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`
`TRANSPLANTATION
`
`Genetic modification of primary natural killer cells overcomes inhibitory signals
`and induces specific killing of leukemic cells
`Chihaya Imai, Shotaro Iwamoto, and Dario Campana
`
`Natural killer (NK) cells hold promise for
`improving the therapeutic potential of al-
`logeneic hematopoietic transplantation,
`but their effectiveness is limited by inhibi-
`tory HLA types. We sought to overcome
`this intrinsic resistance by transducing
`CD56ⴙCD3ⴚ NK cells with chimeric recep-
`tors directed against CD19, a molecule
`widely expressed by malignant B cells.
`An abundance of NK cells for transduc-
`tion was secured by culturing peripheral
`blood mononuclear cells with K562 cells
`expressing the NK-stimulatory molecules
`4-1BB ligand and interleukin 15, which
`Introduction
`
`yielded a median greater than 1000-fold
`expansion of CD56ⴙCD3ⴚ cells at 3 weeks
`of culture, without T-lymphocyte expan-
`sion. Expression of anti-CD19 receptors
`linked to CD3␨ overcame NK resistance
`and markedly enhanced NK-cell–medi-
`ated killing of leukemic cells. This result
`was significantly improved by adding the
`4-1BB costimulatory molecule to the chi-
`meric anti-CD19-CD3␨ receptor; the cyto-
`toxicity produced by NK cells expressing
`this construct uniformly exceeded that of
`NK cells whose signaling receptors lacked
`4-1BB, even when natural cytotoxicity
`
`was apparent. Addition of 4-1BB was also
`associated with increased cell activation
`and production of interferon ␥ and granu-
`locyte-macrophage colony-stimulating
`factor. Our findings indicate that enforced
`expression of signaling receptors by NK
`cells might circumvent inhibitory signals,
`providing a novel means to enhance the
`effectiveness of allogeneic stem cell
`transplantation. (Blood. 2005;106:376-383)
`
`© 2005 by The American Society of Hematology
`
`B-cell malignancies of children and adults, such as acute lympho-
`blastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and
`non-Hodgkin lymphoma (NHL), are often incurable even with
`intensive chemotherapy. For many patients, bone marrow ablation
`followed by allogeneic hematopoietic stem cell transplantation is
`the only potentially curative option, but the disease may return after
`transplantation.1 The well-documented association between T-cell–
`mediated graft-versus-host disease (GvHD) and a delay or suppres-
`sion of leukemic relapse after allogeneic stem cell transplanta-
`tion2-4 has led some investigators to manipulate GvHD by infusion
`of donor T lymphocytes. Although this procedure can induce a
`measurable antineoplastic response,5-8 it carries the risk of severe
`GvHD, particularly in those patients (⬎ 70%) who lack an
`HLA-identical donor. Moreover, in some B-cell malignancies, such
`as ALL, the effect of lymphocyte infusions is often inadequate.6,9,10
`Besides T lymphocytes, natural killer (NK) cells also exert
`cytotoxicity against cancer cells.11 Recent studies have emphasized
`the potential of NK-cell
`therapy in recipients of allogeneic
`hematopoietic stem cell transplants. In animal models of transplan-
`tation, donor NK cells could lyse leukemic cells and host lympho-
`hematopoietic cells without affecting nonhematopoietic tissues,12
`suggesting that NK-mediated graft-versus-leukemia responses may
`occur in the absence of systemic disease. Because NK cells are
`inhibited by self-HLA molecules, which bind to killer immunoglobu-
`lin-like receptors (KIRs), these findings have led to the clinical
`practice of selecting hematopoietic stem cell transplantation donors
`with an HLA and KIR type that favors NK-cell activation and thus
`
`could be expected to promote an antileukemic effect.13-15 However,
`selection of the “best” donor is limited to patients who have more
`than one potential donor and the capacity of NK cells to lyse
`lymphoid cells is generally low and difficult to predict.13,15-17
`Emerging evidence indicates that T lymphocytes genetically
`modified with chimeric receptors able to recognize a surface
`molecule of target cells and transduce activation signals can
`specifically enhance T-cell cytotoxicity against cancer cells both in
`vitro and in vivo.18-21 The studies presented here are based on the
`concept that expression of chimeric receptors on NK cells could
`overcome HLA-mediated inhibitory signals, thus endowing the
`cells with cytotoxicity against otherwise NK-resistant cells. To test
`this hypothesis, we first developed a novel method that allows
`specific and vigorous expansion of NK cells lacking T-cell
`receptors (CD56⫹CD3⫺ cells) and their highly efficient transduc-
`tion with chimeric receptors. Then, we tested the relative antileuke-
`mic effects of genetically modified NK cells bearing chimeric
`receptors (directed against CD19, a molecule widely expressed
`by malignant B cells) that deliver different primary and costimula-
`tory signals.
`
`Materials and methods
`
`Cells
`
`The CD19⫹ human B-lineage ALL cell lines RS4;11, OP-1, 380, 697, and
`KOPN57bi; the T-cell line CEM-C7; and the myeloid cell lines K562 and
`
`From the Departments of Hematology-Oncology and Pathology, St Jude
`Children’s Research Hospital, Memphis; and Department of Pediatrics,
`University of Tennessee College of Medicine, Memphis.
`
`Reprints: Dario Campana, Department of Hematology-Oncology, St Jude
`Children’s Research Hospital, 332 N Lauderdale, Memphis TN 38105; e-mail:
`dario.campana@stjude.org.
`
`Submitted December 16, 2004; accepted March 2, 2005. Prepublished online
`as Blood First Edition Paper, March 8, 2005; DOI 10.1182/blood-2004-12-4797.
`
`Supported by grants CA58297 and CA21765 from the National Cancer Institute
`and by the American Lebanese Syrian Associated Charities (ALSAC).
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
`
`© 2005 by The American Society of Hematology
`
`376
`
`BLOOD, 1 JULY 2005 䡠 VOLUME 106, NUMBER 1
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`BLOOD, 1 JULY 2005 䡠 VOLUME 106, NUMBER 1
`
`NK-CELL THERAPY FOR LEUKEMIA
`
`377
`
`U-937 were available in our laboratory.21 Cells were maintained in RPMI
`1640 (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum
`(FCS; BioWhittaker, Walkersville, MD) and antibiotics.
`Primary leukemia cells were obtained from 9 patients with B-lineage
`ALL, with appropriate informed consent and Institutional Review Board
`(IRB) approval; from 4 of these patients, we also studied (with Saint Jude
`IRB approval) cryopreserved peripheral blood samples obtained during
`clinical remission. An unequivocal diagnosis of B-lineage ALL was
`established by morphologic, cytochemical, and immunophenotypic criteria;
`in each case, more than 95% of the cells were positive for CD19. Peripheral
`blood was obtained from 8 healthy adult donors. Mononuclear cells
`collected from the samples by centrifugation on a Lymphoprep density step
`(Nycomed, Oslo, Norway) were washed twice in phosphate-buffered saline
`(PBS) and once in AIM-V medium (Gibco).
`
`Plasmids and retrovirus production
`
`The anti-CD19-␨, anti-CD19-BB-␨, and anti-CD19-truncated (control)
`plasmids are described elsewhere.21 The pMSCV-IRES-GFP, pEQ-
`PAM3(–E), and pRDF constructs were obtained from the Saint Jude
`Vector Development and Production Shared Resource. The cDNA
`encoding the intracellular domains of human DAP10 and 4-1BB ligand
`(4-1BBL), and interleukin-15 (IL-15) with long signal peptide were
`subcloned by polymerase chain reaction (PCR) with a human spleen
`cDNA library (from Dr G. Neale, St Jude Children’s Research Hospital)
`used as a template. An anti-CD19-DAP10 plasmid was constructed by
`replacing the sequence encoding CD3␨ with that encoding DAP10,
`using the splicing by overlapping extension by PCR (SOE-PCR)
`method. The cDNA encoding the signal peptide of CD8␣, the mature
`IL-15 and the transmembrane domain of CD8␣ were
`peptide of
`assembled by SOE-PCR to encode a “membrane-bound” form of IL-15.
`The resulting expression cassettes were subcloned into EcoRI and XhoI
`sites of murine stem-cell virus–internal ribosome entry site–green
`fluorescent protein (MSCV-IRES-GFP).
`The RD114-pseudotyped retrovirus was generated as previously de-
`scribed.21 We used calcium phosphate DNA precipitation to transfect 293T
`cells with anti-CD19-␨, anti-CD19-DAP10, anti-CD19-BB-␨, or anti-CD19-
`truncated; pEQ-PAM3(–E); and pRDF. Conditioned medium containing
`retrovirus was harvested at 48 hours and 72 hours after transfection,
`immediately frozen in dry ice, and stored at ⫺80°C until use.
`
`Development of K562 derivatives, expansion of NK cells,
`and gene transduction
`
`K562 cells were transduced with the construct encoding the “membrane-
`bound” form of IL-15. Cells were cloned by limiting dilution, and a
`single-cell clone with high expression of GFP and of surface IL-15
`(K562-mb15) was expanded. This clone was subsequently transduced with
`human 4-1BBL and designated as K562-mb15-41BBL. K562 cells express-
`ing wild-type IL-15 (K562-wt15) or 4-1BBL (K562-41BBL) were pro-
`duced by a similar procedure. Peripheral blood mononuclear cells (1.5 ⫻ 106)
`were incubated in a 24-well tissue-culture plate with or without 106
`K562-derivative stimulator cells in the presence of 10 IU/mL human IL-2
`(National Cancer Institute BRB Preclinical Repository, Rockville, MD) in
`RPMI 1640 and 10% FCS.
`Mononuclear cells stimulated with K562-mb15-41BBL were trans-
`duced with retroviruses, as previously described for T cells.21 Briefly, 14
`mL polypropylene centrifuge tubes (Falcon, Lincoln Park, NJ) were coated
`with human fibronectin (100 ␮g/mL; Sigma, St Louis, MO) or RetroNectin
`(50 ␮g/mL; TaKaRa, Otsu, Japan). Prestimulated cells (2 ⫻ 105) were
`resuspended in the tubes in 2 to 3 mL virus-conditioned medium with
`Polybrene (4 ␮g/mL; Sigma) and centrifuged at 2400g for 2 hours
`(centrifugation was omitted when RetroNectin was used). The multiplicity
`of infection (4-6) was identical in each experiment comparing the activity
`of different chimeric receptors. After centrifugation, cells were left
`undisturbed for 24 hours in a humidified incubator at 37°C, 5% CO2. The
`
`transduction procedure was repeated on 2 successive days. After a second
`transduction, the cells were restimulated with K562-mb15-41BBL in the
`presence of 10 IU/mL IL-2. Cells were maintained in RPMI 1640, 10%
`FCS, and 10 IU/mL IL-2.
`
`Detection of chimeric receptor expression and
`immunophenotyping
`
`Transduced NK cells were stained with goat anti–mouse (Fab)2 polyclonal
`antibody conjugated with biotin (Jackson ImmunoResearch Labs, West
`Grove, PA) followed by streptavidin conjugated to peridinin chlorophyll
`protein (PerCP; Becton Dickinson, San Jose, CA). For Western blotting,
`cells were lysed in RIPA buffer (PBS, 1% Triton-X100, 0.5% sodium
`deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) containing 3 ␮g/mL
`pepstatin, 3 ␮g/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF),
`2 mM ethylenediaminetetraacetic acid (EDTA), and 5 ␮g/mL aprotinin.
`Centrifuged lysate supernatants were boiled with an equal volume of
`loading buffer with or without 0.1 M dithiothreitol (DTT), and then
`separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) on a
`precast 10% to 20% gradient acrylamide gel (Bio-Rad, Hercules, CA). The
`proteins were transferred to a polyvinylidene fluoride (PVDF) membrane,
`which was incubated with primary mouse anti–human CD3␨ monoclonal
`antibody (clone 8D3; PharMingen, San Diego, CA). Membranes were then
`washed, incubated with a goat anti–mouse IgG horseradish peroxidase–
`conjugated second antibody, and developed by using the enhanced chemilu-
`minescence system (Amersham, Little Chalfont, United Kingdom).
`The following antibodies were used for immunophenotypic characteriza-
`tion of expanded and transduced cells: anti-CD3 conjugated to fluorescein
`isothiocyanate (FITC), to PerCP, or to energy-coupled dye (ECD); anti-
`CD10 conjugated to phycoerythrin (PE); anti-CD19 PE; anti-CD22 PE;
`anti-CD56 FITC, PE, or allophycocyanin (APC); anti-CD16 CyChrome
`(antibodies from Becton Dickinson, PharMingen, or Beckman-Coulter,
`Miami, FL); and anti-CD25 PE (Dako, Carpinteria, CA). Surface expres-
`sion of KIR and NK activation molecules was determined with specific
`antibodies conjugated to FITC or PE (from Beckman-Coulter or Becton
`Dickinson), as previously described.15 Antibody staining was detected with
`a FACScan or an LSR II flow cytometer (Becton Dickinson).
`
`Cytotoxicity assays and cytokine production
`
`Target cells (1.5 ⫻ 105) were placed in 96-well U-bottomed tissue-culture
`plates (Costar, Cambridge, MA) and incubated with primary NK cells
`transduced with chimeric receptors at various effector-target (E/T) ratios in
`RPMI 1640 supplemented with 10% FCS; NK cells were cultured with
`1000 U/mL IL-2 for 48 hours before the assay. Cultures were performed in
`the absence of exogenous IL-2. After 4 hours and 24 hours, cells were
`harvested, labeled with CD10 PE or CD22 PE and CD56 FITC, and assayed
`by flow cytometry as previously described.21-23 The numbers of target cells
`recovered from cultures without NK cells were used as a reference.
`For cytokine production, primary NK cells (2 ⫻ 105 in 200 ␮L)
`expressing chimeric receptors were stimulated with various target cells at a
`1:1 ratio for 24 hours. The levels of interferon ␥ (IFN-␥) and granulocyte-
`macrophage colony-stimulating factor (GM-CSF) in cell-free culture
`supernatants were determined with a Bio-Plex assay (Bio-Rad).
`
`Statistical analysis
`
`A test of equality of mean NK expansion with various stimuli was
`performed using analysis of variance for a randomized complete block
`design with each donor considered a random block. The Tukey honest
`significant difference procedure was used to compute simultaneous confi-
`dence intervals for each pairwise comparison of the differences of treatment
`means. Differences in cytotoxicities and cytokine production among NK
`cells bearing different chimeric receptors were analyzed by the paired
`Student t test.
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`IMAI et al
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`BLOOD, 1 JULY 2005 䡠 VOLUME 106, NUMBER 1
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`
`Expression of IL-15 on the surface of K562 cells was more than 5
`times higher with the IL-15–CD8␣ construct than with wild-type
`IL-15 (not shown).
`To test whether the modified K562 cells expressing both
`4-1BBL and IL-15 (K562-mb15-41BBL cells) promote NK-cell
`expansion, we cultured peripheral blood mononuclear cells from 7
`donors in the presence of low-dose (10 U/mL) IL-2 as well as
`irradiated K562 cells transduced with 4-1BBL or IL-15 (or both),
`or with an empty control vector. As shown in Figure 1, expression
`of either 4-1BBL or IL-15 by K562 cells improved the stimulation
`of NK-stimulatory capacity of K562 in some cases but not overall,
`whereas simultaneous expression of both molecules led to a
`consistent and striking amplification of NK cells (median recovery
`of CD56⫹CD3⫺ cells at 1 week of culture, 2030% of input cells
`[range, 1020%-2520%] compared with a median recovery of 250%
`[range, 150%-640%] for K562 cells lacking 4-1BBL and IL-15;
`P ⬍ .001). In 24 experiments with cells from 8 donors, NK-cell
`expansion after 3 weeks of culture with K562 cells expressing both
`stimulatory molecules ranged from 309-fold to 12 409-fold (me-
`dian, 1089-fold). Importantly, neither the modified nor unmodified
`K562 cells caused an expansion of T lymphocytes (Figure 1).
`Among expanded CD56⫹CD3⫺ NK cells, expression of CD56
`was higher than that of unstimulated cells (Figure 2); expression of
`CD16 was similar to that seen on unstimulated NK cells (median
`CD16⫹ NK cells in 7 donors: 89% before expansion and 84% after
`expansion). We also compared the expression of KIR molecules on
`the expanded NK cells with that on NK cells before culture, using
`the monoclonal antibodies CD158a (against KIR 2DL1), CD158b
`(2DL2), NKB1 (3DL1), and NKAT2 (2DL3). The prevalence of
`NK subsets expressing these molecules after expansion resembled
`that of their counterparts before culture, although the level of
`expression of KIR molecules was higher after culture (Figure 2).
`Similar results were obtained for the inhibitory receptor CD94,
`whereas expression of the activating receptors NKp30 and NKp44
`became detectable on most cells after culture (not shown). In sum,
`the immunophenotype of expanded NK cells reiterated that of
`activated NK cells,
`indicating that contact with K562-mb15-
`41BBL cells had stimulated expansion of all subsets of NK cells.
`
`Transduction of NK cells with chimeric receptors
`
`Before transducing peripheral blood mononuclear cells with retro-
`viral vectors containing chimeric receptor constructs and GFP
`(Figure 3), we stimulated them with K562-mb15-41BBL cells. In
`
`Figure 2. Immunophenotypic features of NK cells before and after expansion
`with K562-mb15-41BBL cells. Expression of CD3 and CD56, as well as expression
`of the KIRs CD158a (2DL1), CD158b (2DL2), NKB1 (3DL1), and NKAT2 (2DL3) on
`CD56⫹CD3⫺ cells were examined in peripheral blood mononuclear cells from a
`healthy donor before (top row) and after (bottom row) 3 weeks of coculture with
`K562-mb15-41BBL cells and low-dose (10 U/mL) IL-2.
`
`Figure 1. Expansion of NK cells after 1 week of culture with genetically modified
`K562 cells. Peripheral blood mononuclear cells from 7 healthy individuals (repre-
`sented by different symbols) were cultured with various preparations of K562 at a 1:1
`ratio in the presence of low-dose (10 U/mL) IL-2. Percentages of CD56⫹CD3⫺ NK
`cells and CD3⫹ T lymphocytes after 7 days of culture relative to the number of input
`cells are shown. Each data point represents the average of 2 measurements; bars
`correspond to the median expansion in each group. K562 cells expressing both
`membrane-bound IL-15 and 4-1BBL (K562-mb15-41BBL) induced a markedly
`superior expansion of NK cells (P ⬍ .001 by the Tukey honest significant difference
`test) without inducing T-cell proliferation; there were no significant differences among
`other pairwise comparisons of NK expansions obtained with K562, K562-41BBL, and
`K562-mb15.
`
`Results
`
`Culture conditions that favor the expansion of primary NK cells
`
`To transduce chimeric receptors into primary NK cells, we
`searched for stimuli that would induce specific NK-cell prolifera-
`tion. In preliminary experiments, we depleted peripheral blood
`mononuclear cells of CD3⫹ T lymphocytes and stimulated the
`remaining cells with IL-2 (1000 U/mL) or IL-15 (10 ng/mL).
`Under these culture conditions there was no expansion of NK cells,
`which, in fact, progressively declined in numbers. With phytohe-
`magglutinin (PHA; 7 ␮g/mL) and IL-2 (1000 U/mL) as stimuli, we
`observed a 2- to 5-fold expansion of CD56⫹ CD3⫺ NK cells after
`1 week of culture. However, despite the low proportion of
`contaminating CD3⫹ cells (⬍ 2% in 2 experiments) at the begin-
`ning of the cultures, these cells expanded more than NK cells
`(⬎ 30-fold expansion), and after 1 week of culture represented
`approximately 35% of the cell population.
`NK cells can be stimulated by contact with the human leukemia
`cell line K562, which lacks HLA-antigen expression,24 and geneti-
`cally modified K562 cells have been used to stimulate cytotoxic T
`lymphocytes.25 We therefore tested whether the NK-stimulatory
`capacity of K562 cells could be increased through enforced
`expression of additional NK-stimulatory molecules, using 2 mol-
`ecules that are not expressed by K562 cells and are known to
`stimulate NK cells. One molecule, the ligand for 4-1BB, triggers
`activation signals after binding to 4-1BB (CD137), a signaling
`molecule expressed on the surface of NK cells.26 The other
`molecule, IL-15, is a cytokine known to promote NK-cell develop-
`ment and the survival of mature NK cells.27-30 Because IL-15 has
`greater biologic activity when presented to NK cells bound to IL-15
`receptor ␣ (IL-15R␣) on the cell membrane of stimulatory cells,
`rather than in its soluble form,31-35 we made a construct containing
`the human IL15 gene fused to the gene encoding the human CD8␣
`transmembrane domain and used it
`to transduce K562 cells.
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`NK-CELL THERAPY FOR LEUKEMIA
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`Figure 5. Chimeric receptors bearing CD3␨ overcome the NK resistance of
`leukemic cells. The results shown for 3 cell
`lines, 380, RS4;11, and 697, are
`expressed as the mean (⫾ SD; n ⫽ 4) percentage of leukemic cell recovery after 24
`hours of culture at a 1:1 E/ T ratio relative to cultures without NK cells, as measured by
`flow cytometry. NK cells expressing anti-CD19-␨ receptors were more cytotoxic than
`NK cells expressing anti-CD19-DAP10 receptors, anti-CD19 receptors without
`signaling capacity (anti-CD19-trunc), or NK cells transduced with GFP control vector
`(P ⬍ .001).
`
`Expression of receptors without signaling molecules did not
`increase NK-mediated cytotoxicity over that exerted by NK cells
`transduced with the vector containing only GFP (Figure 5). By
`contrast, expression of anti-CD19-␨ receptors markedly enhanced
`NK cytotoxicity in all experiments (Figure 5), regardless of the
`intrinsic ability of donor NK cells to kill leukemic targets. For
`example, 380 cells were highly resistant to NK cells from donors 2
`and 3, but were killed when these donor cells expressed anti-
`CD19-␨ receptors. Similar observations were made for RS4;11
`cells and the NK cells of donor 1 and for 697 cells and NK cells of
`donor 2. Moreover, the anti-CD19-␨ receptors led to improved
`killing of target cells even when natural cytotoxicity was present. In
`all experiments, the cytotoxicity triggered by the anti-CD19-␨
`receptor was enhanced over that achieved by replacing CD3␨ with
`DAP10 (P ⬍ .001; Figure 5).
`
`4-1BB–mediated costimulatory signals enhance
`NK cytotoxicity
`
`Previous studies have shown that the addition of costimulatory
`molecules to chimeric receptors enhances the proliferation and
`cytotoxicity of T lymphocytes.21,38-43 Of the 2 best known costimu-
`latory molecules in T lymphocytes, CD28 and 4-1BB, only 4-1BB
`is expressed by NK cells.26,44,45 We therefore determined whether
`the addition of 4-1BB to the anti-CD19-␨ receptor would enhance
`NK cytotoxicity. In a 4-hour cytotoxicity assay, cells expressing the
`4-1BB–augmented receptor showed a markedly better ability to kill
`CD19⫹ cells than did cells lacking this modification (Figure 6A-B).
`The superiority of NK cells bearing the anti-CD19-BB-␨ receptor
`was also evident in 24-hour assays with NK cells from different
`donors cultured at a 1:1 ratio with the leukemia cell lines 697,
`KOPN57bi, and OP-1 (not shown).
`Next, we determined whether the antileukemic activity of NK
`cells expressing anti-CD19-BB-␨ receptors extended to primary
`leukemic samples. In 5 samples from children with different
`molecular species of ALL, NK cells expressing the 4-1BB recep-
`tors exerted strong cytotoxicity that was evident even at low E/T
`ratios (eg, ⬍ 1:1; Figure 7) and uniformly exceeded the activity of
`NK cells expressing signaling receptors that lacked 4-1BB. Even
`
`Figure 3. Schematic representation of the chimeric receptors used in this
`study. LTR indicates long terminal repeat; AMP, ampicillin resistance; and bp, base pair.
`
`27 experiments, the median percentage of NK cells that were GFP⫹
`at 7 to 11 days after transduction was 69% (43%-93%). Chimeric
`receptors were expressed at high levels on the surface of NK cells
`and, by Western blotting, were in both monomeric and dimeric
`configurations (Figure 4).
`To identify the specific signals required to stimulate NK cells
`with chimeric receptors, and overcome inhibitory signals mediated
`by KIR molecules and other NK inhibitory receptors that bind to
`HLA class I molecules, we first compared 2 types of chimeric
`receptors containing different signaling domains: CD3␨, a signal-
`transducing molecule containing 3 immunoreceptor tyrosine–based
`activation motifs (ITAMs) and linked to several activating recep-
`tors expressed on the surface of NK cells,11,36 and DAP10, a signal
`transducing molecule with no ITAMs linked to the activating
`receptor NKG2D and previously shown to trigger NK cytotoxic-
`ity.11,36,37 As a control, we used NK cells transduced with a vector
`containing an anti-CD19 receptor but no signaling molecules or
`containing GFP alone. NK cells were challenged with the CD19⫹
`leukemic cell lines 380, 697, and RS4;11, all of which express high
`levels of HLA class I molecules by antibody staining. By
`genotyping, RS4;11 is Cw4/Cw3, Bw4 and A3; 380 is Cw4/
`Cw4, Bw4; and 697 is Cw3/Cw3. Hence, these cell lines were
`fully capable of inhibiting NK-cell cytotoxicity via binding to
`NK-inhibitory receptors.
`
`Figure 4. Expression of chimeric receptors by NK cells expanded from
`peripheral blood mononuclear cells. (A) Surface receptor expression was visual-
`ized by flow cytometry after staining with a goat anti–mouse (Fab)2 polyclonal
`antibody conjugated with biotin followed by streptavidin PerCP (y-axes); expression
`of GFP is also shown (x-axes). (B) Western blot analysis of chimeric receptor
`expression in NK cells, under reducing or nonreducing conditions. Filter membranes
`were labeled with an antihuman CD3␨ antibody and a goat anti–mouse IgG
`horseradish peroxidase–conjugated second antibody.
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`Figure 8. Chimeric receptors bearing 4-1BB induce a superior NK-cell activa-
`tion. (A) Expanded primary NK cells expressing chimeric receptors were incubated
`for 24 hours with the ALL cell line RS4;11 at a 1:1 ratio. Flow cytometric dot plots
`illustrated CD25 expression (in the y-axes) and side scatter (SSC; in the x-axes) of
`GFP⫹ cells before and after culture. The percentages of CD25⫹ NK cells are
`indicated. (B) Production of IFN-␥ and GM-CSF by NK cells expressing different
`chimeric receptors after 24 hours of culture with 697 cells at a 1:1 ratio (mean ⫾ SD of
`3 measurements). The 4-1BB receptors elicited a significantly higher production of
`both cytokines (P ⬍ .005).
`
`BB-␨ receptors induced a much higher production of IFN-␥ and
`GM-CSF on contact with CD19⫹ cells than did receptors without
`4-1BB (Figure 8B).
`We asked whether the expression of signaling chimeric recep-
`tors would affect spontaneous NK activity against NK-sensitive
`cell lines not expressing CD19. Spontaneous cytotoxicity of NK
`cells from 3 donors against the CD19⫺ leukemia cell lines K562,
`U937, and CEM-C7 was not diminished by expression of chimeric
`receptors, with or without 4-1BB (not shown).
`
`Anti-CD19 chimeric receptors induce NK cytotoxicity against
`autologous leukemic cells
`
`To determine whether the NK-cell expansion and transduction
`system that we developed would be applicable to clinical samples,
`we studied peripheral blood samples that had been obtained (and
`cryopreserved) from 4 patients with childhood B-lineage ALL in
`clinical remission, 25 to 56 weeks from diagnosis. NK-cell
`expansion occurred in all 4 samples; after 1 week of culture with
`K562-mb15-41BBL cells, recovery of CD56⫹CD3⫺ NK cells
`ranged from 1350% to 3680% of the input.
`
`Figure 7. NK cells expressing 4-1BB–augmented chimeric recep-
`tors show powerful cytotoxicity against leukemic cells from
`patients. Expanded primary NK cells expressing chimeric receptors
`were incubated for 4 hours with leukemic cells from children with
`different subtypes of B-lineage ALL (patient [Pt] 1, hyperdiploid 47-50;
`Pt 2 and Pt 5, t(4;11)(q21;q23); Pt 3, t(14;?)(q32;?); Pt 4, der8, t(8;?))
`at the indicated E/ T ratios. Each data point represents the mean (⫾
`SD; n ⫽ 4) percentage of ALL cell killing after culture as compared to
`that of parallel cultures without NK cells. With the exception of the
`results obtained in patient 2 at a 1:1 ratio, the cytotoxicity of NK cells
`expressing chimeric receptors containing 4-1BB was significantly
`higher than that induced by receptors without 4-1BB (P ⬍ .005).
`
`Figure 6. Addition of the 4-1BB costimulatory molecule to the chimeric
`receptors augments their capacity to induce NK cytotoxicity against NK-
`resistant leukemic cells. (A) Expanded primary NK cells expressing chimeric
`receptors were incubated for 4 hours with the B-lineage ALL cell lines 380 and RS4;11
`at the indicated E/ T ratios. Each data point represents the mean (⫾ SD; n ⫽ 4)
`percentage of ALL cell killing after culture as compared to that of parallel cultures
`without NK cells. At all E/ T ratios, cytotoxicity of NK cells expressing chimeric
`receptors containing 4-1BB was significantly higher than that induced by receptors
`without 4-1BB (P ⬍ .001). (B) Flow cytometric dot plots show staining with anti-CD56
`and anti-CD22 after a 4-hour coculture of NK cells (CD56⫹) and ALL cells (380;
`CD22⫹) at a 2:1 ratio. The percentage of cell killing obtained with NK cells expressing
`different chimeric receptors (% kill) was calculated by comparing the number of viable
`CD22⫹ ALL cells recovered after the test culture to that of parallel cultures without NK
`cells.
`
`when donor NK cells had natural cytotoxicity against ALL cells
`and CD3␨ receptor did not improve it (patient no. 3 in Figure 7),
`addition of 4-1BB to the receptor significantly enhanced cytotoxic-
`ity. Consistent with their increased cytotoxicity, NK cells express-
`ing anti-CD19-BB-␨ mediated more vigorous activation signals. As
`shown in Figure 8A, 46% of NK cells bearing this receptor
`expressed the IL-2R␣ chain CD25 after 24 hours of coculture with
`CD19⫹ ALL cells, compared with only 17% of cells expressing the
`anti-CD19-␨ receptor and less than 1% for cells expressing
`receptors that lacked stimulatory capacity. Moreover, anti-CD19-
`
`UPenn Ex. 2065
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`BLOOD, 1 JULY 2005 䡠 VOLUME 106, NUMBER 1
`
`NK-CELL THERAPY FOR LEUKEMIA
`
`381
`
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`tion method for the present study. Most investigators have demon-
`strated efficient gene transfer only in continuously growing NK-
`cell lines48-54 or reported methods yielding only transient gene
`expression.37,55,56 We achieved stable expression of chimeric recep-
`tors in primary CD56⫹CD3⫺ NK cells by using an RD114-
`pseudotyped retroviral vector and specifically expanding primary
`CD56⫹CD3⫺ NK cells before they were exposed to the retrovirus,
`a step that allowed highly efficient gene expression. Although
`several cytokines such as IL-2, IL-12, and IL-15 stimulate NK
`cells,27,57,58
`their capacity to induce proliferation of
`resting
`CD56⫹CD3⫺ cells has been poor, unless accessory cells are present
`in the cultures.24 Perussia et al59 found that contact with irradiated
`B-lymphoblastoid cells induced as high as a 25-fold expansion of
`NK cells after 2 weeks of stimulation, and Miller et al60 reported an
`approximate 30-fold expansion of NK cells after 18 days of culture
`with 1000 U/mL IL-2 and monocytes. However, these culture
`conditions are likely to promote the growth of CD3⫹ T lympho-
`cytes as well as NK cells. Because our ultimate aim is to generate
`pure preparations of donor NK cells devoid of CD3⫹ T lympho-
`cytes that can be infused into recipients of allogeneic hematopoi-
`etic stem cell transplants, we searched for methods that would
`maximize NK-cell expansion without producing T-cell mitogenicity.
`Contact with K562 cells (which lack major histocompatibility
`complex [MHC] class I molecule expression and hence do not
`trigger KIR-mediated inhibitory signals in NK cells) is known to
`augment NK-cell proliferation in response to IL-15.24 We found
`that membrane-bound IL-15 and 4-1BBL, coexpressed by K562
`cells, acted synergistically to augment K562-specific NK-
`stimulatory capacity, resulting in vigorous expansion of peripheral
`blood CD56⫹CD3⫺ NK cells without concomitant growth of T
`lymphocytes. After 2 to 3 weeks of culture, we observed NK-cell
`expansions of up to 10 000-fold, and virtually pure populations of
`NK cells could be obtained, even without the need for T-cell
`depletion in some cases. NK cells expanded in this system retained
`the immunophenotypic diversity seen among peripheral blood
`subsets of NK cells, as well as their natural cytotoxicity against
`sensitive target cells, even after transductio