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GENE THERAPY
From the Medical Clinic III, University Hospital
Grosshadern and Gene Center, Ludwig-Maximilians-University; KKG Gene
Therapy, GSF, National Research Center for Environment and Health; IBE,
Institute for Biometry and Epidemiology, Ludwig-Maximilians-University,
Munich, Germany.
B cells of chronic lymphocytic leukemia (B-CLL) are resistant to
transduction with most currently available vector systems. Using an
optimized adenovirus-free packaging system, recombinant adeno-associated virus (rAAV) vectors coding for the enhanced green
fluorescent protein (AAV/EGFP) and CD40 ligand (AAV/CD40L) were
packaged and highly purified resulting in genomic titers up to
3 × 1011/mL. Cells obtained from 24 patients with B-CLL
were infected with AAV/EGFP or AAV/CD40L at a multiplicity of infection
(MOI) of 100 resulting in transgene expression in up to 97% of cells as detected by flow cytometry 48 hours after infection. Viral transduction could be specifically blocked by heparin. Transduction with AAV/CD40L resulted in up-regulation of the costimulatory molecule
CD80 not only on infected CLL cells but also on noninfected bystander
leukemia B cells, whereas this effect induced specific proliferation of
HLA-matched allogeneic T cells. Vaccination strategies for patients
with B-CLL using leukemia cells infected ex vivo by rAAV vectors now
seems possible in the near future.
(Blood. 2002;100:1655-1661) B cells of chronic lymphocytic leukemia (B-CLL) are
inefficient antigen-presenting cells because they lack costimulatory
molecules for efficient T-cell activation.1 Moreover, CD40
ligand (CD40L), a critical molecule for T-cell activation, is
down-regulated on T cells in patients with CLL.2 This
defect can be partially corrected by gene transfer of CD40L into B-CLL
cells. This strategy, using recombinant adenoviral vectors, induced an
autologous immune recognition of B-CLL cells in vitro and in
patients.3,4 However, transduction by recombinant
adenovirus requires a high concentration of viral particles per cell
(multiplicity of infection [MOI] up to 2000) because B-CLL cells lack
the fiber receptor essential for adenoviral attachment.5
Furthermore, the use of adenovirus has raised the question whether
adenoviral fiber structures mediate an unspecific immune
stimulation.6 In contrast, adeno-associated virus (AAV), a
nonenveloped human parvovirus, has gained attention for human gene
therapy, because it does not seem to be pathogenic. AAV vectors have
been proven to be efficient gene transfer vehicles, in particular for
solid tumors.7-10 However, sufficient gene transfer into
primary B-CLL cells with AAV vectors has not been achieved so far.
Previous efforts to transduce primary B-CLL cells with recombinant AAV
(rAAV) were inefficient with transgene expression levels below
3%.11 A recent improvement in the AAV packaging process
was the introduction of a plasmid that encodes the required adenoviral
gene products for the helper virus.12,13 In addition, concentration of viral particles by density-gradient steps using iodixanol, a nonionic dimeric x-ray contrast solution, instead of
cesium chloride yielded higher titers.14 By using these
improvements, we could prepare high-titer rAAV stocks free of
adenovirus to reassess the susceptibility of primary B-CLL cells for
rAAV vectors.
Patients, cells, and cell culture
HeLa cells were obtained from the American Type Culture Collection
(Rockville, MD); the 293 cells were a gift from M. Lohse, Max-Planck-Institute of Biochemistry (Martinsried, Germany). Cells were
cultured at 37°C in 5% CO2 in air, in culture medium
consisting of Dulbecco modified Eagle medium (DMEM; Biochrom, Berlin,
Germany) supplemented with 10% fetal calf serum (FCS; Biochrom), 2 mM
L-glutamine (Biochrom), 100 U/mL penicillin (Biochrom), and
100µg/mL streptomycin (Biochrom).
HeLa/SF cells transfected with human CD40L cDNA were produced
as previously described.16 Cells were Antibodies and reagents
Plasmids The adenoviral pXX6 plasmid was a kind gift of R. Samulski and described previously.17 The gene for enhanced green fluorescent protein (EGFP) was obtained by excising the Asp718-NotI fragment of pEGFP-N1 (Clontech, Heidelberg, Germany) and inserted into the Asp718-NotI site of psub/CEP4(Sal invers). psub/CEP4(Sal invers) is a derivative of psub201(+), which was digested with XbaI, blunt ended, and ligated to the blunt-ended 3923-bp SalI-NruI-fragment of pCEP4(Sal invers).18 pCEP4(Sal invers) differs from pCEP4 (Invitrogen, Groningen, Netherlands) by inversion of the SalI8-SalI3-1316-fragment. The construct pAAV/mCD40L contains the murine CD40L encoding gene driven by the cytomegalovirus (CMV) promoter. To generate pAAV/mCD40L, the 0.8-kb open reading frame (ORF) was released by BamHI digestion from pCEP4/mCD40L and ligated into an Asp718/NotI-digested psubCEP4(Sal invers).rAAV vector production and purification The 293 cells were seeded at 80% confluency and cotransfected by calcium phosphate with a total of 37.5 µg vector plasmid (pAAV/EGFP or pAAV/mCD40L), packaging plasmid pRC, and adenoviral plasmid pXX6 at a 1:1:1 molar ratio.19 Then, 48 hours after transfection cells were harvested and pelleted by low-speed centrifugation. Cells were resuspended in 150 mM NaCl, 50 mM Tris-HCl (pH 8.5), freeze-thawed several times, and treated with Benzonase (50 U/mL) for 30 minutes at 37°C.20 Cell debris was spun down at 3700g for 20 minutes at 4°C. Supernatant was purified by ammonium sulfate precipitation. The pellet was resuspended in PBS-MK buffer (1 × PBS, 1 mM MgCl2, and 2.5 mM KCl) and loaded onto an iodixanol gradient and purified as previously described.20 The iodixanol fraction was further purified by heparin affinity column chromatography and the virus dialyzed against PBS-MK before being used.21Viral vector titering assays The vector particle titer for AAV/EGFP and AAV/CD40L was determined by treating vector dilutions with DNAse I (end concentration 0.5 µg/µL; Boehringer Mannheim, Mannheim, Germany) for 60 minutes at 25°C to remove putatively free viral genomes that could be subsequently hybridized with the probe. The viral preparations were then blotted in a serial 2-fold dilution and finally hybridized with a random-primed transgene specific probe by standard methods. Particle titers were determined by comparing the intensity of the hybridization signals with that obtained for the vector plasmid standard of known concentration blotted on the same membrane. The transducing titer was determined as follows: 7 × 104 HeLa cells per well were seeded in a 12-well plate (4 cm2); 24 hours later the cells were irradiated with 100 Gy and serial dilutions of the recombinant virus were added to the cells. After 48 hours, the number of transgene-expressing cells was quantified by flow cytometry.AAV transduction Primary CLL cells (5 × 105 cells/well in 96-well plates) were incubated in a total of 50 µL Iscove modified Dulbecco medium (IMDM) supplemented with 20% FCS and infectious AAV was added resulting in an MOI between 1 and 500. Cells were incubated for 2 hours at 37°C in 5% CO2 in air. Thereafter, infected cells were transferred on a -irradiated feeder layer expressing CD40L
(HeLa/SF) as outlined above and 150 µL Iscove medium was added.
Flow cytometry Forty-eight hours after AAV transduction, CLL cells were harvested, purified by Ficoll density gradient centrifugation, and washed. Specific, directly conjugated antibodies were applied to cells for 30 minutes in PBS, 4% FCS, 0.1% sodium azide, 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), and 5 mM EDTA (ethylenediaminetetraacetic acid), pH 7.3, on ice and washed. Nonspecific binding was controlled by incubation with isotypic controls (murine isotype IgG1 mAb and hamster isotype IgG3 mAb, BD Pharmingen). Fluorescence was measured with a Coulter Epics XL-MCL (Beckman Coulter). A minimum of 5000 cells was analyzed for each sample. The percentage of positive cells is defined as the fraction beyond the region of 99% of the control-stained cells. Data were analyzed with the use of WinMDI2.8 FACS software. We calculated the mean fluorescence intensity ratio (MFIR) to compare the relative staining intensities of 2 or more stained cell populations. The MFIR is the mean fluorescence intensity (MFI) of cells stained with a fluorochrome-conjugated antigen-specific mAb divided by the MFI of cells stained with a fluorochrome-conjugated isotype control mAb.3Transactivation assay CD40L-transduced or mock-infected B-CLL cells were prelabeled with a green fluorescent dye (CellTracker Green CMFDA, Molecular Probes, Eugene, OR) at a concentration of 1 µM for 15 minutes at 37°C. After extensive washing, labeled stimulator cells were cocultured with noninfected, nonstained CLL cells from the same patient at 37°C for another 48 hours. Expression of CD80 on noninfected, nonlabeled naive CLL cells was assessed by PE-conjugated anti-CD80 mAb (Beckman Coulter).Mixed lymphocyte reaction T cells derived from HLA.A2+ healthy donors were isolated to more than 95% purity by depleting CD19+, CD14+, CD56+, and CD16+ cells from blood mononuclear cells using the Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and magnetic separation columns (Miltenyi Biotec). CLL cells isolated from HLA.A2+ patients were transduced with AAV (AAV/EGFP, AAV/CD40L) and after separation from HeLa/SF feeder cells coincubated for 72 hours with untransduced CLL cells (ratio 1:10) derived from the same patient. Irradiated (200 Gy) CLL cells were used as stimulators, cocultured at 1 × 104 cells/well in a final volume of 200 µL with allogeneic T cells at 1 × 105 cells/well in 96-well round-bottom plates, and incubated for 4 days in IMDM medium (Biochrom) supplemented with interleukin 2 (IL-2; 20 IU/mL; Panbiotech, Aidenbach, Germany) at 37°C in a 5% CO2 humidified atmosphere. During the last 6 hours of the 96-hour culture period, cells were pulsed with 0.5 µCi (0.0185 MBq) [3H]-thymidine (Amersham, Braunschweig, Germany). Cells were harvested onto glass fiber filters and dried, and the [3H]-thymidine incorporation was measured by scintillation spectrophotometry in a Wallac Microbeta Plus 1450 scintillation counter (Turku, Finland).Statistics Statistical associations between dependent subgroups were analyzed by the t test for paired samples; statistical associations between independent subgroups were carried out using the median 2-sample test. In the case of multiple comparisons P values were adjusted by the Bonferroni method. A statistical significance was accepted for P < .05. The calculations were determined by the statistical software package SAS, version 8.2.
Efficient packaging of high-titer, purified rAAV vectors coding for EGFP and CD40L with an improved packaging system Using a new helper virus-free packaging protocol, efficient production of rAAV virions coding for EGFP and CD40L was achieved. The peak fraction of rAAV preparations contained a concentration of 3.8 × 1011 (SEM, 0.6 × 1011) viral particles per milliliter for AAV/EGFP and 2.4 × 1011 (SEM, 0.8 × 1011) for AAV/CD40L, respectively. To exclude the possibility that contaminating DNA accounted for hybridization signals, viral stocks were preincubated with DNAse. No significant decrease in titer was seen, suggesting that the majority of viral particles detected was intact (Figure 1). In several viral preparations infectious titers of 3.7 × 109/mL (SEM, 0.3 × 109) for AAV/EGFP and 4.7 × 109/mL (SEM, 0.3 × 109) for AAV/CD40L were achieved. This corresponds to an infectious-to-particle titer ratio of about 1:100 for AAV/EGFP and 1:50 for AAV/CD40L.
High expression of EGFP and CD40L on primary B-CLL after AAV transduction Freshly isolated CD5/CD19+ cells from patients with established diagnosis of B-CLL were infected with AAV/EGFP and AAV/CD40L at an MOI of 100 and assessed for transgene expression by flow cytometry after 48 hours. In Figure 2, results with cells double-stained for the lineage marker CD19 and the transgene are shown for 4 representative patients (nos. 4, 5, 21, 22 in Table 1). Whereas green fluorescence was detected in only 1% of untransduced cells, expression increased to 42.4% (MFIR, 5.3), 44.9% (MFIR, 6.6), 50.3% (MFIR, 5.4), and 63.0% (MFIR, 15.2), respectively, after transduction with AAV/EGFP. Leukemic cells of the same patients were transduced with AAV/CD40L, and CD40L expression showed a marked shift to 77.8% (MFIR, 209.0), 97.7% (MFIR, 312.2), 78.9% (MFIR, 80.2), and 81.5% (MFIR, 164.9), respectively.
Cumulative data for 24 different patients with B-CLL studied for AAV
infection are given in Table 1. The mean age of the study population
was 62.3 years (SEM, 1.9 years) with a median of 62 years. Ten patients
presented with early disease (Binet stage A) and 14 with advanced
disease (Binet stage B, n = 4; Binet stage C, n = 10). Eighteen
patients were studied for EGFP transduction; the mean percentage of
EGFP-transduced cells was 30.3% (SEM, 3.6%). For CD40L-transduced
samples (n = 15), 44.0% (mean) of CLL cells could be infected (SEM,
7.4%). Using the median 2-sample test, there was no correlation seen
between transduction efficiency with AAV/EGFP or AAV/CD40L,
respectively, and clinical stage of disease (P = .36 and
P = .48), sex (P = .61 and
P = .35), or age (P = .36 and
P = .20). Furthermore, the association between the clinical stage of disease and susceptibility to AAV/EGFP or AAV/CD40L infection, respectively, was examined by the median 2-sample test using
the Bonferroni method with adjustment for sex (male, female) and age
(median age or younger, older than median age). Therefore, P < .0125 (0.05 divided by 4, due to 4 subgroups) would
indicate a significant association at the .05 level. For all 4 subgroups (male, female, age Studying the patients' samples, which were both transduced for EGFP and CD40L (n = 9), values for transduction efficiency were not statistically different (t test for paired samples, P = .17); a mean percentage of 37.3% (SEM, 5.3%) of infected cells could be transduced by AAV/EGFP and 50.6% of CLL cells by AAV/CD40L (SEM, 11.3%). Transduction efficiencies of both viral vectors correlated in these patients with each other (Spearman correlation coefficient test: P = .01). Using the Bonferroni method for adjustment with respect to clinical stage and age of patients (adjustment for sex was not performed because of only one female patient studied both for EGFP and CD40L transduction), a significant P value in the Spearman correlation coefficient test (P < .0001) was only achieved for samples derived from patients with advanced clinical stage (Binet B or C). Concentration and time kinetics of AAV transduction in CLL cells Primary B-CLL cells were infected with AAV/EGFP at different MOIs ranging from 10 to 500. Forty-eight hours after infection cells were analyzed for expression of EGFP by flow cytometry. At an MOI of 10, an average of 8.3% of cells (SEM, 3.9%) was positive for transgene expression. At an MOI of 100, a significant increase of the fraction of transgene-positive cells was observed (t test for paired samples: P = .02); an average of 32.2% of cells (SEM, 4.8%) showed an EGFP expression. At an MOI of 500, only a slight, but insignificant (t test, P = .31) increase in the fraction of EGPF+ cells was detectable (38.3%; SEM, 3.5%; Figure 3).
Recently, membrane-associated heparan sulfate proteoglycans (HSPGs) were identified as the primary receptors for AAV virions, thus mediating both AAV attachment to and infection of target cells.21 Inhibition of AAV attachment and infection was shown to be achieved in competition experiments using heparin, a molecule chemically very similar to heparan sulfate glycosaminoglycan thus functioning as a soluble receptor analog. Therefore, viral supernatants coding for EGFP (AAV/EGFP) were mixed with heparin and incubated with B-CLL cells. As seen in Figure 3, viral transduction of EGFP at a high MOI of 500 was significantly decreased by heparin (t test, P = .007) resulting in background level of EGFP expression; a mean percentage of only 0.05% of cells (SEM, 0.05%) were EGFP+. Transgene expression was assessed over time using an MOI of 100. As
seen in Figure 4, 12 hours after EGFP
infection only small amounts of EGFP+ cells were detected
by flow cytometry (mean, 0.4%; SEM, 0.1%). Twenty-four hours after
transduction a mean of 4.6% of cells was positive (SEM, 1.3%). A
significant (t test, P = .005) shift in the
fraction of EGFP-expressing cells was seen after 48 hours (mean,
32.2%; SEM, 4.8%). A further although insignificant (t test: P = .11) increase was detectable another 48 hours
later (ie, 96 hours after infection) with an average EGFP+
cell population of 53.0% (SEM, 8.6%). The mean percentage of EGFP+ cells was decreased 1 week after infection at
significant levels in comparison to transduction data obtained 48 hours
after infection (20.9%; SEM, 9.1%; t test:
P = .04).
Up-regulation of CD80 on B-CLL cells after AAV/CD40L transduction To prove that CLL cells transduced with CD40L became highly proficient antigen-presenting cells, expression of the costimulatory molecule CD80 was assessed before and 8 days after infection with AAV/CD40L. In CLL samples derived from 3 different patients, CD80 expression could be induced from 6.9% (MFIR, 1.6) to 19.4% (MFIR, 2.3) in patient 3, from 1.7% (MFIR, 1.2) to 10% (MFIR, 2.8) in patient 6, and from 4% (MFIR, 0.8) to 27% (MFIR, 2.4) in patient 7. CD80 expression on CD40L-transduced CLL cells (mean, 18.8%; SEM, 4.9%) was significantly higher in comparison to uninfected control CLL cells (mean, 4.2%; SEM, 1.5%; median 2-sample test, P = .03). Incubation with an anti-murine CD40L mAb could significantly inhibit CD80 up-regulation (mean, 4.8%; SEM, 0.9%) in these 3 patients with an expression of 5.4% (MFIR, 0.7), 3% (MFIR, 1.8), and 6% (MFIR, 1.9), respectively (median 2-sample test: P = .03; Figure 5). Primary CLL cells infected with AAV/EGFP did not result in any up-regulation of CD80 in comparison to uninfected controls (data not shown).
Transactivation of noninfected bystander leukemia cells by CD40L-transduced CLL cells The transactivation capacity of AAV/CD40L-infected CLL cells was assessed after they were labeled with a green fluorescent dye (CellTracker) and used as stimulator cells for equal numbers of nonlabeled CLL B cells from the same patient. Uninfected nonlabeled bystander CLL cells of patient 16 (Table 1) were induced to express CD80 when cocultured with CD40L-infected CLL cells (29.4%; MFIR, 4.0), but not after coincubation with uninfected control CLL cells (1.3%; MFIR, 1.0). Similar results were obtained when naive CLL cells were stained and coincubated with CD40L-transduced nonlabeled leukemia cells; CD80 was up-regulated on naive bystander cells (29.7%; MFIR, 4.2), incubation with uninfected control CLL cells resulted in very low CD80 expression (0.3%; MFIR, 0.9). This stimulatory effect on bystander CLL cells could be abrogated by coincubation of stimulator cells with an anti-murine CD40L mAb (3.2%; MFIR 1.5). Furthermore, stimulation of naive CLL cells by mock-infected (wild-type AAV) CLL cells did not affect CD80 expression levels (0.7%; MFIR, 0.8). Specific up-regulation of CD80 on bystander CLL cells was also observed in 2 other patient samples studied (nos. 12 and 15; Figure 6). CD80 expression on bystander cells after coincubation with CD40L-transduced CLL cells (mean, 18.4%; SEM, 6.1%) was significantly higher for all samples studied in comparison to data obtained after coincubation with wild-type AAV-infected (mean, 1.4%; SEM, 0.5%) or uninfected CLL cells (mean, 1.3%; SEM, 0.8%; median 2-sample test: P = .03).
CD40L-transduced CLL cells induce a proliferative T-cell response We examined whether AAV/CD40L-infected CLL cells could stimulate allogeneic T cells in an HLA.A2-matched mixed lymphocyte reaction (MLR). For this purpose, we used highly purified allogeneic T cells from HLA.A2+ healthy donors and incubated them with-irradiated CLL cells from HLA.A2+ CLL patients. To
minimize unspecific T-cell stimulatory effects by HeLa/SF-feeder
cocultivation of infected CLL cells, transduced CLL cells were mixed
1:10 with naive bystander cells derived from the same CLL patient and
used as mixture in allogeneic T-cell proliferation assays. On coculture
with AAV/CD40L-infected CLL cells, allogeneic T cells were induced to
undergo proliferation at rates significantly greater (mean, 13 274
cpm; SEM, 3493 cpm) than that observed with CLL cells infected with
AAV/EGFP (mean, 2467 cpm; SEM, 142 cpm; median 2-sample test,
P = .03; Figure 7). The
experiments shown are representative for different allogeneic T-cell
donors and different CLL samples examined.
By use of a novel adenovirus-free packaging method we were able to
generate very high-titer and pure viral preparations of rAAV. The
infectious-to-particle titer achieved is quite favorable in comparison
to other packaging procedures for AAV because eradication of
contaminating helper virus particles is unnecessary.7
Using this purified helper virus-free rAAV, we were able to transduce primary B-CLL cells at low MOIs with high efficiency. In comparison to
adenoviral transgene vectors, rAAV vectors were efficient to transduce
CLL cells at much lower MOIs.3 We have shown that the
transduction potency of AAV vectors is independent of the transgene
used. Differences observed between individual patients are independent
of clinical stage of disease, age, or sex. We were able to provide
evidence that the transduction of CLL cells by rAAV was specific and
not due to a pseusotransduction because viral transduction could be
significantly abrogated by heparin. Because membrane-associated HSPGs
were identified as the primary receptors for AAV virions mediating both
AAV attachment and infection, the blockade of infection by heparin, a
chemical analog of heparan sulfate glycosaminoglycan, is thought to be
specific for this type of vector transduction and has been demonstrated
by others.21,24 After attachment of the virus it is
assumed that AAV is internalized by clathrin-mediated endocytosis
promoted by the interaction of the virus with the
Besides expression of specific receptors on target cells, transduction
efficiency of AAV vectors correlates with the phosphorylation state of
cellular proteins, especially the single-stranded D-sequence-binding protein (ssD-BP). This protein interacts with D( During AAV transduction primary B-CLL cells were incubated on a feeder layer expressing CD40L itself. By this CD40 activation, malignant B cells are shifted in a more proliferative status as has been shown previously.32 Based on cell cycle profile assessment using propidium iodide staining and flow cytometric analysis, Granziero and colleagues demonstrated an increase in the proliferative pool with 0.5% to 5.3% of CLL cells in S phase after 3 days of CD40 stimulation by human soluble CD40L.33 In our preliminary experiments we could show an increase in the fraction of CLL cells in S phase (< 5%) after cultivating them on CD40L-expressing feeder cells (data not shown). Therefore, besides an improved packaging protocol the preactivation of B cells by CD40 cross-linking remains a prerequisite for efficient transgene transduction by AAV. For adenoviral infection of primary CLL cells this CD40 stimulus was shown to improve transduction efficiency.3 A future goal for AAV-mediated transduction of CLL cells will be to replace this time-consuming cellular feeder system; besides AAV-2 other serotypes of AAV will be studied for their B-cell tropism. Serotypes 1, 3, 4, and 5 have been shown to transduce muscle cells more efficiently than recombinant AAV-2 particles.34 Furthermore, packaging of self-complementary rAAVs (scAAVs) could enable a feeder-independent transduction by rAAV. These are AAV vectors with about half the size of wild-type AAV and are therefore preferentially packaged to dimeric molecules, that is, scAAVs contain 2 complementary sequences of the genome in an inverted repeat configuration. Thus the limiting step of conversion of a single-stranded AAV vector to a double-stranded transgene sequence in the target cell is not necessary. In a murine model scAAV particles coding for erythropoietin showed a faster and higher transgene expression in comparison to monomeric AAV particles after intramuscular injection.35 Finally, retargeting of AAV vectors by modification of their capsid proteins could enable an improved transduction of malignant B cells. For recombinant AAV-2 vectors a region I-587 was recently defined at the capsid that allowed insertion of a peptide ligand thus allowing efficient retargeting of AAV vectors to cells with specific integrin receptors.19 Using an immunoglobulin-binding domain at this insertion site, B cell-specific antibodies could redirect via their Fc region AAV particles to B-CLL cells. By infection with viral particles encoding murine CD40L, we modified primary human B-CLL cells to express a functional ligand for CD40. We used murine CD40 ligand because it was shown that recombinant soluble murine CD40L is able to bind human CD40 on B cells and induces a T-cell activation.36 Furthermore, this heterologous ligand could be distinguished from endogenously expressed human CD40L on infected human cells because no cross-reactivity of murine and human antibodies was observed. Therefore, it can be excluded that putatively unspecific stimulation of endogenous human CD40L during AAV infection or cytophilic soluble human CD40L made by the HeLa/SF feeder cells account for false-positive transduction signals. In a clinical phase 1 trial using murine CD40L, no unspecific immune reactions were observed in treated patients. CD40L transduction of B-CLL cells resulted not only in expression of the immune accessory molecule CD80 on infected cells, but also in activation of noninfected bystander leukemia B cells. Similar results with an up-regulation of CD54 and CD86 on leukemic bystander cells were observed previously using an adenoviral transfer system.3 We could show that this bystander effect was specific because incubation with an antibody directed against murine CD40L abrogated the induction of costimulatory molecules. Furthermore, coincubation of bystander cells with mock-infected or untransduced CLL cells did not affect the expression of CD80. Infection with AAV/CD40L significantly improves the functional antigen-presenting capacity of CLL cells in vitro because allogeneic T cells can be induced to proliferate at higher levels in comparison to mock-infected CLL cells. Similar results have also been shown for CLL cells transduced by adenoviral vectors coding for CD40L.3,4 In our experimental setting this T-cell proliferative response is mainly mediated by activated CLL bystander cells that were coincubated with CD40L-transduced CLL cells, thus resembling an in vivo vaccination situation where the number of resident nontransduced leukemia cells would greatly outnumber the infused CD40L-infected CLL cells.4 Having a nonimmunogenic vector transfer system like AAV in hand, it is now possible in future experiments to exclude any unspecific T-cell induction and define CLL-specific cytolytic T-cell responses using putative CLL-associated antigens.37,38 In conclusion, in the present study we have shown that an efficient transduction of primary B-CLL cells with the costimulatory molecule CD40L can be achieved by high-titer, adenovirus-free rAAV vectors. CD40L gene transfer resulted in specific up-regulation of the costimulatory molecule CD80 on infected B-CLL cells and on leukemic bystander cells and induced an allogeneic T-cell response. Therefore transduction of immunostimulatory molecules into CLL cells is now possible with these biologically active preparations of rAAV and enables vaccination strategies with this safe vector system in the near future.
The authors gratefully acknowledge the assistance and help of many colleagues who enabled the preparation of this report: Dr J. Samulski for providing us with the pXX6 plasmid and Dr G. Kröner-Lux for stimulating discussions; Dr A. Girod, F. Gerner, and M. Hutter for their instruction in the initial stage of this study and their support in vector preparations; and S. Anton for her technical assistance. We gratefully acknowledge our colleagues, especially Dr A. Gerl, Dr R. Forstpointner, and Dr M. Mempel, and the nursing staff from the Medical Clinic III at the KGMC who took care of the patients on the wards and in the outpatient clinic.
Submitted May 9, 2001; accepted April 20, 2002.
C.-M.W. and M.H. were supported in part by grants from the Deutsche Forschungsgemeinschaft (We 2346/1-1 and SFB 455) and the Matthias-Lackas-Stiftung.
C.-M.W. and D.M.K. contributed equally to this study.
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.
Reprints: Michael Hallek, Medizinische Klinik III, Klinikum Grosshadern, Ludwig-Maximilians-Universität, Marchioninistr 15, D-81377 München, Germany; e-mail: michael.hallek{at}med3.med.uni-muenchen.de.
1.
Ranheim EA, Kipps TJ.
Activated T cells induce expression of B7/BB1 on normal or leukemic B cells through a CD40-dependent signal.
J Exp Med.
1993;177:925-935 2. Cantwell MJ, Hua T, Pappas J, Kipps TJ. Acquired CD40-ligand deficiency in chronic lymphocytic leukemia. Nat Med. 1997;3:984-989[CrossRef][Medline] [Order article via Infotrieve]. 3. Kato K, Cantwell MJ, Sharma S, Kipps TJ. Gene transfer of CD40-ligand induces autologous immune recognition of chronic lymphocytic leukemia B cells. J Clin Invest. 1998;101:1131-1141.
4.
Wierda G, Cantwell MJ, Woods SJ, Rassenti LZ, Prussak CE, Kipps TJ.
CD40-ligand (CD154) gene therapy for chronic lymphocytic leukemia.
Blood.
2000;96:2917-2924
5.
Cantwell MJ, Sharma S, Friedmann T, Kipps TJ.
Adenovirus vector infection of chronic lymphocytic leukemia B cells.
Blood.
1996;88:4676-4683
6.
Yang R, Nunes FA, Berencsi K.
Cellular immunity to vital antigens limits E1-deleted adenoviruses for gene therapy.
Proc Natl Acad Sci U S A.
1994;91:4407-4411 7. Chiorini JA, Wendtner CM, Urcelay E, Safer B, Hallek M, Kotin RM. High-efficiency transfer of the T cell co-stimulatory molecule B7-2 to lymphoid cells using high-titer recombinant adeno-associated virus vectors. Hum Gene Ther. 1995;6:1531-1541[Medline] [Order article via Infotrieve]. 8. Wendtner C-M, Nolte A, Mangold E, et al. Gene transfer of the costimulatory molecules B7-1 and B7-2 into human multiple myeloma cells by recombinant adeno-associated virus enhances the cytolytic T cell response. Gene Ther. 1997;4:726-735[CrossRef][Medline] [Order article via Infotrieve]. 9. Maass G, Bogedain C, Scheer U, et al. Recombinant adeno-associated virus for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells. Hum Gene Ther. 1998;9:1049-1059[Medline] [Order article via Infotrieve].
10.
Hanazono Y, Brown KE, Handa A, et al.
In vivo marking of rhesus monkey lymphocytes by adeno-associated viral vectors: direct comparison with retroviral vectors.
Blood.
1999;94:2263-2270 11. Rohr UP, Kronenwetter R, Haas R. Transduction efficiencies of primary normal and malignant cells using a recombinant AAV-2 vector depend on cell type and cell cycle [abstract]. Blood. 1999;94:181a.
12.
Xiao X, Li J, Samulski RJ.
Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.
J Virol.
1998;72:2224-2232 13. Grimm D, Kern A, Rittner K, Kleinschmidt JA. Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum Gene Ther. 1998;9:2745-2760[Medline] [Order article via Infotrieve]. 14. Hermens WTJMC, Brake OT, Dijkhuizen PA, et al. Purification of recombinant adeno-associated virus by iodixonal gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system. Hum Gene Ther. 1999;10:1885-1891[CrossRef][Medline] [Order article via Infotrieve].
15.
Cheson B, Bennett JM, Grever M, et al.
National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment.
Blood.
1996;87:4990-4997
16.
Buhmann R, Nolte A, Westhaus D, Emmerich B, Hallek M.
CD40-activated B-cell chronic lymphocytic leukemia cells for tumor immunotherapy: stimulation of allogeneic versus autologous T cells generates different types of effector cells.
Blood.
1999;93:1992-2002
17.
Nathwani AC, Hanawana H, Vandergriff J, Kelly P, Vanin EF, Nienhuis AW.
Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38 18. Samulski RJ, Chang LS, Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol. 1989;61:3096-3101. 19. Girod A, Ried M, Wobus C, et al. Genetic capsid modifications allow efficient retargeting of adeno-associated virus type 2. Nat Med. 1999;5:1052-1056[CrossRef][Medline] [Order article via Infotrieve]. 20. Zolotukhin S, Byrne BJ, Mason E, et al. Utilization of AAV receptor affinity to develop an efficient and novel purification protocol for vector production. Gene Ther. 1999;6:973-985[CrossRef][Medline] [Order article via Infotrieve].
21.
Summerford C, Samulski RJ.
Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.
J Virol.
1998;72:1438-1445
22.
Alexander IE, Russell DW, Miller AD.
DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors.
J Virol.
1994;68:8282-8287
23.
Russell DW, Alexander IE, Miller AD.
DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.
Proc Natl Acad Sci U S A.
1995;92:5719-5723 24. Hacker UT, Gerner FM, Büning H, et al. Standard heparin, low molecular weight heparin, low molecular weight heparinoid, and recombinant hirudin differ in their ability to inhibit transduction by recombinant adeno-associated virus type 2 vectors. Gene Ther. 2001;8:966-968[CrossRef][Medline] [Order article via Infotrieve].
25.
Summerford C, Bartlett JS, Samulski RJ.
26. Qiu J, Brown KE. Integrin alphaVbeta5 is not involved in adeno-associated virus type 2 (AAV2) infection. Virology. 1999;264:436-440[CrossRef][Medline] [Order article via Infotrieve]. 27. Qing K, Mah C, Hansen J, Zhou S, Dwarki V, Srivastava A. Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nat Med. 1999;5:71-77[CrossRef][Medline] [Order article via Infotrieve].
28.
Qing K, Wang XS, Kube DM, et al.
Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression.
Proc Natl Acad Sci U S A.
1997;94:10879-10884
29.
Qing K, Khuntirat B, Mah C, et al.
Adeno-associated virus type 2-mediated gene transfer: correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein with transgene expression in human cells in vitro and murine tissues in vivo.
J Virol.
1998;72:1593-1599
30.
Mah C, Qing K, Khuntirat B, et al.
Adeno-associated virus 2-mediated gene transfer: role of epidermal growth factor receptor protein tyrosine kinase in transgene expression.
J Virol.
1998;72:9835-9843 31. Hansen J, Qing K, Kwon H-J, Mah C, Srivastava A. Impaired intracellular trafficking of adeno-associated virus 2 vectors limits efficient transduction of murine fibroblasts [abstract]. Blood. 1999;94:410b.
32.
Teoh G, Tai Y-T, Urashima M, et al.
CD40 activation mediates p53-dependent cell cycle regulation in human multiple myeloma cell lines.
Blood.
2000;95:1039-1046
33.
Granziero L, Ghia P, Circosta P, et al.
Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia.
Blood.
2001;97:2777-2783 34. Chao HC, Liu Y, Rabinowitz J, Li C, Samulski RJ. Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors. Mol Ther. 2000;2:619-623[CrossRef][Medline] [Order article via Infotrieve]. 35. McCarty DM, Monahan PE, Samulski RJ. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther. 2001;8:1248-1254[CrossRef][Medline] [Order article via Infotrieve].
36.
Lane P, Brocker T, Hubele S, Padovan E, Lanzavecchia A, McConnell F.
Soluble CD40 ligand can replace the normal T cell-derived CD40 ligand signal to B cells in T cell-dependent activation.
J Exp Med.
1993;177:1209-1213 37. Trojan A, Schultze JL, Witzens M, et al. Immunoglobulin framework-derived peptides function as cytotoxic T-cell epitopes commonly expressed in B-cell malignancies. Nat Med. 2000;6:667-672[CrossRef][Medline] [Order article via Infotrieve]. 38. Vonderheide RH, Hahn WC, Schultze JL, Nadler LM. The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity. 1999;10:673-679[CrossRef][Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
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  C. Frecha, C. Costa, C. Levy, D. Negre, S. J. Russell, A. Maisner, G. Salles, K.-W. Peng, F.-L. Cosset, and E. Verhoeyen Efficient and stable transduction of resting B lymphocytes and primary chronic lymphocyte leukemia cells using measles virus gp displaying lentiviral vectors Blood, October 8, 2009; 114(15): 3173 - 3180. [Abstract] [Full Text] [PDF]
|
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C. P. Pallasch, A. Schulz, N. Kutsch, J. Schwamb, S. Hagist, H. Kashkar, A. Ultsch, C. Wickenhauser, M. Hallek, and C.-M. Wendtner Overexpression of TOSO in CLL is triggered by B-cell receptor signaling and associated with progressive disease Blood, November 15, 2008; 112(10): 4213 - 4219. [Abstract] [Full Text] [PDF]
|
|
|
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 |
|
 C. Zuber, G. Mitteregger, N. Schuhmann, C. Rey, S. Knackmuss, W. Rupprecht, U. Reusch, C. Pace, M. Little, H. A. Kretzschmar, et al. Delivery of single-chain antibodies (scFvs) directed against the 37/67 kDa laminin receptor into mice via recombinant adeno-associated viral vectors for prion disease gene therapy J. Gen. Virol., August 1, 2008; 89(8): 2055 - 2061. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Du, G. Zhao, Y. Lin, H. Sui, C. Chan, S. Ma, Y. He, S. Jiang, C. Wu, K.-Y. Yuen, et al. Intranasal Vaccination of Recombinant Adeno-Associated Virus Encoding Receptor-Binding Domain of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Spike Protein Induces Strong Mucosal Immune Responses and Provides Long-Term Protection against SARS-CoV Infection J. Immunol., January 15, 2008; 180(2): 948 - 956. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Palena, K. A. Foon, D. Panicali, A. G. Yafal, J. Chinsangaram, J. W. Hodge, J. Schlom, and K. Y. Tsang Potential approach to immunotherapy of chronic lymphocytic leukemia (CLL): enhanced immunogenicity of CLL cells via infection with vectors encoding for multiple costimulatory molecules Blood, November 15, 2005; 106(10): 3515 - 3523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Mayr, D. M. Kofler, H. Buning, D. Bund, M. Hallek, and C.-M. Wendtner Transduction of CLL cells by CD40 ligand enhances an antigen-specific immune recognition by autologous T cells Blood, November 1, 2005; 106(9): 3223 - 3226. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Jahrsdorfer, J. E. Wooldridge, S. E. Blackwell, C. M. Taylor, T. S. Griffith, B. K. Link, and G. J. Weiner Immunostimulatory oligodeoxynucleotides induce apoptosis of B cell chronic lymphocytic leukemia cells J. Leukoc. Biol., March 1, 2005; 77(3): 378 - 387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Mayr, D. Bund, M. Schlee, A. Moosmann, D. M. Kofler, M. Hallek, and C.-M. Wendtner Fibromodulin as a novel tumor-associated antigen (TAA) in chronic lymphocytic leukemia (CLL), which allows expansion of specific CD8+ autologous T lymphocytes Blood, February 15, 2005; 105(4): 1566 - 1573. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kashiwakura, K. Tamayose, K. Iwabuchi, Y. Hirai, T. Shimada, K. Matsumoto, T. Nakamura, M. Watanabe, K. Oshimi, and H. Daida Hepatocyte Growth Factor Receptor Is a Coreceptor for Adeno-Associated Virus Type 2 Infection J. Virol., January 1, 2005; 79(1): 609 - 614. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hojo, T. Abe, E. Sugano, Y. Yoshioka, Y. Saigo, H. Tomita, R. Wakusawa, and M. Tamai Photoreceptor Protection by Iris Pigment Epithelial Transplantation Transduced with AAV-Mediated Brain-Derived Neurotrophic Factor Gene Invest. Ophthalmol. Vis. Sci., October 1, 2004; 45(10): 3721 - 3726. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. von Bergwelt-Baildon, B. Maecker, J. Schultze, and J. G. Gribben CD40 activation: potential for specific immunotherapy in B-CLL Ann. Onc., June 1, 2004; 15(6): 853 - 857. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, anti-
(Beckman Coulter, Krefeld, Germany). Fluorescein-conjugated mAbs specific for murine CD40L were purchased from BD Pharmingen (Heidelberg, Germany) and expression was controlled by an isotype hamster IgG3 mAb (BD Pharmingen). Heparin
(10 000 U/mL; Braun, Melsungen, Germany) was used for blocking
experiments with infectious AAV.
62 years, and > 62 years),
P > .0125 was found, demonstrating no association between
clinical stage and susceptibility to AAV/EGFP and AAV/CD40L infection, respectively.
5-subunit of 
v
) sequences within the inverted terminal repeat (ITR) of AAV.
subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV.
Gene Ther.
2000;7:183-195