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. 2007 Jul 31;57(2):233–246. doi: 10.1007/s00262-007-0370-8

Selective induction of apoptosis in leukemic B-lymphoid cells by a CD19-specific TRAIL fusion protein

Julia Stieglmaier 1,✉, Edwin Bremer 2, Christian Kellner 1, Tanja M Liebig 3, Bram ten Cate 2, Matthias Peipp 4, Hendrik Schulze-Koops 5, Matthias Pfeiffer 6, Hans-Jörg Bühring 7, Johann Greil 8, Fuat Oduncu 9, Bertold Emmerich 9, Georg H Fey 1,✉, Wijnand Helfrich 2
PMCID: PMC11030665  PMID: 17665197

Abstract

Although the treatment outcome of lymphoid malignancies has improved in recent years by the introduction of transplantation and antibody-based therapeutics, relapse remains a major problem. Therefore, new therapeutic options are urgently needed. One promising approach is the selective activation of apoptosis in tumor cells by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study investigated the pro-apoptotic potential of a novel TRAIL fusion protein designated scFvCD19:sTRAIL, consisting of a CD19-specific single-chain Fv antibody fragment (scFv) fused to the soluble extracellular domain of TRAIL (sTRAIL). Potent apoptosis was induced by scFvCD19:sTRAIL in several CD19-positive tumor cell lines, whereas normal blood cells remained unaffected. In mixed culture experiments, selective binding of scFvCD19:sTRAIL to CD19-positive cells resulted in strong induction of apoptosis in CD19-negative bystander tumor cells. Simultaneous treatment of CD19-positive cell lines with scFvCD19:sTRAIL and valproic acid (VPA) or Cyclosporin A induced strongly synergistic apoptosis. Treatment of patient-derived acute B-lymphoblastic leukemia (B-ALL) and chronic B-lymphocytic leukemia (B-CLL) cells resulted in strong tumoricidal activity that was further enhanced by combination with VPA. In addition, scFvCD19:sTRAIL prevented engraftment of human Nalm-6 cells in xenotransplanted NOD/Scid mice. The pre-clinical data presented here warrant further investigation of scFvCD19:sTRAIL as a potential new therapeutic agent for CD19-positive B-lineage malignancies.

Keywords: TRAIL, CD19, Leukemia, Antibody-derived therapeutics, Apoptosis

Introduction

Antibody-derived agents have greatly improved the therapeutic options for lymphoid malignancies, with the CD20-specific chimeric antibody Rituximab now being routinely used for the treatment of several B-cell cancers [13]. However, many patients still relapse for example due to CD20-negative recurrences and intrinsic or acquired resistance to Rituximab [21, 28]. Therefore, novel therapeutic strategies are urgently required for B-cell malignancies. In this respect, various antibody-based therapeutics are being investigated for application in B-cell leukemia and lymphoma including approaches targeting CD19 [11, 29, 35, 41, 47, 56].

CD19 is a type I membrane protein of the immunoglobulin superfamily, which is expressed in nearly all stages of B-cell development [46] and also on the majority of chronic B-lymphocytic leukemia (CLL), non-Hodgkin lymphoma and acute B-lymphoblastic leukemia (ALL) cells [50]. The antigen is rarely lost during malignant transformation and is not shed from the cell surface [45]. These properties have established CD19 as an attractive target antigen for new antibody-based therapeutics for B-lymphoid malignancies.

A promising candidate for cancer therapy is the TNF-related apoptosis-inducing ligand (TRAIL) [48]. TRAIL possesses potent pro-apoptotic activity for tumor cells, with no or minimal activity towards normal cells [42, 55]. TRAIL is expressed as a homotrimeric type II transmembrane protein (memTRAIL), but can also be proteolytically cleaved into a soluble form (sTRAIL). TRAIL induces apoptosis by cross-linking of the agonistic receptors TRAIL-R1 and TRAIL-R2 [37, 38, 52]. Engagement of these receptors leads to assembly of the death-inducing signaling complex (DISC), resulting in the subsequent activation of initiator- and effector-caspases and the induction of apoptosis (reviewed in [1, 54]). Two antagonistic receptors, TRAIL-R3 and TRAIL-R4 probably act as decoy receptors [16, 31], but so far no correlation between receptor density and sensitivity for, or resistance to apoptosis has been observed [14, 30]. Therefore, the precise mechanism regulating the preferential induction of apoptosis of tumor cells by TRAIL-derived agents remains unknown.

Several preparations of recombinant sTRAIL demonstrated potent antitumor effects towards cultured tumor cells, and in xenograft mouse models [2, 53]. These promising results have led to a phase I clinical trial assessing the effect of sTRAIL (AMG-951). However, the efficacy of sTRAIL may be negatively affected by the widespread expression of TRAIL-receptors, which may limit the accumulation of sTRAIL on tumor cells. This may further be limited by the typically rapid off-rate of cytokine/receptor interactions. In addition, sTRAIL cannot efficiently activate the agonistic and high-affinity receptor TRAIL-R2 [34, 49].

These limitations can be overcome by genetically fusing sTRAIL to a tumor-specific single-chain Fv antibody fragment (scFv) [7, 9, 10, 51]. The scFv component mediates specific binding to a pre-selected target antigen, thus leading to accumulation of scFv:sTRAIL at the cell surface. Subsequently, the membrane bound sTRAIL component induces apoptosis by cross-linking and activation of both TRAIL-R1 and TRAIL-R2 in a cis-acting manner on the same cell or a trans-acting manner on neighboring cells.

In this study a novel CD19-targeted scFv:sTRAIL fusion protein was designed and pre-clinically characterized with the intent to provide a novel agent to target a variety of B-lymphoid malignancies.

Materials and methods

Monoclonal antibodies

The murine IgG3 monoclonal antibody N6B1 with specificity for human CD19 (HJB, unpublished data) was purified from hybridoma supernatant via protein A beads (Sigma–Aldrich, Taufkirchen, Germany) under high salt conditions (3 M sodium chloride, 1.5 M glycine). The CD7-specific mIgG1 antibody TH69 was provided by M. Gramatzki (University Clinic Schleswig-Holstein, Kiel, Germany [4]). TRAIL neutralizing monoclonal antibody 2E5 was purchased from Alexis (Lausen, Switzerland), TRAIL-receptor-specific antibodies from Diaclone SAS (Besançon, France) and mIgG1 or mIgG3 antibody from DAKO Diagnostica (Hamburg, Germany) and Sigma, respectively.

Culture of eukaryotic cells

The human CD19-positive B-ALL cell lines Nalm-6 (DSMZ; German Collection of Microorganisms and Cell lines; Braunschweig, Germany), Reh (DSMZ), SEM [19] and the B-cell lymphoma cell line Ramos (ATCC; American Type Culture Collection; Manassas, VA, USA), the CD19-negative T-ALL cell lines Jurkat (ATCC) and CEM (DSMZ), as well as the murine hybridoma N6B1, were cultured in RPMI-1640 medium (Invitrogen, Karlsruhe, Germany) containing 10% fetal calf serum (FCS; Invitrogen) and 1% penicillin and streptomycin (P/S; Invitrogen) at 37°C in a humidified 5% CO2 atmosphere. CHO-S cells (Invitrogen) were cultured according to manufacturer’s recommendations in serum-free CD CHO medium (Invitrogen) supplemented with 50 nM hypoxanthine and 8 nM thymidine (Invitrogen) and 0.5% P/S with or without the dipeptide l-alanyl-l-glutamine (Glutamax I, Invitrogen).

Isolation of mononuclear cells from leukemia patients and healthy donors

Heparinized peripheral blood and bone marrow samples from B-ALL and B-CLL patients and healthy donors were obtained after receiving written informed consent and with the approval of the Ethics Committee of the University of Erlangen-Nuremberg. Mononuclear cells (MNC) were prepared by Lymphoflot (Biotest, Dreieich, Germany) Ficoll density centrifugation in Leukosep tubes (Greiner, Frickenhausen, Germany) according to manufacturers’ instructions, and maintained in RPMI-1640 medium supplemented with 10% FCS and 1% P/S. The size of the CD19-positive fraction was evaluated by flow cytometry after staining with a CD19-specific antibody phycoerythrin (PE)-conjugate (BD Biosciences, Heidelberg, Germany). ZAP-70 expression and p53 deletion, two clinically relevant parameters for B-CLL, were analyzed following standard procedures [17, 43].

Animals

Animal experiments were performed in female NOD/Scid mice (M&B, Ry, Denmark). Mice were maintained under sterile and standardized environmental conditions (22 ± 1°C, 50 ± 10% relative humidity, 12 h light–dark-cycle) and received autoclaved food and bedding (ssniff Spezialdiäten, Soest, Germany) and acidified (pH 4.0) drinking water ad libitum. All experiments were performed according to the German Animal Protection Law with permission from the local government authorities. In compliance with such regulations, mice were euthanized at the onset of paralysis and/or loss of >20% of body weight.

Construction, expression and purification of scFvCD19

ScFvCD19 was subcloned from the hybridoma N6B1 as previously described [39]. The cDNA coding for the CD19-specific scFv was cloned into the expression vector pAK400, and the plasmids were propagated in E. coli HB2151 (from G. Winter; MRC, Cambridge, UK). Expression and purification of hexa-histidine-tagged scFvCD19, using nickel-nitrilotriacetic agarose beads (Qiagen, Hilden, Germany), was performed as described [40].

Determination of KD

Equilibrium binding constants (K D) were determined by flow cytometry using published procedures [5]. Experiments were repeated four times and mean values are reported. Numerical values were calculated and graphic analyses were performed using GraphPad Prism 3.0 software (Graph Pad software, San Diego, CA, USA).

Construction and production of scFv:sTRAIL

The eukaryotic expression plasmid pEE14scFv:sTRAIL [20], designed for the rapid construction and stable expression of scFv:sTRAIL fusion proteins, was used [7]. This vector employs the strong cytomegalovirus promotor/enhancer to drive expression of the fusion protein. The expression cassette carries sequences coding for the murine kappa light-chain leader peptide upstream of a multiple cloning site, followed by coding sequences for a 26 amino-acid linker and sTRAIL. The cDNA fragment coding for scFvCD19 was amplified by PCR, whereby 5′ SfiI and 3′ NotI restriction sites were introduced. Subsequently, scFvCD19 cDNA was inserted using SfiI and NotI. The scFvCD19:sTRAIL fusion protein was expressed in CHO-S cells after transfection with Lipofectamine 2000 (Invitrogen). Stable transfectants were generated by the glutamine synthetase selection method [15]. Supernatant was harvested and stored at −80°C, and recombinant protein concentration in serum-free CD CHO medium (routinely 1.5–2 μg/ml) was analyzed using a solid phase sandwich TRAIL ELISA according to manufacturer’s recommendations (Diaclone SAS). ScFvCD19:sTRAIL preparations are active only after trimerization, therefore supernatants were monitored for absence of inactive monomers and dimers using size-exclusion FPLC as described previously [7]. For control experiments the fusion protein scFv425:sTRAIL, with specificity for epidermal growth factor receptor (EGFR), was used [10]. ScFv425:sTRAIL induced EGFR-restricted apoptosis in EGFR-expressing tumor cells but not in human leukemic B-cells.

Flow cytometric analyses

Specific binding to CD19

Specific binding of scFvCD19 and scFvCD19:sTRAIL was analyzed using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA, USA). After washing with PBA buffer [phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin], 0.5 × 106 CD19-positive SEM cells were incubated with 4 μg/ml scFvCD19 and 2 μg/ml scFvCD19:sTRAIL fusion protein, respectively. Bound scFvCD19 and scFvCD19:sTRAIL were detected with an anti-penta histidine antibody AF555-conjugate (Qiagen) or 2.5 μl anti-human sTRAIL PE-conjugate (Diaclone SAS), respectively. For blocking experiments, cells were pre-incubated with a tenfold molar excess of parental N6B1 antibody, after which staining was performed as described above. All incubations were performed for 30 min on ice.

Expression of CD19 and TRAIL-receptors on cell lines

Expression profiles of CD19 and TRAIL-receptors were determined using saturating concentrations of 1 μg/ml parental CD19 antibody N6B1 or TRAIL-receptor-specific antibodies according to manufacturer’s recommendations, respectively. Bound antibody was detected using an anti-murine IgG PE-conjugate (DAKO). Staining and subsequent analyses were performed as described above.

CD19-restricted induction of cell death by scFvCD19:sTRAIL

Cell lines were seeded at a concentration of 0.3 × 106 cells/ml and treated with varying concentrations of scFvCD19:sTRAIL. For blocking experiments, cells were incubated 1 h prior to treatment with a 100-fold molar excess of parental N6B1 antibody, a tenfold molar excess of 2E5 TRAIL neutralizing antibody, or respective antibody isotype controls. MNCs (0.5 × 106/ml) from healthy donors were incubated with 100 μg/ml scFvCD19:sTRAIL. Where indicated, MNCs were treated with 8 μg/ml Actinomycin D (Sigma) or with 300 μg/ml of valproic acid (Sigma).

Assays for cell death and apoptosis

Hypotonic PI staining of nuclei

Cell death was measured by staining of nuclei with a hypotonic solution of propidium iodide (PI; Sigma). The extent of cell death was determined by measuring the fraction of nuclei with subdiploid DNA content [36].

Monitoring the exposure of phosphatidylserine by AnnexinV-FITC/PI staining

Whole cells were washed, stained with fluorescein-isothiocyanate (FITC)-conjugated AnnexinV (Caltag Laboratories, Hamburg, Germany) and PI according to the manufacturer’s protocol, and analyzed by flow cytometry. AnnexinV-positive or AnnexinV/PI-double-positive cells were considered apoptotic.

Loss of mitochondrial membrane potential (ΔΨ)

ΔΨ was analyzed with the cell-permanent green-fluorescent lipophilic dye DiOC6 (Molecular Probes, Eugene, OR, USA). After treatment, cells were incubated for 20 min at 37°C with 0.1 μM DiOC6 in fresh medium, washed once with PBS, and analyzed by flow cytometry.

Analysis of poly(ADP-ribose) polymerase (PARP)-cleavage and caspase-3 activation

Cell lysates were prepared 48 h after treatment of cells (0.5 × 106 cells/ml) with scFvCD19:sTRAIL (100 ng/ml), and protein concentrations were determined as described [3]. Sodium dodecyl sulphate polyacrylamide gels (10 and 15%) were loaded with 30 and 50 μg of total protein for PARP-cleavage and caspase-3 activation, respectively, and electrophoresis was performed according to standard procedures. Proteins were blotted and PARP was detected with a PARP-specific antibody (BD Biosciences), pro-caspase-3 with a caspase-3-specific antibody (BD Biosciences). Actin was detected as a loading control with pan-actin Antibody-5 (Dianova, Hamburg, Germany). All antibodies were used according to manufacturers’ protocols and detected with appropriate horseradish peroxidase-coupled secondary antibodies and enhanced chemiluminescence reagents (GE Healthcare, Freiburg, Germany).

Determination of serum stability of scFvCD19 and scFvCD19:sTRAIL

ScFvCD19 (4 μg/ml) and scFvCD19:sTRAIL (2 μg/ml) were incubated in human serum at 37°C for variable time intervals. Residual binding capacity to SEM cells was assessed as described [11]. To determine residual apoptotic potential, samples were applied to CD19-positive Reh cells and antigen-negative CEM cells at a subsaturating concentration (50 ng/ml) and incubated for 96 h. The extent of apoptosis was determined by AnnexinV/PI staining relative to maximum apoptosis (100%) resulting from treatment for 96 h with freshly prepared fusion protein. Four and three independent experiments were performed for residual binding studies to CD19 and apoptotic potential, respectively, and numerical values were calculated using GraphPad Prism 3.0 software.

Differential quantification of apoptosis in target and bystander cells in mixed culture experiments

CD19-positive target cells were labeled with the red fluorescent dye DiI (Molecular Probes) as previously described [9]. DiI-labeled target cells, and non-labeled bystander cells were mixed at indicated ratios with a final cell concentration of 0.5 × 106 cells per well. After treatment, differential fluorescent characteristics of target and bystander cells were used to separately evaluate apoptosis by exposure of phosphatidylserine to the outer cell membrane or by measurement of ΔΨ.

Synergistic induction of cell death by scFvCD19:sTRAIL plus Cyclosporin A or valproic acid

Synergistic apoptotic effects, induced by treatment of cells with scFvCD19:sTRAIL plus either Cyclosporin A (CsA, Sigma) or valproic acid, were determined by evaluating the cooperativity index (C-index), defined as the ratio of the sum of specific cell death induced by single agent treatments and specific cell death achieved by treatment with the combination. C-Index values <1 were taken to indicate synergy. Cells were co-treated with 10 ng/ml of scFvCD19:sTRAIL. Cell death was determined by AnnexinV/PI staining after treatment for 24 h.

Induction of cell death in blasts from leukemia patients

MNCs from patients were seeded at either 0.5 × 106 or 0.4 × 106/ml in 48-well plates for B-CLL and B-ALL patients, respectively, and treated with 100 ng/ml of scFvCD19:sTRAIL or scFv425:sTRAIL control. Specific cell death was calculated by subtraction of spontaneous cell death in untreated control samples. To determine synergistic effects between an antileukemic agent and the scFvCD19:sTRAIL, B-CLL cells were treated with 300 μg/ml of valproic acid alone or valproic acid in combination with scFvCD19:sTRAIL. After treatment for 24 h, cell death was monitored by exposure of phosphatidylserine and C-index values were calculated as described above. All experiments were performed in triplicates.

Inhibition of tumor engraftment in mice

Nalm-6 B-ALL cells were washed once with PBS. One million cells in 100 μl PBS were injected intravenously into the lateral tail vein on day 0 of the experiment. Mice were then treated via intravenous injections with 4 μg of scFvCD19:sTRAIL in medium, scFv425:sTRAIL or medium control, respectively (days 3, 5 and 7). Mice were investigated daily for health status and moribund mice were euthanized according to regulations. Survival times were determined for evaluation of therapeutic efficacy by Kaplan-Meier-Analysis and to evaluate median survival. Statistical analyses were performed using a log-rank test.

Statistical analysis

At least three independent experiments were performed for all assays using cell lines. Data reported are mean values and their standard error of the mean (SEM). For dose–response experiments of CD19-positive cells EC50 values were calculated. Where appropriate, P values were determined using two-sided, un-paired Student’s t test. In the murine leukemia model 4 and 5 mice per group were analyzed. Survival curves were generated by the Kaplan–Meier method. Therapeutic efficacy was compared using the log-rank test and median survival of mice was calculated. In all statistical analyses a difference was defined as statistically significant for P < 0.05.

Results

CD19-restricted binding and serum stability of scFvCD19:sTRAIL

The scFvCD19:sTRAIL fusion protein consists of sTRAIL genetically fused to a CD19-specific scFv, derived from the N6B1 hybridoma (Fig. 1a). The parental scFvCD19 antibody fragment efficiently bound to CD19-positive SEM leukemic cells (Fig. 1b) with an equilibrium binding constant (K D) of 142 nM. Binding was CD19-specific, because it was blocked in competition experiments by the parental antibody. The scFvCD19:sTRAIL fusion protein displayed similar antigen-restricted binding (Fig. 1b). Both proteins had a comparable serum binding stability (P = 0.0657) when incubated in human serum at 37°C (Fig. 1c). Half-maximum residual binding was reached after 35.2 and 29.5 h for scFvCD19 and scFvCD19:sTRAIL, respectively. Interestingly, scFvCD19:sTRAIL retained potent apoptotic activity for CD19-positive Reh leukemia cells after incubation for 73.6 h in human serum (Fig. 1d). In a control experiment with CD19-negative cells, scFvCD19:sTRAIL did not induce apoptosis.

Fig. 1.

Fig. 1

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CD19-restricted binding and serum stability of scFvCD19 and scFvCD19:sTRAIL. a Scheme of homotrimeric scFvCD19:sTRAIL consisting of the N-terminal (N-) hemagglutinin-tag (HA) genetically fused to scFvCD19 and C-terminal (C-) sTRAIL. The intrinsic trimerization domain of sTRAIL leads to formation of active homotrimers. b CD19-positive SEM cells were stained with scFvCD19 or scFvCD19:sTRAIL (dark grey), respectively. PBS or CD CHO medium served as control for background staining (light grey). Binding was assessed by flow cytometry. Bold black lines blocked binding after pre-incubation with tenfold molar excess of parental antibody. c and d scFvCD19 and scFvCD19:sTRAIL were kept in human serum at 37°C for the indicated time intervals. c Samples were analyzed by flow cytometry and residual binding to SEM cells was calculated from 4 independent experiments. Values are normalized to binding of freshly prepared samples. d CD19-positive Reh cells were treated with subsaturating concentrations of scFvCD19:sTRAIL after incubation in human serum for the indicated length of time. Cell death was measured by AnnexinV/PI staining after 96 h. Experiments were performed 4 times independently. Percent cell death was calculated relative to maximum apoptosis obtained with freshly prepared samples. Error bars standard error of the mean (SEM)

Induction of potent, CD19-restricted apoptosis by scFvCD19:sTRAIL

The scFvCD19:sTRAIL fusion protein induced potent cell death in the precursor (pre) B-ALL cell lines Reh (EC50 = 37.5 ng/ml) and Nalm-6 (EC50 = 12.4 ng/ml), while the pro B leukemia cell line SEM, carrying a t(4;11) translocation to the MLL gene, was moderately sensitive to induction of cell death. No correlation between differences in sensitivity for scFvCD19:sTRAIL and expression levels of CD19 or TRAIL-receptors was observed for the cell lines investigated here (Table 1). Similar treatment of CD19-negative CEM cells, derived from T-ALL, did not induce cell death (Fig. 2a). Cell death was CD19-specific, because pre-incubation with the parental N6B1 antibody strongly inhibited cell death by scFvCD19:sTRAIL (Fig. 2b). This result was further supported by the fact that treatment of these cell lines with non-targeted scFv425:sTRAIL did not induce cell death. In addition, cell death was TRAIL-mediated, because it was abrogated by the TRAIL-neutralizing antibody 2E5 (Fig. 2b). Cell death by scFvCD19:sTRAIL was characterized by classical features of apoptosis, including a progressive exposure of phosphatidylserine on the outer cell membrane, as assayed by AnnexinV binding, and accessibility of nuclear DNA, as evidenced by binding of PI (Fig. 2c). Moreover, caspase-3 was activated and subsequently the caspase-3 target protein PARP was cleaved (Fig. 2d).

Table 1.

Expression profile of CD19 and TRAIL receptors on cell lines

Cell line CD19a TRAIL-R1a TRAIL-R2a TRAIL-R3a TRAIL-R4a
Reh 94.2 ± 5.9 4.2 ± 1.2 7.0 ± 0.7 – –
Nalm-6 193.0 ± 23.4 9.8 ± 1.4 3.2 ± 0.8 – –
SEM 256.6 ± 61.7 – 3.0 ± 0.7 – –
Ramos 80.2 ± 5.5 9.6 ± 1.7 4.0 ± 1.8 – –
CEM – – 2.1 ± 0.4 ND ND
Jurkat – – 6.0 ± 0.9 ND ND
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CD19 expression on the cell lines used in this study was determined by flow cytometric analyses after staining with CD19-specific antibody PE-conjugate. Expression of TRAIL-receptors was determined after staining with the respective receptor-specific antibody and an appropriate PE-conjugated secondary antibody. Data are mean values ± SEM of 3 independent experiments

ND not determined

aMean fluorescence intensity relative to isotype

– not detectable/mean fluorescence intensity relative to isotype < 1

Fig. 2.

Fig. 2

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Induction of CD19-restricted apoptosis by scFvCD19:sTRAIL in leukemia-derived cell lines, but not in MNCs from healthy donors. a Various CD19-positive cell lines (Reh, Nalm-6 and SEM) and the CD19-negative cell line CEM were treated with increasing concentrations of scFvCD19:sTRAIL. After treatment for 96 h, cell death was measured by hypotonic PI staining of nuclei. Data are mean values ± SEM from 3 independent experiments. b Reh, Nalm-6 and CEM control cells were treated with scFvCD19:sTRAIL (100 ng/ml) in the absence or presence of 100-fold molar excess of parental CD19 antibody N6B1, tenfold molar excess of TRAIL neutralizing antibody 2E5, or the respective isotype antibodies. (+): treatment with scFvCD19:sTRAIL plus the respective antibody. Cell death was measured 96 h after treatment by hypotonic PI staining. Values shown are mean values + SEM from 3 independent experiments. c Reh, Nalm-6 and CEM cells were treated with 10 ng/ml of scFvCD19:sTRAIL or CD CHO medium. After treatment for 24 h, exposure of phosphatidylserine to the outer cell membrane was assessed by AnnexinV/PI staining. Percentages in the lower right quadrant indicate early apoptotic cells. d Cells were treated with 100 ng/ml of scFvCD19:sTRAIL (+) or medium control (−) for 48 h. Characteristic indicators of apoptosis were revealed by immunoblotting: PARP and its cleavage product (arrow) and disappearance of pro-caspase 3 with actin as a loading control. e Isolated MNCs from healthy donors were treated with 100 ng/ml of scFvCD19:sTRAIL, medium control, 8 μg/ml Actinomycin D or 300 μg/ml VPA for 24 h. Apoptosis was assessed by loss of ΔΨ and values are mean values + SEM from 3 independent experiments

To investigate potential unwanted apoptotic effects towards normal hematopoietic cells, scFvCD19:sTRAIL was applied to MNCs from healthy donors. Treatment of these cells with scFvCD19:sTRAIL for 24 h did not cause significant apoptosis. Moreover, treatment with valproic acid (VPA) as a potential candidate for combination therapy revealed no significant sensitivity (P = 0.07) of MNCs towards this agent. In contrast, treatment with Actinomycin D as a positive control induced apoptosis in 54.6% of cells (Fig. 2e).

Induction of bystander apoptosis by scFvCD19:sTRAIL in CD19-negative tumor cells

Binding of scFvCD19:sTRAIL to CD19-positive target cells was exploited to cross-link TRAIL receptors on neighboring CD19-negative tumor cells and to eliminate these cells by the so-called bystander effect, as previously described [8]. Therefore, CD19-positive Ramos target cells and CD19-negative Jurkat bystander cells were mixed at varying ratios and treated with scFvCD19:sTRAIL. Significant apoptosis compared to sTRAIL-blocked controls was detected in Ramos cells at all tested target to bystander (T:B) ratios (Fig. 3a, open bars). Importantly, at a T:B ratio of 1:1, 71.2% of the Jurkat bystander cells were eliminated. Significant bystander apoptosis of 41.7% occurred even at the low T:B ratio of 1:10 (Fig. 3b, open bars). Apoptosis of both target and bystander cells was abrogated by incubation with the TRAIL-neutralizing antibody 2E5 (Fig. 3a–b, black bars).

Fig. 3.

Fig. 3

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Induction of apoptosis in CD19-negative bystander tumor cells. Mixed culture experiments with CD19-positive Ramos cells a and CD19-negative Jurkat cells b were performed at varying T:B ratios. Cells were treated with 100 ng/ml scFvCD19:sTRAIL in the presence or absence of the TRAIL neutralizing antibody 2E5. The differential fluorescent labeling of target and bystander cell populations was used to separately evaluate apoptosis induction by ΔΨ after 24 h of treatment. Values given are mean values + SEM from 3 independent experiments

Synergistic tumoricidal effect of scFvCD19:sTRAIL and antileukemic drugs for various B-ALL cell lines

Various agents, including the modulator of mitochondrial permeability transition pores CsA [12, 25, 58] and the histone deacetylase inhibitor (HDACi) VPA [26, 33, 44], are currently under investigation for their potential use as antileukemic drugs. Previously VPA has been shown to sensitize cells to TRAIL-mediated apoptosis [57]. Consequently, the effect of co-treatment of various tumor-derived cell lines with either CsA or VPA in combination with scFvCD19:sTRAIL was investigated (Fig. 4a–b). Combination of scFvCD19:sTRAIL and CsA resulted in synergistic induction of cell death for the CD19-positive cell lines (Fig. 4a; C-index Reh: 0.77; Nalm-6: 0.73; SEM: 0.69). Similar data were obtained when cells were co-treated with VPA and scFvCD19:sTRAIL, including synergistic induction of cell death (Fig. 4b; C-index Reh: 0.79; Nalm-6: 0.74; SEM: 0.67). The synergistic effect of co-treatment with VPA and scFvCD19:sTRAIL was not correlated with upregulation of either of the agonistic receptors TRAIL-R1/2, since no upregulation of TRAIL-R1/2 was observed after incubation with VPA (Fig. 4c).

Fig. 4.

Fig. 4

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Synergistic tumoricidal effect of scFvCD19:sTRAIL in combination with CsA or VPA for various B-ALL cell lines. Treatment of Reh, Nalm-6, SEM and CEM control cells with antileukemic drugs CsA (a) or VPA (b), respectively, was performed in combination with scFvCD19:sTRAIL (10 ng/ml). For VPA treatment, combination of VPA plus scFv425:sTRAIL was included as an additional control. Final concentrations of 4 μg/ml for Reh, Nalm-6 and CEM cells or 10 μg/ml of CsA for SEM cells and 100 μg/ml for Reh, SEM and CEM cells or 150 μg/ml of VPA for Nalm-6 cells, respectively, were applied. Cell death was determined by AnnexinV/PI staining after 24 h. Specific cell death was defined as percentage of dead cells over background of spontaneous cell death in untreated control samples. C-Index values were calculated as described in “Materials and methods” and synergism (C-Index < 1.0) is indicated by (*). Values given are mean values + SEM from 3 independent experiments. c After combination treatment with VPA, expression levels of activating TRAIL-R1/2 were monitored by flow cytometry for the cell lines investigated here. Cells were stained with TRAIL-R1- (left panel) or TRAIL-R2-specific antibody (right panel), respectively. Murine IgG1 antibody served as control for background staining (light grey). Binding of receptor-specific antibody in untreated samples is depicted in dark grey. Black lines represent receptor expression of VPA-treated samples, dotted lines of scFvCD19:sTRAIL treated samples. Receptor expression of combination-treated samples is depicted in bold black lines

Induction of cell death by scFvCD19:sTRAIL in primary cells from pediatric B-ALL patients

To further define the therapeutic potential of scFvCD19:sTRAIL towards CD19-positive pediatric malignancies, MNCs from 5 pediatric B-ALL patients (patient 1: pro B-ALL; 2–5: c-ALL) were treated with scFvCD19:sTRAIL or scFv425:sTRAIL, directed against EGFR (Fig. 5a). Samples 1–3 contained more than 75%, and samples 4 and 5, 25 and 44% CD19-positive cells, respectively. Specific cell death was observed for MNCs from 3 patients (#2: scFvCD19:sTRAIL 24.5% and scFv425:sTRAIL 7.6%; #4: scFvCD19:sTRAIL 14.2% and scFv425:sTRAIL 0.1%; #5: scFvCD19:sTRAIL 34.8% and scFv425:sTRAIL 8.8%), whereas cells from patients 1 and 3 were largely refractory to scFvCD19:sTRAIL (#1: scFvCD19:sTRAIL 8.6% and scFv425:sTRAIL 0.1% #3: scFvCD19:sTRAIL 7.5% and scFv425:sTRAIL 2.6%). Specific binding to CD19 was crucial for the apoptotic activity of scFvCD19:sTRAIL, because treatment with the control protein scFv425:sTRAIL resulted in only moderate apoptosis.

Fig. 5.

Fig. 5

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Induction of cell death in B-ALL-derived MNCs by scFvCD19:sTRAIL, and synergistic effects on primary B-CLL cells by co-treatment with VPA. a MNCs from pediatric B-ALL patients were treated with 100 ng/ml of scFvCD19:sTRAIL or scFv425:sTRAIL, respectively. Specific cell death was determined after 24 h by AnnexinV/PI staining. Several B-CLL samples sensitive for scFvCD19:sTRAIL (b) and the only resistant sample (c) were treated for 24 h with 300 μg/ml of VPA in the presence or absence of scFvCD19:sTRAIL. Specific cell death was determined using AnnexinV/PI staining. C-Index values were calculated as described in “Materials and methods”; synergism is indicated by (*). All experiments were performed in triplicates and values given are mean values ± SEM

Induction of cell death by scFvCD19:sTRAIL in primary cells from adult B-CLL patients

Treatment of primary cells from adult B-CLL patients resulted in specific cell death ranging from 10 to 42% for all samples tested, with the exception of cells from patient 5, which were resistant to treatment (Table 2). For 11/12 patients, the EGFR-targeted fusion protein scFv425:sTRAIL did not induce apoptosis, indicating that binding of scFvCD19:sTRAIL to CD19 was essential for the induction of apoptosis. Interestingly, in one patient, scFv425:sTRAIL induced apoptosis (7.2% indicated by superscript “a” in Table 2). All B-CLL samples monitored for prognostic parameters were ZAP-70 negative. Moreover, no correlation was observed between sensitivity to scFvCD19:sTRAIL and response to treatment with chemotherapeutics. Of note, 2/3 patients (#1 and #7) with deletion of p53 remained sensitive to induction of apoptosis by scFvCD19:sTRAIL.

Table 2.

Induction of cell death by scFvCD19:sTRAIL in MNCs from different adult B-CLL patients and determination of prognostic factors for clinical outcome

Patient % CD19+ % Specific apoptosis ZAP-70 p53-deletion Clinically refractory to alkylators/fludarabin
1 83 20 ND pos. ND/ND
2 87 24 ND ND ND/ND
3 71 31 Neg. Neg. ND/ND
4 73 38 Neg. Neg. No/no
5 30 0 Neg. Pos. ND/ND
6 81 19 Neg. Neg. No/yes
7 63 11 Neg. Pos. No/yes
8 87 42 Neg. Neg. No/no
9 66 21 Neg. ND Yes/yes
10 80 20a Neg. Neg. No/no
11 67 25 Neg. Neg. No/ND
12 42 10 Neg. Neg. No/no
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MNCs were prepared from whole blood and the CD19-positive fraction was estimated by flow cytometric analyses after staining with CD19-specific antibody PE-conjugate. Cells were treated with 100 ng/ml scFvCD19:sTRAIL or scFv425:sTRAIL, respectively. Specific cell death above background level was assessed by AnnexinV FITC/PI staining after incubation for 24 h. ZAP-70 negative indicates intracellular ZAP-70 levels below 20% as measured by flow cytometry after intracellular staining. Deletion of p53 was determinded by FISH analysis according to standard procedures

ND not determined

a7% dead cells were detected after treatment with scFv425:sTRAIL for this patient

Synergistic apoptosis of primary B-CLL cells by combined treatment with scFvCD19:sTRAIL and VPA

To further evaluate the potential benefit of combined treatment of cells with VPA and scFvCD19:sTRAIL, primary cells from 4 patients were subjected to treatment with scFvCD19:sTRAIL and VPA. For all of these samples combination treatment resulted in a synergistic induction of apoptosis (Fig. 5b; C-index #1: 0.74; #2: 0.5; #3: 0.79; #4: 0.6). Cells from patient 5, which did not respond to treatment with scFvCD19:sTRAIL, showed a small degree of VPA-induced apoptosis. Remarkably, combination treatment of cells from this resistant patient with VPA plus scFvCD19:sTRAIL induced cell death in a synergistic manner (Fig. 5c; C-index: 0.37). In contrast to primary B-CLL cells, treatment of MNCs from healthy donors with scFvCD19:sTRAIL and VPA did not result in induction of apoptosis.

Inhibition of tumor engraftment in mice xenotransplanted with human Nalm-6 B-ALL cells by scFvCD19:sTRAIL

Previously, various sTRAIL preparations have shown potent effects in murine models of human tumors without systemic toxicity [2, 22, 27, 53]. To investigate the in vivo potential of scFvCD19:sTRAIL, a NOD/Scid mouse model of xenotransplanted human pre B-ALL cells was established. Intravenous transplantation of Nalm-6 cells led to the development of a disseminated tumor with hind leg paralysis and involvement of bone marrow and spleen in control mice treated with medium only. Median survival of this group was 28 days (Fig. 6). However, treatment of groups of 4 and 5 mice with 3 consecutive intravenous doses of 4 μg each of scFvCD19:sTRAIL (days 3, 5 and 7 after inoculation) resulted in a significant therapeutic effect compared with medium-treated mice (P < 0.0001) with 7/9 mice surviving for 112 days. In contrast, similar treatment with scFv425:sTRAIL did not prolong survival (median survival: 27 days) compared with medium-treated control mice (P = 0.1893).

Fig. 6.

Fig. 6

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Inhibition of engraftment in a murine model of xenotransplanted human B-ALL cells by scFvCD19:sTRAIL. Female NOD/Scid mice were injected intravenously into the lateral tail vein with 106 Nalm-6 cells on day 0. On days 3,5 and 7 mice were treated with 4 μg of scFvCD19:sTRAIL per dose intravenously, or with scFv425:sTRAIL, and one group received medium as a control. Mice were monitored for 112 days. At first signs of paralysis of hindlegs and/or loss of > 20% of body weight, mice were euthanized. Percent survival was calculated for 9 mice from 2 independent experiments, which were treated with medium, scFv425:sTRAIL and scFvCD19:sTRAIL, respectively. P values were calculated using log rank test and the median survival was calculated for each group

Discussion

The principal new finding reported in this pre-clinical study is that selective apoptosis was induced in leukemic B-lymphoid cells by targeting CD19 with a novel scFv:sTRAIL fusion protein. ScFvCD19:sTRAIL showed potent apoptotic activity on primary CD19-positive malignant cells from patients and prevented engraftment of human leukemic cells in a murine model of pre B-ALL.

Specific binding of scFvCD19:sTRAIL to CD19 resulted in cell surface accumulation. Cross-linking of agonistic TRAIL-receptors was not detectable after blocking with the parental CD19 antibody, which is presumably due to the fast-on/fast-off rates of death receptor-polypeptide ligand interactions. Although the scFvCD19 antibody fragment had a low affinity of 142 nM, it possessed a high functional activity as part of the scFvCD19:sTRAIL fusion protein. This is most likely a result of the typical trimeric form of sTRAIL, which for scFvCD19:sTRAIL leads to the presence of 3 scFvCD19 molecules. The resulting gain in avidity probably compensated for the low affinity of the monovalent scFv.

The binding capacity of the scFv component of scFvCD19:sTRAIL decayed with a half-life of 29.5 h in human serum at 37°C, whereas the half-life of the apoptosis-inducing activity of scFvCD19:sTRAIL in human serum was approximately 3 days. Importantly, no increase of CD19-independent apoptosis was observed, which indicates that no secondary aggregation of unfolded or denatured molecules occurred. This favorable discrepancy between half-life of binding and half-life of apoptotic activity may be due to the continued presence of active trimerized scFvCD19:sTRAIL, which is retained at the cell surface by one or two remaining functional scFvs.

ScFvCD19:sTRAIL eliminated various leukemia-derived CD19-positive cell lines by induction of apoptosis. Different levels of cell death were observed for Reh, Nalm-6 and SEM cells, which were most likely due to intrinsic, genetically determined sensitivity for, or resistance to, apoptosis, with no correlation between cell death and expression of CD19 or activating TRAIL-R1/2. Reh and Nalm-6 cells are derived from pre B-ALL cells, whereas SEM was established from pro B-ALL cells with translocation into the MLL gene. Translocation-activated target genes may play a role in the reduced sensitivity of SEM cells to induction of apoptosis by scFvCD19:sTRAIL. Cell death was strictly CD19-dependent, because pre-incubation of tumor cells with the parental CD19 antibody completely inhibited induction of apoptosis and no cell death was induced when treating the cells with non-targeted scFv425:sTRAIL. Therefore, in this fusion protein sTRAIL is only active after accumulation on the cell surface, resulting from binding to CD19. In addition, cell death was fully TRAIL-mediated, because treatment with the TRAIL neutralizing antibody 2E5 abrogated the apoptotic activity. This is in agreement with previous findings that CD19 is not involved in apoptotic signaling [18]. ScFvCD19:sTRAIL did not induce apoptosis in MNCs derived from healthy donors. This resistance of normal cells to TRAIL-mediated apoptosis is in accordance with previous reports [2, 9] and suggests that therapeutic application of scFvCD19:sTRAIL may only minimally affect normal hematopoietic cells.

Consistent with a previous report on EGP2-targeted scFv:sTRAIL [8], CD19-bound scFvCD19:sTRAIL induced potent apoptosis in neighboring CD19-negative bystander tumor cells. This anti-tumor bystander activity may be a potentially valuable property, because during antibody-based therapy, with for example Rituximab, tumor cells can lose the expression of the target antigen [28]. Whether CD19-negative tumor cells will arise during therapy is currently unknown, because no CD19-directed therapeutics are in routine clinical use. However the anti-tumor bystander activity of scFvCD19:sTRAIL may be of potential clinical benefit.

ScFvCD19:sTRAIL was also effective in inducing apoptosis in primary pediatric precursor B-ALL cells. The fact that scFvCD19:sTRAIL was effective in 3/5 samples indicates its potential for precursor B-ALL, despite the limited number of samples available. In addition, treatment of primary MNCs from adult B-CLL patients with scFvCD19:sTRAIL induced significant apoptosis in the majority of B-CLL samples (11/12), which is encouraging, because primary B-CLL cells often are resistant to treatment with TRAIL [32]. The extent of cell death showed considerable variation for the different samples, which may be due to individual susceptibilities to TRAIL-induced apoptosis, variations in TRAIL-R1/R2 and/or CD19 densities. The relative composition in clonogenic progenitors, proliferating blasts and quiescent cells may also have greatly varied among these patient-derived samples.

Poor clinical outcome of B-CLL correlates with elevated intracellular concentrations of ZAP-70 [43]. Unfortunately, all patients studied were ZAP-70 negative, thus precluding any prediction with regard to scFvCD19:sTRAIL activity in the ZAP-70 subgroup. In addition, p53 gene deletion is associated with poor clinical outcome [17]. In 2/3 patient samples with p53 deletion, scFvCD19:sTRAIL induced apoptosis, confirming reports, that TRAIL functions independently from p53 status. However, a larger panel of patient-derived samples will need to be analyzed to determine, whether scFvCD19:sTRAIL may be of use for patients with ZAP-70 expression or deletion of p53.

Combinatorial treatment of tumor cells with sTRAIL and several experimental anti-cancer drugs has been shown to strongly augment its apoptotic activity. In this respect, HDACis are currently under clinical investigation for potential application in leukemia therapy. VPA induced apoptosis in human leukemic cells [26] and sensitized cultured B-CLL cells to TRAIL [23]. However, VPA was effective only at high concentrations, which would limit its potential as a single-agent therapeutic [44]. Here we found strong synergistic effects of scFvCD19:sTRAIL in combination with low doses of VPA for leukemic B-cell lines and primary patient-derived B-CLL cells, whereas MNCs from healthy donors showed no sensitivity for VPA alone or the combination with scFvCD19:sTRAIL. Synergism was CD19-dependent, because combination treatment with scFv425:sTRAIL and VPA showed no synergistic induction of apoptosis. In agreement with Inoue et al. [24], TRAIL-R1/2 upregulation was not involved in VPA mediated sensitization of scFvCD19:sTRAIL-mediated apoptosis. Thus synergism is most likely due to intracellular modulation of pro- and anti-apoptotic proteins. Interestingly, cells from one patient, that were resistant to treatment with scFvCD19:sTRAIL alone, responded to the combination with VPA. This synergistic effect suggests that such a treatment strategy may be promising for therapy of leukemia and lymphoma. Furthermore, we identified CsA as an agent synergizing with scFvCD19:sTRAIL. It has also been known that CsA mediates synergistic apoptosis of B-ALL cells in combination with the plant-derived antioxidant resveratrol [58]. Taken together, our observations suggest, that clinical application of scFvCD19:sTRAIL may benefit from combination with VPA and/or CsA.

Treatment with scFvCD19:sTRAIL in experiments with xenografted human Nalm-6 cells in NOD/Scid mice clearly prevented engraftment of tumor cells and significantly prolonged survival. This anti-tumor activity of scFvCD19:sTRAIL was dependent on specific binding to CD19, because treatment with an identical scFv:sTRAIL fusion protein, targeting the irrelevant antigen EGFR, did not prolong survival. As previously reported for other sTRAIL preparations [2, 27], scFvCD19:sTRAIL displayed no toxicity. However, murine TRAIL receptors only weakly interact with human TRAIL [6]. Therefore, to reliably determine toxicity of scFvCD19:sTRAIL including immunogenicity of the murine scFv component, studies in animals, closer related to humans are likely to be informative. In this respect, sTRAIL has previously been found to be safe and non-immunogenic in cynomolgus monkeys and chimpanzees.

In conclusion, the pre-clinical data presented here on the targeted delivery of sTRAIL to CD19, using scFvCD19:sTRAIL, warrant further evaluation of scFvCD19:sTRAIL towards potential clinical applications for B-cell malignancies.

Author’s contributions

JS, CK and EB participated in designing and performing the research and writing the paper; TML, BC and M. Peipp performed research; HJB contributed the hybridoma, M Pfeiffer, JG, FO and BE contributed leukemia patient material; HSK contributed mice and supported the experiment; GHF and WH controlled data and wrote the paper; all authors checked the final version of the manuscript.

Acknowledgments

We thank Domenica Saul, Kristin Mentz, Linda van Genne and Douwe Samplonius for excellent technical assistance and Thorsten Haferlach for determining the p53 deletion status of B-CLL samples. The personnel of the University of Erlangen animal research facility is kindly acknowledged for breeding and taking care of the mice and Teresa M. Allen for commenting on the manuscript.

Abbreviations

TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand

scFv

Single-chain Fv antibody fragment

sTRAIL

Soluble extracellular domain of TRAIL

CsA

Cyclosporin A

B-ALL

Acute B-lymphoblastic leukemia

B-CLL

Chronic B-lymphocytic leukemia

MNC

Peripheral blood mononuclear cells

EGFR

Epidermal growth factor receptor

HDACi

Histone deacetylase inhibitor

VPA

Valproic acid

Footnotes

This work was supported by Schickedanz KinderKrebs Stiftung (JS) and grants from the Association “Kaminkehrer helfen krebskranken Kindern” (CK, GHF), the Association of supporters of the University of Erlangen Childrens’s Hospital (GHF) and the Dutch Cancer Society (RUG 2002-2668 and 2005-3358) (EB, BC, WH).

Julia Stieglmaier and Edwin Bremer contributed equally.

Contributor Information

Julia Stieglmaier, Email: jstiegl@biologie.uni-erlangen.de.

Georg H. Fey, Phone: +49-91318528493, FAX: +49-91318528526, Email: gfey@biologie.uni-erlangen.de

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