5 days,
`1 x 104 co19+ target cells (in case of cell lines) or 2 x 105
`CD19 + target cells (in case of primary ALL cells) were plated on
`the wells and allowed to rest for 2 h. After extensive washing to
`remove residual IL-2-containing medium, receptor-transduced
`primary T cells were added to the wells at the proportion
`indicated in Results. Cultures were performed in the absence of
`exogenous IL-2. Plates were incubated at 37°C in 5% CO2 for
`5-7 days. Ce! Is were harvested, passed through a 19-gauge
`needle to disrupt residual mesenchymal-cell aggregates, stained
`with anti-CD19-PE antibody, and assayed by flow cytometry
`with a method specifically designed to enumerate ALL cells
`from culture, as previously described. 46
`47
`49
`52
`recovered
`•
`•
`-
`
`Table 1
`lmmunophenotype of peripheral blood lymphocytes
`expressing chimeric receptors•
`
`Cell marker (number
`of experiments)
`
`Percentage of GFP+cells expressing the listed
`marker'
`
`Anti-CD19-(
`
`Anti-CD19-BB-(
`
`CD3 (n=6)
`85.4± 11.0
`89.6±2.3
`CD4 (n =6)
`21.1 ±8.8
`18.0±8.7
`CD8 (n =6)
`68.1 ±8.1
`66.1 ±11.7
`CD28 (n=3)
`41.2± 12.2
`38.1 ±16.1
`CD8+CD28 (n = 3)
`24.2+11.2
`20.6±11.3
`"Transduced lymphocytes were cultured for 14 days in the presence of
`IL-2 (200IU/ml).
`bMedian (range) percent of GFP+ cells was 65% (31-86%) for anti(cid:173)
`CD19-( and 65% (37-83%) for anti-CD19-BB-(.
`
`l
`
`Expression of DsRed served as a marker of residual K562 cells.
`Experiments were carried out in triplicate.
`
`Results
`
`Transduction of primary human T lymphocytes with
`anti-CD 19 chimeric receptors
`
`In preliminary experiments, stimulation of peripheral blood
`mononuclear cells with PHA (7 µg!ml) and IL-2 (200 IU/ml) for
`48 h, followed by centrifugation (at 2400 x g) with retroviral
`supernatant
`in
`tubes coated with
`fibronectin, consistently
`yielded a high percentage of chimeric receptor and GFP
`expression: in 75 transduction experiments, 31-86% (median,
`64%) of mononuclear cells expressed GFP. This method was
`used in all subsequent experiments. The immunophenotypes of
`the cells transduced with anti-CD19-BB-( receptors and of those
`transduced with the anti-CD19-( receptors lacking 4-1 BB were
`similar (Table 1 ).
`The surface expression of the chimeric receptors on GFP+
`cells was confirmed by staining with a goat anti-mouse antibody
`that reacted with the scFv portion of anti-CD19. Expression was
`detectable on most GFP+ cells and was not detectable on GFP(cid:173)
`cells and vector-transduced cells (Figure 2a). The level of surface
`expression of anti-CD19-BB-( was identical to that of the
`receptor lacking 4-1 BB (Figure 2a). Expression was confirmed by
`Western blot analysis (Figure 2b); under nonreducing condi(cid:173)
`tions, peripheral blood mononuclear cells transduced with the
`chimeric receptors expressed
`them mostly as monomers,
`although dimers could be detected. The expression level of
`the receptors in primary lymphocytes was stable for at least 8
`weeks after transduction; expression in Jurkat cells has remained
`stable for more than 50 passages.
`
`Cytotoxicity triggered by anti-CD 19 chimeric receptor/
`
`Lymphocytes transduced with anti-CD19 signaling receptors
`exerted dose-dependent cytotoxicity, as shown by a 5-h LOH
`release assay using the CD19 + OP-1 cell line as a target
`(Figure 3a). Although no lysis of target cells was apparent at a
`1: 1 f: T ratio in the 5-h LOH assay, most leukemic eel Is were
`specifically killed by lymphocytes expressing the receptors
`when the cultures were examined at 16 h by flow cytometry
`(Figure 3b) and by inverted microscopy (Figure 3c). Similar
`results were seen in experiments using other CD19 + cells as a
`target: in cultures with RS4;11 B-lineage ALL cells or with K562
`(a CD19-negative myeloid cell line that lacks HLA antigens)
`transduced with CD19 ('K562-CD19'), T lymphocytes expres(cid:173)
`sing the chimeric receptors virtually eliminated all CD19 + cells
`when present in the cultures at a 1 :1 f: T ratio, whereas vector(cid:173)
`transduced lymphocytes did not. In these experiments, the
`effects of lymphocytes transduced with anti-CD19-BB-( and
`anti-CD19-( were equivalent.
`We next determined whether T cells expressing anti-CD19
`chimeric receptors would exert significant anti leukemic activity.
`when present at a lower E: T ratio (ie, 0.1 :1) and, if so, whether
`there were differences in the cytotoxicity mediated by receptors
`with and without the 4-1 BB signaling molecules. Lymphocytes
`from various donors were expanded in vitro for 14 days after
`transduction with the receptors (transduction efficiency range:
`62-73% for anti-CD19-( and from 60-70% for anti-CD19-BB-()
`and used as such or after purification of CD8 + GFP + cells.
`Lymphocytes were mixed at different ratios with K562-CD19
`
`• 678
`
`Leukemia
`
`Miltenyi Ex. 1022 Page 6
`
`

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