Original Manuscript

Leukemia (2004) 18, 676–684. doi:10.1038/sj.leu.2403302 Published online 12 February 2004

Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia

C Imai1, K Mihara1, M Andreansky1, I C Nicholson2, C-H Pui1,3,4, T L Geiger3 and D Campana1,3,4

  1. 1Department of Hematology-Oncology, St Jude Children's Research Hospital, Memphis, TN, USA
  2. 2Child Health Research Institute, Women's and Children's Hospital, Adelaide, South Australia
  3. 3Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA
  4. 4Department of Pediatrics, University of Tennessee College of Medicine, Memphis, TN, USA

Correspondence: Dr D Campana, Department of Hematology-Oncology, St Jude Children's Research Hospital, 332 North Lauderdale, 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.



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 CD8alpha, and the signaling domain of CD3zeta. We compared the antileukemic activity mediated by a novel receptor ('anti-CD19-BB-zeta') containing the signaling domain of 4-1BB (CD137; a crucial molecule for T-cell antitumor activity) to that of a receptor lacking costimulatory molecules. Retroviral transduction produced efficient and durable receptor expression in human T cells. Lymphocytes expressing anti-CD19-BB-zeta receptors exerted powerful and specific cytotoxicity against ALL cells, which was superior to that of lymphocytes with receptors lacking 4-1BB. Anti-CD19-BB-zeta 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-zeta-transduced T cells eliminated 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 4-1BB. We conclude that anti-CD19 chimeric receptors containing 4-1BB are a powerful new tool for T-cell therapy of B-lineage ALL and other CD19+ B-lymphoid malignancies.


T-cell receptor, CD137, acute lymphoblastic leukemia, B-cell lymphoma



In approximately 20% of children and 65% of adults with acute lymphoblastic leukemia (ALL), drug-resistant leukemic cells survive intensive chemotherapy and cause disease recurrence. 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 allogeneic stem cell transplantation.4,5,6 Infusions of donor lymphocytes can have antileukemic effects,7,8,9,10 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 transduction of artificial immune receptors, which typically consist of an extracellular antibody-derived single-chain variable domain (scFv) and an intracellular signal transduction molecule (eg, CD3zeta).13,14,15 Allogeneic or autologous T lymphocytes expressing these receptors can be activated by cell surface epitopes targeted by the scFv and kill the epitope-presenting cells. In ALL, CD19 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-lineage lymphoid cells.16,17,18,19 However, CD3zeta signaling may not be sufficient to produce a durable immune response; without a second signal, or costimulus, T cells rapidly undergo apoptosis after stimulation.19,20,21,22 This is a central issue for T-cell therapy of ALL because ALL cells generally lack the ligands of CD28,23 and of 4-1BB (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-CD3zeta receptor to that of T cells expressing a novel chimeric receptor that contains the signal transduction domain of 4-1BB (CD137) as well as anti-CD19 scFv and CD3zeta (anti-CD19-BB-zeta). 4-1BB, a tumor necrosis factor-receptor family member, was selected because it prevents activation-induced death of T cells,24,25,26,27 induces expansion of CD8+ cells,28 and enhances CD8+ T-cell responses during viral infection and allograft rejection.28,29,30,31 Most importantly, extensive experimental evidence with animal models of cancer points to a crucial role of 4-1BB signaling for effective antitumor responses.32,33,34,35,36 We found that anti-CD19-BB-zeta-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 approximate the in vivo microenvironment where leukemic cells grow.


Materials and methods


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 K56241 and U-93742 were available in our laboratory. Cells were maintained in RPMI-1640 (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.

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 Lymphoprep density step (Nycomed, Oslo, Norway) and were washed two times in phosphate-buffered saline (PBS) and once in AIM-V medium (Gibco).


The plasmid encoding anti-CD19 scFv was previously reported.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 CD8alpha, and intracellular domains of 4-1BB, CD3zeta, 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 extension by PCR (SOE-PCR) to assemble several genetic fragments.44 The sequence of each genetic fragment was confirmed by direct sequencing. The expression cassettes were subcloned into EcoRI and XhoI sites of MSCV-IRES-GFP vector

Figure 1.
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Schematic representation of the chimeric receptor constructs used in this study.

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To transduce CD19-negative K562 cells with CD19, we constructed an MSCV-IRES-DsRed vector. The IRES and DsRed sequences were subcloned from MSCV-IRES-GFP and pDsRedN1 (Clontech, Palo Alto, CA, USA), respectively, and assembled by SOE-PCR. The IRES-DsRed cassette was digested and ligated into XhoI and NotI sites of MSCV-IRES-GFP. The expression cassette for CD19 was subsequently ligated into EcoRI and XhoI 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 times 106 293T cells, maintained in 10-cm tissue culture dishes (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) for 24 h, with 8 mug of one of the vectors, anti-CD19-zeta, anti-CD19-BB-zeta, or anti-CD19-truncated, 8 mug of pEQ-PAM3(-E), and 4 mug of pRDF. After 24 h, the medium was replaced with RPMI-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 cells were collected and prestimulated for 48 h with 7 mug/ml PHA-M (Sigma, St Louis, MO, USA) and 200 IU/ml human IL-2 (National Cancer Institute BRB Preclinical Repository, Rockville, MD, USA) in RPMI-1640 and 10% FCS. Cells were then transduced as follows. A 14-ml polypropylene centrifuge tube (Falcon) was coated with 0.5 ml of human fibronectin (Sigma) diluted to 100 mug/ml for 2 h at room temperature and then incubated with 2% bovine serum albumin (Sigma) for 30 min. Prestimulated cells (2 times 105) were resuspended in the fibronectin-coated tube in 2–3 ml of virus-conditioned medium with polybrene (4 mug/ml; Sigma) and centrifuged at 2400 g for 2 h. The multiplicity of infection (4–8) was identical in each experiment comparing the activity of different chimeric receptors. After centrifugation, cells were left undisturbed for 24 h in a humidified incubator at 37°C, 5% CO2. The transduction procedure was repeated on two successive days. Cells were then washed twice with RPMI-1640 and maintained in RPMI-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 times 105 K562 cells in 3 ml of MSCV-CD19-IRES-DsRed virus medium with 4 mug/ml polybrene in a fibronectin-coated tube; the tube was centrifuged at 2400 g for 2 h and 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 CD19 and of DsRed alone were selected for further experiments.

Detection of chimeric receptor expression

Cells were stained with goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin (Jackson Immunoresearch, 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 to PE and anti-CD8 conjugated to PerCP (from Becton 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 times 107 cells were lysed in 1 ml RIPA buffer (PBS, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% SDS) containing 3 mug/ml of pepstatin, 3 mug/ml of leupeptin, 1 mM of PMSF, 2 mM of EDTA, and 5 mug/ml of aprotinin. Cell lysates were separated by SDS-PAGE on a 12% acrylamide gel (BioRad, Hercules, CA, USA). After transfer to a PVDF membrane, this was incubated with a mouse anti-human CD3zeta (clone 8D3; Pharmingen) and then with a goat anti-mouse IgG horseradish peroxidase-conjugated antibody. Antibody binding was revealed by using the ECL kit (Pharmacia, Piscataway, NJ, USA).

Expansion of receptor-transduced primary T cells and IL-2 production

Receptor-transduced lymphocytes (3 times 105) were cocultured with 1.5 times 105 irradiated OP-1 cells in RPMI-1640 with 10% FCS with or without exogenous IL-2. Cells were pulsed weekly with irradiated target cells at an E: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 the absence of CD19-positive cells. To prepare pure populations of CD8+ cells expressing chimeric receptors, we labeled cells with a PE-conjugated anti-CD8 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 times 105 in 200 mul) expressing chimeric receptors were stimulated with OP-1 cells at a 1:1 E: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 (LDH) 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) times 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 LDH release value of lymphocytes alone, and 'low control' is the spontaneous LDH release value of target cells alone; background control (the value obtained from medium alone) was subtracted from each value before the calculation.

The antileukemic 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.45,46,47,48 Briefly, 2 times 104 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 times 104 CD19+ target cells (in case of cell lines) or 2 times 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. Cells 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 recovered from culture, as previously described.46,47,49,50,51,52 Expression of DsRed served as a marker of residual K562 cells. Experiments were carried out in triplicate.



Transduction of primary human T lymphocytes with anti-CD19 chimeric receptors

In preliminary experiments, stimulation of peripheral blood mononuclear cells with PHA (7 mug/ml) and IL-2 (200 IU/ml) for 48 h, followed by centrifugation (at 2400 times 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-zeta receptors and of those transduced with the anti-CD19-zeta receptors lacking 4-1BB 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- cells and vector-transduced cells (Figure 2a). The level of surface expression of anti-CD19-BB-zeta was identical to that of the receptor lacking 4-1BB (Figure 2a). Expression was confirmed by Western blot analysis (Figure 2b); under nonreducing conditions, 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.

Figure 2.
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Expression of chimeric receptors in T cells. (a) Expression of chimeric receptors in Jurkat cells (top panels) and in peripheral blood lymphocytes (bottom panels) after transduction. Surface receptor expression was visualized with a goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin and streptavidin PerCP (Y axes); expression of GFP is also shown (X axes). (b) Western blot analysis of chimeric receptor expression in primary lymphocytes, under reducing (left panel) and nonreducing conditions (right panel). Filter membranes were labeled with an anti-human CD3zeta antibody and a goat anti-mouse IgG horseradish peroxidase-conjugated second antibody.

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Cytotoxicity triggered by anti-CD19 chimeric receptors

Lymphocytes transduced with anti-CD19 signaling receptors exerted dose-dependent cytotoxicity, as shown by a 5-h LDH 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 E:T ratio in the 5-h LDH assay, most leukemic cells 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 expressing the chimeric receptors virtually eliminated all CD19+ cells when present in the cultures at a 1:1 E:T ratio, whereas vector-transduced lymphocytes did not. In these experiments, the effects of lymphocytes transduced with anti-CD19-BB-zeta and anti-CD19-zeta were equivalent.

Figure 3.
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Cytotoxicity of lymphocytes expressing anti-CD19 chimeric receptors. (a) Lymphocytes from two donors were transduced and mixed with OP-1 cells. Cytotoxicity was measured using a 5-h LDH-release assay (see Materials and methods). The mean percent (plusminuss.d.; n=3)-specific lysis is shown. (b) Transduced lymphocytes were incubated with OP-1 cells for 16 h at 1:1 ratio. After culture cells were labeled with anti-CD19 PE (Y axes); transduced lymphocytes were GFP+ (X axes). In cultures with lymphocytes transduced with vector alone or with the CD19-truncated receptor, CD19+ leukemic cells expanded (upper left quadrants in dot plots); cells in the upper right quadrants, prominent in the cultures containing the anti-CD19-truncated receptor, represent aggregates of leukemic cells and GFP+ lymphocytes. CD19+ ALL cells were eliminated by lymphocytes expressing anti-CD19-zeta and anti-CD19-BB-zeta. (c) Cultures with lymphocytes transduced with vector alone or anti-CD19-BB-zeta receptor.

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We next determined whether T cells expressing anti-CD19 chimeric receptors would exert significant antileukemic 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 4-1BB 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-zeta and from 60–70% for anti-CD19-BB-zeta) and used as such or after purification of CD8+ GFP+ cells. Lymphocytes were mixed at different ratios with K562-CD19 and with RS4;11 cells. Cocultures were maintained for 7 days, and viable leukemic cells were counted by flow cytometry. The results are shown in Figure 4. At a 0.1:1 ratio, T cells transduced with anti-CD19-BB-zeta markedly reduced leukemic cell recovery and were significantly more powerful than cells transduced with anti-CD19-zeta receptors. At this ratio, cells transduced with vector-control only had little, if any, effect on ALL cell recovery. Importantly, chimeric receptor-transduced T cells had no effect on the recovery of cells lacking CD19 such as CEM-C7, U-937, and K-562 (Figure 4), demonstrating the CD19 specificity of the antileukemic activity.

Figure 4.
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Cytotoxicity of lymphocytes transduced with chimeric receptors against CD19+ cell lines. Results are expressed as percentage of leukemia cell recovery after 7 days of culture relative to culture without lymphocytes. Lymphocytes from the same donors transduced with vector alone were used as controls. For each donor, four measurements were performed by flow cytometry. Data are meanplusminuss.d. of pooled results obtained with five donors for K562-CD19, two donors for RS4;11, U937 and CEM-C7, and one donor for K562.

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Effects of the bone marrow microenvironment on chimeric receptor-mediated cytotoxicity

Cells present in the bone marrow microenvironment may decrease T-cell proliferation in a mixed lymphocyte reaction.53,54,55 To test whether these cells would also affect T-cell-mediated antileukemic activity, we performed experiments with OP-1 on plastic and in the presence of bone marrow-derived mesenchymal-cell layers.48 Surprisingly, T-cell cytotoxicity under the latter condition was even greater than that observed in cultures without mesenchymal cells: T cells transduced with anti-CD19-BB-zeta were markedly cytotoxic even at a ratio of 0.01:1 in this assay (Figure 5).

Figure 5.
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Cytotoxicity of lymphocytes transduced with chimeric receptors in the presence of mesenchymal cells. Cytotoxicity was tested against the CD19+ cell line OP-1 in the absence or presence of bone marrow-derived mesenchymal cells. Results are expressed as percentage of CD19+ cell recovery after 7 days of culture relative to cultures without lymphocytes. Lymphocytes from the same donors transduced with vector alone were used as controls. For each donor, four measurements were performed by flow cytometry. Data are meanplusminuss.d. of pooled results obtained with four donors for cultures on plastic and with three donors from the same group for cultures on mesenchymal cells.

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Effect of receptor-transduced T cells on primary leukemic cells

To determine whether the antileukemic activity mediated by anti-CD19-BB-zeta receptors at 0.01:1 E:T ratio was also operative against primary leukemia, we performed tests using primary B-lineage ALL cells and bone marrow-derived mesenchymal cells, which are essential to preserve their viability in vitro.45,48,49 Leukemic cells were obtained from five B-lineage ALL patients at diagnosis; these patients included three who had ALL with 11q23 abnormalities, a karyotype associated with drug resistance.3 Indeed, all three patients had resistant disease: they had persistent minimal residual disease while in clinical remission and, ultimately, relapsed. Mesenchymal cells supported primary ALL cell survival in vitro: in cultures not exposed to exogenous T cells, recovery of leukemic cells from the five patients after 5 days of culture ranged from 100.1 to 180.7% of the input cell number. As little as 1% T cells expressing anti-CD19-BB-zeta receptors added to the cultures were sufficient to produce mean leukemic cytoreductions ranging from 66.6 to 93.7% after only 5 days (Figure 6). At this concentration, lymphocytes expressing anti-CD19-BB-zeta were consistently more cytotoxic than those expressing the anti-CD19-zeta receptor without 4-1BB (Figure 6).

Figure 6.
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Cytotoxicity of lymphocytes transduced with chimeric receptors against primary ALL cells. Bone marrow samples were obtained at diagnosis from children with B-lineage ALL; in all samples, >95% of cells were CD19+. Cells in patients 2–4 had 11q23 abnormalities and MLL gene rearrangements. Results are expressed as percentage of CD19+ cell recovery after 5 days of culture relative to cultures without lymphocytes. Lymphocytes from the same donors transduced with vector alone were used as controls. Data are meansplusminuss.d. of four measurements by flow cytometry; for each patient, results obtained with lymphocytes from two donors were pooled. In all cases, differences between T cells transduced with anti-CD19-zeta and anti-CD19-BB-zeta receptors were significant (P<0.001 in Patients 1, 2 and 5; <0.005 in Patient 3, and P<0.02 in Patient 4).

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Expansion and IL-2 production of receptor-transduced T cells

We hypothesized that the superior cytotoxicity exerted by T cells expressing anti-CD19-BB-zeta receptors at a low E:T ratio was due, at least in part, to a higher capacity to expand in the presence of target cells. To test this, we studied primary T cells (obtained from two donors), 7 days after transduction. When cocultured in the absence of exogenous IL-2 with the irradiated OP-1 cells, lymphocytes transduced with anti-CD19-BB-zeta expanded: after 1 week of culture, GFP+ cells recovered were 320 and 413% of input cells. T cells that expressed the anti-CD19-zeta receptor but lacked 4-1BB signaling capacity remained viable but showed little expansion (cell recovery: 111 and 160% of input cells, respectively), whereas those that expressed a control anti-CD19 receptor lacking 4-1BB and CD3zeta molecules underwent apoptosis (<10% of input cells were viable after 1 week). Lymphocytes transduced with anti-CD19-BB-zeta continued to expand in the presence of irradiated OP-1 cells: after 3 weeks of culture, they had expanded by more than 16-fold; 98% of cells at this point were GFP+. By contrast, cells transduced with only anti-CD19-zeta survived for less than 2 weeks of culture. The initial transduction efficiency with the three receptors was similar: 72 and 67% for anti-CD19-BB-zeta, 63 and 66% for anti-CD19-zeta, and 67 and 68% for the truncated anti-CD19 receptor. Therefore, 4-1BB-mediated costimulation confers a survival advantage to antileukemic lymphocytes.

Increased expansion of anti-CD19-BB-zeta-bearing T cells could be explained by their higher capacity to secrete IL-2. Indeed, after exposure to CD19+ OP-1 target cells, primary lymphocytes expressing anti-CD19-BB-zeta chimeric receptors produced much higher levels of IL-2 than cells expressing anti-CD19-zeta receptors (P<0.005; Figure 7).

Figure 7.
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Anti-CD19 receptors trigger IL-2 production in T lymphocytes to those by other chimeric receptors. Peripheral blood lymphocytes were cultivated alone or with OP-1 at 1:1 ratio for 24 h and IL-2 concentration was measured in the supernatants. Bars represent meanplusminuss.d. of three experiments. Transduction efficiency was 78% with anti-CD19-truncated, 81% with anti-CD19-zeta and 76% with anti-CD19-BB-zeta receptors.

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Results of this study demonstrate that T cells expressing anti-CD19-BB-zeta receptors can exert powerful cytotoxicity on ALL cells. Lymphocytes from different donors transduced with anti-CD19-BB-zeta receptors could consistently kill B-lineage ALL cells from patients at E:T ratios as low as 0.01:1. Lymphocytes expressing anti-CD19-BB-zeta were particularly effective in the presence of bone marrow-derived mesenchymal cells, which form the microenvironment critical for B-lineage ALL cells growth. The presence of 4-1BB in the chimeric receptor did not obstruct receptor expression and did not increase non-CD19-mediated cytotoxicity. These results, together with the capacity of anti-CD19-BB-zeta T cells to secrete IL-2 and vigorously expand in the presence of target cells, suggest that the infusion of relatively low numbers of these T cells could have a measurable antileukemic effect in patients.

Two recent studies have shown that receptors composed of anti-CD19 scFv and CD3zeta endow T cells with the capacity to proliferate when mixed with CD19+ cells and lyse CD19+ targets in vitro and in vivo.18,19 Brentjens et al19 reported that T cells bearing such receptors could significantly improve the survival of immunodeficient mice engrafted with the Raji B-cell lymphoma cell line. Their results demonstrated the requirement for costimulation in T-cell-mediated antileukemic activity in vivo: only cells expressing the B7 ligands of CD28 such as Raji elicited effective T-cell responses. However, this costimulatory pathway is not operative when B-lineage ALL cells are the target, because these cells typically do not express B7-1(CD80) and only a subset expresses B7-2 (CD86) molecules.23 Likewise, all leukemia cell lines that we tested lacked 4-1BB ligand expression (C Imai, D Campana, unpublished observations), further explaining their inadequate capacity to stimulate T cells. Our results indicate that addition of 4-1BB to the chimeric receptor could bypass the limitation posed by the lack of ligands of costimulatory molecules in ALL.

Although we cannot exclude that receptors containing other costimulatory molecules, such as CD28, could also be effective in mediating T-cell antileukemic activity, a large body of experimental evidence indicates that harnessing 4-1BB signaling is important for effective antitumor therapy. Melero et al32 found that antibodies to 4-1BB significantly improved long-lasting remission and survival rates in mice inoculated with the immunogenic P815 mastocytoma cell line. Moreover, immunogenic murine tumor cells made to express 4-1BB ligand were readily rejected and induced long-term immunity.33 Dramatic results were also observed in vaccination experiments using other tumor cell lines expressing 4-1BB ligands.34,35,36 Of note, experiments with the poorly immunogenic Ag104A fibrosarcoma cell line provided some evidence that 4-1BB could be superior to CD28 in eliciting antitumor responses: 80% of mice showed tumor regression with 4-1BB stimulation and 50% of mice with widespread metastasis were cured,32 whereas CD28 costimulation was not effective alone and required simultaneous CD2 stimulation.56

Clinical precedents, such as administration of T-cell clones that target cytomegalovirus epitopes or Epstein Barr virus-specific antigens,57,58 attest to the clinical feasibility of adoptive T-cell therapy. Transfer of chimeric receptor-modified T cells has the added advantage of permitting immediate generation of tumor-specific T-cell immunity. In view of the limited effectiveness and the high risk of the currently available treatment options for chemotherapy-refractory B-lineage ALL and other CD19+ B cell malignancies, the results of our study justify clinical trials using T cells expressing anti-CD19-BB-zeta receptors. Donor-derived T cells endowed with chimeric receptors could replace infusion of nonspecific lymphocytes post-transplant. The reinfusion of autologous T cells collected during clinical remission could also be considered in patients with persistent minimal residual disease. In clinical studies, we envisage the coexpression of anti-CD19-BB-zeta receptors with 'suicide genes'59,60,61 to ensure that the elimination of normal CD19+ B-lineage cells is temporary.



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We thank Dr Elio Vanin and the St Jude Vector Development and Production Shared Resource for retroviral vectors, and Geoffrey Neale for the human spleen cDNA library. This work was supported by Grants CA58297 and CA21765 from the National Cancer Institute, by a Center of Excellence grant from the State of Tennessee, and by the American Lebanese Syrian Associated Charities (ALSAC). Ching-Hon Pui is the FM Kirby Clinical Research Professor of the American Cancer Society.