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GENE THERAPY
From the Institute for Genetic Medicine, University of
Southern California Keck School of Medicine; the Department of Adult
Oncology, Dana Farber Cancer Institute; and the Divisions of
Research Immunology/Bone Marrow Transplantation and
Hematology/Oncology, Childrens Hospital Los Angeles.
Cell vaccines engineered to express immunomodulators have shown
feasibility in eliminating leukemia in murine models. Vectors for
efficient gene delivery to primary human leukemia cells are required to
translate this approach to clinical trials. In this study,
second-generation lentiviral vectors derived from human immunodeficiency virus 1 were evaluated, with the cytomegalovirus (CMV)
promoter driving expression of
granulocyte-macrophage-colony-stimulating factor (GM-CSF) and CD80 in
separate vectors or in a bicistronic vector. The vectors were
pseudotyped with vesicular stomatitis virus G glycoprotein and
concentrated to high titers (108-109 infective
particles/mL). Human acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), and chronic myeloid leukemia cell lines
transduced with the monocistronic pHR-CD80 vector or the bicistronic
pHR-GM/CD vector became 75% to 95% CD80 positive (CD80+).
More important, transduction of primary human ALL and AML blasts with
high-titer lentiviral vectors was consistently successful (40%-95%
CD80+). The average amount of GM-CSF secretion by the
leukemia cell lines transduced with the pHR-GM-CSF monocistronic vector
was 2182.9 pg/106 cells per 24 hours. Secretion was
markedly lower with the bicistronic pHR-GM/CD vector (average, 225.7 pg/106 cells per 24 hours). Lower amounts of CMV-driven
messenger RNA were detected with the bicistronic vector, which may
account for its poor expression of GM-CSF. Primary ALL cells transduced
to express CD80 stimulated T-cell proliferation in an autologous mixed
lymphocyte reaction. This stimulation was specifically blocked with
monoclonal antibodies reactive against CD80 or by recombinant cytotoxic
T-lymphocyte antigen 4-immunoglobulin fusion protein. These results
show the feasibility of efficiently transducing primary leukemia cells
with lentiviral vectors to express immunomodulators to elicit
antileukemic immune responses.
(Blood. 2000;96:1317-1326) Two signals are necessary to activate T lymphocytes
and elicit a response against antigens, including those expressed by
tumor cells. The first is the tumor-associated or specific antigen, which is delivered in the context of the major histocompatibility complex to the T-cell receptor.1 The second signal is
the costimulation of the T cell by means of one or more
membrane-bound ligands on the antigen-presenting cells (APC) or
tumor cell. Antigen presentation in the absence of costimulation is
likely to induce anergy. For example, lack of expression of
costimulatory molecules by tumor cells may allow escape from
immune recognition. Once a productive immune response has been
generated, cytotoxic T lymphocytes can detect and destroy
circulating cells presenting the antigen or antigens, whereas memory T
cells can be reactivated by re-exposure to the antigen or
antigens.2
CD80 (B7.1), a member of the B7 family, is a membrane-bound
costimulatory ligand that triggers strong T-cell activation through its
interaction with CD28.3 Human pre-B acute lymphoblastic leukemia (ALL) cells usually lack expression of CD80,4
which may be sufficient for ALL cells to induce alloantigen-specific T-cell unresponsiveness in vitro.5 However, this anergy
can be prevented by stimulation of the ALL cells by means of CD40, which up-regulates CD80 expression of the leukemia cell.5
Granulocyte-macrophage-colony-stimulating factor (GM-CSF) has been
shown to stimulate in vitro growth, maturation, and function of APC
such as dendritic cells and macrophages.6 The increased antitumor immune response observed when transduced tumor cells producing GM-CSF were injected into mice is presumed to be mediated by
the role of GM-CSF in inducing the maturation and function of APC such
as dendritic cells.7
We previously examined the in vivo effects of coexpression of the
immune stimulatory molecules interleukin (IL)-2, GM-CSF, and CD80 by
BM185, a murine transplantable ALL model.8 We found CD80
to be the most potent at inducing antileukemic responses. Furthermore,
we observed that BM185 cells expressing both CD80 and GM-CSF were
consistently more effective at inducing rejection of a high dose of
BM185 cells injected intravenously after vaccination and could induce
rejection of pre-established subcutaneous leukemia.9 This
strong immune reaction is probably the result of the interaction among CD4+ T-helper, CD8+ T-cytotoxic, and
dendritic cells.9 Therefore, transduction of a patient's
leukemia cells to express the combination of CD80 and GM-CSF may be
considered for immunotherapy.
Different types of gene-delivery vectors have been tested in human
leukemia cells, including those based on Moloney murine leukemia
viruses,10,11 herpesviruses,12
adeno-associated viruses,13 and
plasmids.10,14 We showed that lentiviral vectors efficiently transduce human leukemia cells10 and
hematopoietic progenitor cells.15
Lentiviruses are complex retroviruses that infect macrophages and
lymphocytes. The human immunodeficiency virus 1 (HIV-1) is one of these
viruses and is effective in infecting nonproliferating or
growth-arrested cells in vitro.16 One possible mechanism leading to infection of nonreplicating cells is the presence of nuclear
localization signals in the lentiviral preintegration complex that
mediate its active transport through the nucleopores during
interphase.17 Primary leukemia cells show poor
proliferation in vitro,18 which makes them good candidates
for lentiviral vector transduction.
Here, we describe lentiviral constructs expressing CD80 and GM-CSF as
monocistronic and bicistronic vectors. The viruses were packaged after
a tripartite transient transfection procedure and pseudotyped with the
vesicular stomatitis virus G (VSV-G) envelope, which allowed the virus
to be concentrated to high titers.19-21 This report
describes the potential use of these vectors for the delivery and
coexpression of CD80 and GM-CSF genes in primary human leukemia cells
and the autologous T-cell responses stimulated by transduced
CD80-positive (CD80+) ALL cells.
Cell lines
Cell culture
Primary leukemia cells Cryopreserved primary leukemia cells were obtained from cells remaining from diagnostic bone marrow aspirates (with > 70% leukemic blasts) obtained from pediatric patients. The samples were obtained and studies performed in accordance with protocols approved by the Committee on Clinical Investigation of Childrens Hospital Los Angeles and the Dana Farber Cancer Institute. The cells were cryopreserved in 10% dimethyl sulfoxide and 90% FBS. The cells were thawed at 37°C for 1 hour by using a thawing medium containing AIM-V, 30% FBS, 20 U/mL heparin, and 0.2 U/mL DNAase (Roche, Indianapolis, IN). After thawing, primary cells were washed twice with AIM-V medium. Primary ALL cells were cultured in AIM-V medium containing 100 µg/mL CD40L (Immunex, Seattle, WA), and primary AML cells were cultured in AIM-V medium containing 10 ng/mL IL-3 and 50 ng/mL stem cell factor (SCF) (SCF; R&D Systems, Minneapolis, MN).Autologous T cells Peripheral blood mononuclear cells from patients with leukemia were separated by density centrifugation. Cells were then stained with an anti-CD3 monoclonal antibody (mAb; clone UCHT1; Pharmingen, San Diego, CA), and T cells were sorted with a high-speed sorting apparatus (MoFlow; Cytomotion, Denver, CO). The T cells were more than 95% pure. Sorted T cells were used immediately for the mixed lymphocyte reaction (MLR).Construction of pHR-CD80 vector The CD80 complementary DNA (cDNA) was excised from the plasmid LL17722 with EcoRI, and the 900 base-pair fragment was subcloned into the EcoRI site of pIC20H23 to generate the plasmid CD80-pIC20H. The CD80 cDNA was then excised as a BamHI/XhoI fragment and introduced into the backbone of pHR cytomegalovirus (CMV)-neo,19 which had previously been digested with BamHI/XhoI to delete the neor open-reading frame.Construction of pHR-GM-CSF vector The GM-CSF cDNA was excised from plasmid pCSF-124 as an EcoRI fragment and subcloned into the EcoRI site of pIC20H23 generating GM-CSF-20H. This construct was digested with EcoRI and SalI, and the resulting fragment encoding GM-CSF was inserted into the pHR-CD80 backbone previously digested with EcoRI and XhoI to delete the CD80 cDNA.Construction of PHR-GM/CD vector The internal ribosome entry site (IRES) was obtained from plasmid E5m (provided by Dr Kathy Ponder, Washington University, St Louis, MO), which was subcloned as an EcoRI/NcoI fragment into pGEM-7 (Promega, Madison, WI) to generate pGEM-IRES. A NcoI/BamHI fragment encoding CD80 was obtained from plasmid CD80-pIC20H and subcloned into pGEM-IRES generating pGEM-IRES-CD80. The IRES-CD80 cassette was then excised with EcoRI and introduced into the backbone of pHR-CD80 (previously digested with EcoRI to delete CD80), resulting in the construct pHR-IRES-CD80. The GM-CSF gene was obtained as a BamHI/BglII fragment from GM-CSF-20H-2 (a modified version of GM-CSF-20H containing additional cloning sites) and introduced into the BamHI site of pHR-IRES-CD80. All constructs were rechecked by sequencing.Production of lentivirus The packaging construct pCMV- R8.9 encoding HIV-1
gag, pol, tat, and
rev20 was provided by Dr Didier Trono and Dr
Romain Zufferey (University of Geneva, Switzerland). The plasmid
construct driving expression of the VSV-G envelope (pMD.G) and the
vector containing the epidermal growth-factor receptor (EGFR) gene
under control of the CMV promoter (pHR-CMV-EGFP)25 were
provided by Dr Luigi Naldini (University of Torino, Italy). Lentivirus
vectors were produced by transient cotransfection of 293T cells with
the backbone vector plasmid derived from the pHR series, the packaging plasmid pCMV-R8.9, and the plasmid pMD.G encoding the VSV-G
envelope, as described previously.10,19 Viral titer was
determined by assessing expression of the transgene (EGFP or CD80) in
transduced 293 cells by flow cytometry as described
previously.10 Viral p24 antigen concentration was
determined by immunocapture (Coulter Immunotech, Miami, FL). The
minimum detectable p24 concentration for this assay is 3 pg/mL.
Lentivirus-mediated gene transfer Leukemia cells were washed twice with AIM-V and resuspended to obtain a cell density of 1 to 2 × 106 cells/mL. Then, 1 mL of the cell suspension was seeded in each well of 6-well plates and the viral supernatant was added to provide the desired concentration (0.5-1.0 × 107 infective particles/mL). Protamine sulfate was added at the final concentration of 6 µg/mL, and the transduction plates were incubated at 37°C in 5% CO2 for 5 hours. Subsequently, 3 mL of AIM-V medium (plus the corresponding cytokines) was added to each well and the plates were incubated for an additional 16 hours. Leukemia cells were washed twice with AIM-V medium and seeded at a density of 106 cells/mL in AIM-V and cytokines. Twenty-four hours later, the supernatant was collected for enzyme-linked immunosorbent assay (ELISA) and the cells were harvested for fluorescence-activated cell-sorter (FACS) analysis or kept in culture for additional time-point analyses.FACS analysis For analysis of EGFP expression, cells were washed once with phosphate-buffered saline (PBS) and resuspended in 100 µL of 1% paraformaldehyde for fixation. For analysis of expression of CD80, CD19 (B lymphoid marker), and CD33 (myeloid marker), cells were washed once with PBS, blocked with PBS containing mouse IgG (50 µg/mL) for 15 minutes on ice, stained with the corresponding mAb for 20 minutes, washed, and resuspended in 100 µL of 1% paraformaldehyde for fixation. The mAb to CD80 was conjugated with phycoerythrin (Pharmingen), and the mAbs against CD19 and CD33 were conjugated with fluorescein isothiocyanate, conjugated (FITC; Becton Dickinson, San Jose, CA). Detection of CD80, CD19, CD10, or EGFP was accomplished with a FACScan cytometer equipped with a 488-nm argon laser for excitation of the reporter proteins. To establish the background for fluorescence and to set gates for data acquisition, mock-transduced cells were used. Care was taken to analyze cells that were in the lymphoblast gate as indicated by forward- and side-scatter characteristics.Analysis of GM-CSF Expression of GM-CSF was analyzed by ELISA (R&D Systems) conducted according to the manufacturer's instructions. The minimum detectable concentration of GM-CSF after this assay is typically less than 3 pg/mL.Southern and Northern blot analysis Nalm-6 cells were transduced with pHR-GM-CSF, pHR-CD80, or pHR-GM/CD and maintained in culture for 8 days before harvest. Control (mock-transduced) cells were maintained under the same conditions. Cells were washed twice with PBS, and half of the cells were used for extracting DNA and the other half for RNA.Genomic DNA was digested simultaneously with EcoRI and
NcoI and separated on a 0.7% agarose gel in
Tris-acetate-EDTA buffer. The blot was hybridized first to a phosphorus
32 (32P)-labeled DNA fragment encoding human CD80. The
blot was then stripped and subsequently hybridized to a
32P-labeled DNA fragment encoding human GM-CSF. Filters
were washed to a stringency of 0.3 × SSC at 65°C and exposed to
Kodak AR 5 films at Total cellular RNA was extracted with RNA STAT-60 (Tel-test,
Friendswood, TX). Ten-microgram samples of total RNA were separated on
a 1.2% agarose-formaldehyde gel in 3-[N-morpholino] propane sulfonic
acid (MOPS) buffer and transferred to a nylon membrane. The RNA
was transferred to nylon filters and blots were probed with a
32P-labeled 0.9-kilobase (kb) DNA fragment encoding CD80 or
with a 0.6-kb DNA fragment encoding human GM-CSF. Filters were washed to a stringency of 0.3 × SSC and 0.1% sodium dodecyl sulfate at 65°C and exposed to Kodak AR 5 films at Primary and secondary autologous MLR For primary MLR, irradiated (3200 cGy) and nontransduced or transduced primary ALL cells were used as stimulators. For these experiments, ALL cells were transduced without adding exogenous CD40 ligand. ALL cells were cocultured at a 1 to 2 ratio with sorted autologous T cells in 96-well round-bottomed plates containing 105 ALL cells and 2 × 105 T cells in 200 µL of medium (small-scale MLR) or in 6-well plates containing 106 ALL cells and 2 × 106 T cells in 2 mL of medium (large-scale MLR) and incubated for 6 days at 37°C in a 5% CO2 incubator. All MLR cultures used RPMI-1640 supplemented with 4% heat-inactivated human AB serum (NABI, Miami, FL) and 10 U/mL recombinant human IL-2 (National Institutes of Health, Bethesda, MD) for the last 3 days of culture. These stimulator-responder cell ratios and times of incubation were found to represent the optimal culture conditions. All microcultures were performed in triplicate. Cells were pulsed with 0.037 MBq tritium-thymidine (Du Pont, Boston, MA) for the last 18 hours of the culture period and then harvested onto glass-fiber filters. Tritium-thymidine incorporation was measured by liquid scintillation spectrophotometry. Stimulation indexes (SI) were calculated for each individual experiment as SI = cpm (T cells + ALL cells)/cpm (T cells).For the secondary MLR, T cells that were initially primed in the large-scale MLR with either nontransduced or transduced irradiated primary ALL cells were isolated by Ficoll-Hypaque density centrifugation and placed for 12 hours in RPMI-1640 with 4% heat-inactivated human AB serum. Rested, primed T cells were then rechallenged in triplicate with irradiated (3200 cGy) primary nontransduced or transduced ALL cells at stimulator-responder ratios of 1 to 2 in 96-well round-bottomed plates and incubated for 3 days at 37°C in a 5% CO2 incubator. Tritium-thymidine incorporation and stimulation indexes were determined as described above. Blocking assays Blocking experiments were performed by using an anti-CD80 mAb (4B2.C4) at 5 µg/mL or the fusion protein cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig) at 10 µg/mL.5 A control fusion protein and irrelevant mouse IgG were used as negative controls. Antibodies were premixed with T cells before the leukemia cells were added.
Production and titer of lentiviral vectors expressing CD80 and GM-CSF Lentiviral vectors were produced by transient cotransfection of 293T cells with the packaging vector pCMV-D8.9,20 the
VSV-G envelope encoding vector pMD.G,19 and the
corresponding backbone vector pHR-CMV-EGFP,25 pHR-CD80,
pHR-GM-CSF, or PHR-GM/CD (Figure 1).
Supernatant viral titer analysis was done on 293 cells and followed by
FACS analysis as previously described.10 The viral titers
for the crude pHR-CD80 vector ranged from 1.0 to
3.0 × 107 infective particles/mL, whereas the pHR-GM/CD
viral supernatants showed lower titers overall: 0.5 to
3.0 × 106 infective particles/mL. Concentration of the
lentiviral particles by ultracentrifugation increased the titer levels
by 10 fold to 100 fold.
To determine whether the analysis of the CD80 transgene could be used
to assess gene delivery, as was previously done with EGFP,10 Nalm-6 leukemia cells were transduced with
pHR-CMV-EGFP and pHR-CD80 lentiviral vectors, and EGFP expression and
CD80 expression were compared by using flow cytometry analysis (Figure 2). The pHR-CD80 vector produced more
than 90% CD80+ Nalm-6 cells (Figure 2B), comparable to the
number of EGFP-positive cells resulting from the pHR-CMV-EGFP
transduction (Figure 2A). To assess the stability of CD80 expression,
transduced cells were maintained in culture for 10 days and FACS
analysis was repeated. Expression of CD80 remained stable during this
period, thus excluding the possibility that a pseudotransduction
artifact was present. Therefore, CD80 detection in leukemia cells was
used as a marker for gene-delivery efficiency.
Transduction of leukemia cell lines with lentiviral vectors expressing CD80 and GM-CSF The correlation between viral titer and gene delivery was evaluated by titration of pHR-GM/CD lentiviral suspensions (ranging from 105-107 infective particles/mL) in transduction of the pre-B ALL cell line Nalm-6 and the AML cell line ML-1 (Table 1). Transduction efficiency and the level of expression of the transgenes depended on viral titer. At the highest titer evaluated (107 infective particles/mL, equivalent to a multiplicity of infection [MOI] of 10), more than 95% of the ALL and AML cells were transduced. At the 106 titer (MOI of 1), 64.9% of Nalm-6 and 32.6% of ML-1 cells were transduced, whereas at the 105 titer (MOI of 0.1), CD80 expression was at baseline levels. CD80 expression (measured as the mean relative fluorescence of CD80+ cells) and GM-CSF coexpression (measured by ELISA) were also titer dependent. At the highest titer (107 infective particles/mL), GM-CSF production was 862 pg/106 cells per 24 hours and 419 pg/106 cells per 24 hours, respectively, for the Nalm-6 and ML-1 cell lines.
Panels of human lymphoid and myeloid leukemia lines were used for
transduction with pHR-GM-CSF, pHR-CD80, and pHR-GM/CD vectors. Background CD80 and GM-CSF expression values from nontransduced (mock-transduced) cells were subtracted. The CD80 expression by transduced ALL (Nalm-6, Reh, and Sup-B15), AML (AML-5, U937, and ML-1),
and CML (K562) cell lines is shown in Figure
3. We observed high transduction
efficiencies, ranging from 75% to 95%, for both the pHR-CD80 and the
pHR-GM/CD vector.
GM-CSF production by the transduced leukemia cell lines was measured by
ELISA (Figure 4). The pHR-GM-CSF vector
produced an average (± SD) of 2182.9 ± 1287.4 pg/106
cells per 24 hours, whereas the pHR-GM/CD vector produced an average of
225.7 ± 193.1 pg/106 cells per 24 hours. A comparison
between the human pre-B leukemia cell line (Nalm-6) and the human
embryonic kidney fibroblast cell line 293 transduced with the pHR-GM/CD
vector showed that the fibroblasts had GM-CSF production levels that
were 5 to 10 times higher (data not shown).
Molecular analysis of lentivirus integration and messenger RNA (mRNA) expression The integration and mRNA expression of CD80 and GM-CSF genes in transduced Nalm-6 cells were evaluated by Southern and Northern blot analysis, respectively (Figure 5A-B). Nontransduced Nalm-6 cells and Nalm-6 cells transduced with pHR-GM-CSF, pHR-CD80, or pHR-GM/CD vectors were maintained in culture for 8 days after transduction and harvested for DNA and RNA extraction. Genomic DNA was digested with restriction enzymes that cleave the DNA at the flanks of the CD80 and GM-CSF transgenes so that the cDNA integrity could be assessed by Southern blot analysis. The CD80 cDNA (approximately 0.9 kb) and the GM-CSF cDNA (approximately 0.6 kb) were readily detected in the cells transduced with the lentiviral vectors carrying the corresponding genes (Figure 5A). Higher-molecular-weight bands corresponding to fragments of genomic DNA that hybridized with the probes were also detected (Figure 5A).
The mRNA transcripts produced after Nalm-6 transduction with the lentiviral vectors were detected after Northern blotting and hybridization against CD80 or GM-CSF probes (Figure 5B). Two major RNA species were observed for each vector: longer transcripts (generated by transcription from the long terminal repeat [LTR] promoter) and shorter transcripts (generated by transcription from the internal CMV promoter). For the monocistronic mRNAs expressed from the pHR-CD80 and pHR-GM-CSF vectors, the transcripts derived from the CMV promoter were the most abundant RNA species (calculated ratios of CMV-derived transcripts to LTR-derived transcripts were 1.1 and 1.3 fold, respectively). However, reduced levels of mRNA transcribed from the CMV promoter were observed for the bicistronic mRNA expressed from the pHR-GM/CD vector (calculated ratios of CMV-derived transcripts to LTR-derived transcripts resulted in 0.7 fold for the hybridization against CD80 and 0.4 fold for the hybridization against GM-CSF), indicating lower processing, transport, or stability of this transcript. Transduction of fresh primary leukemia cells with pHR-GM/CD promotes high CD80 expression and low GM-CSF secretion Fresh leukemia cells obtained from bone marrow aspirates were transduced with lentiviral vectors (Figure 6). After cell-density separation, the mononuclear fraction was kept overnight in culture. The next day, the nonadherent cells were collected and transduced overnight with the pHR-GM/CD lentiviral vector. CD80 expression and GM-CSF expression were evaluated 48 hours after transduction. During the procedure, ALL and AML cells were maintained in AIM-V medium containing soluble CD40L (ALL cells) or IL-3 and SCF (AML cells). Under these conditions, we obtained good cell viability and more than 90% CD80+ marking in the transduced ALL and AML cells (Figure 6). Secreted GM-CSF levels were low, however, reaching only 11 pg/106 cells per 24 hours for ALL cells, and 137 pg/106 cells per 24 hours for AML cells.
pHR-GM/CD lentiviral vectors efficiently transduce cryopreserved primary ALL and AML cells To evaluate a clinically applicable approach that uses cryopreserved leukemia cells for immunotherapy, transduction of frozen-thawed samples of leukemia cells was performed. We used cryopreserved bone marrow aspirates with a 70% or higher percentage of blasts that were obtained from pediatric patients with ALL or AML at diagnosis or relapse. The phenotypic features of each leukemia sample are shown in Table 2. After thawing, the ALL and AML samples were maintained in AIM-V medium and soluble CD40L (ALL sample) or IL-3 and SCF (AML sample). Under these conditions, we usually obtained 10% to 20% viability for ALL cells and more than 80% viability for AML cells, as assessed by trypan blue exclusion after 24 hours, with or without transduction. Cells were transduced with the pHR-GM/CD vector at a final viral concentration of 1 × 107 infective particles/mL (MOI of 10). CD80 expression and GM-CSF expression were assessed 48 hours after transduction by flow cytometry and ELISA, respectively. The reference cell lines Nalm-6 and ML-1 showed greater than 97% transduction (Figures 7 and 8). The average (± SD) transduction efficiencies for 5 ALL and 5 AML samples from patients were 64.3% ± 23.1% and 77.6% ± 10.0%, respectively (Figures 7 and 8). GM-CSF expression was undetectable in transduced primary ALL cells, reflecting the low GM-CSF expression profile of the pHR-GM/CD vector and the poor viability of the cryopreserved ALL cells in culture. The average production of GM-CSF by transduced primary AML cells was 39.3 ± 36.2 pg/106 cells per 24 hours. Transduction with the lentiviral vectors did not produce signs of cytotoxicity or decreased cell viability during the 48 hours of culture after transduction.
Cotransduction of ALL and AML cells with the pHR-CD80 and pHR-GM-CSF vectors leads to higher levels of GM-CSF production than transduction with the bicistronic pHR-GM/CD vector The low GM-CSF expression levels obtained after transduction of primary leukemia cells with the bicistronic pHR-GM/CD vector prompted us to investigate whether cotransduction of these cells with 2 separate vectors (pHR-CD80 and pHR-GM-CSF) would produce greater GM-CSF production. We therefore produced batches of pHR-CD80, pHR-GM-CSF, and pHR-GM/CD vectors and concentrated the supernatants by using ultracentrifugation. The HIV-1 gag p24 concentrations in the viral preparations were used as a biochemical reference to standardize the amounts of vector used for transductions. Thus, 100 ng/mL of p24 of the pHR-GM/CD bicistronic vector (approximately equivalent to 106 infective particles/mL, a MOI of 1) or a mixture containing 100 ng/mL of p24 of the pHR-CD80 and 100 ng/mL of p24 of the pHR-GM-CSF vectors (MOI of 1 for each) were used for transduction of leukemia cells (Figure 9). Forty-eight hours after transduction, transfer of the CD80 gene was evaluated by FACS.
The presence of CD80+ cells in the transduced ALL cell line (Nalm-6), cryopreserved primary ALL cells (ALL 1), AML cell line (ML-1), and cryopreserved primary AML cells (AML 1) did not differ significantly after cotransduction with pHR-GM-CSF and pHR-CD80 or with pHR-GM/CD (Figure 9A). The relative fluorescence emitted by the CD80+ cells was compared (Figure 9B). In ALL cells, the expression of CD80 after the cotransduction procedure was approximately 2-fold higher than after transduction with the bicistronic vector. The level of CD80 expression by transduced AML cells was lower overall than that by ALL cells, but there was no significant difference between transduction with pHR-CD80 and pHR-GM-CSF and transduction with pHR-GM/CD. Comparison of the GM-CSF production in the 4 cell types studied consistently showed at least a 6-fold higher expression for cotransduced cells than for cells transduced with the bicistronic vector (Figure 9C). Autologous MLR and T-cell proliferation assay We performed functional experiments to evaluate the autologous T-cell response to transduced ALL cells expressing CD80. In the first experiment, we compared the T-cell stimulatory activity of ALL cells that were either nontransduced (mock), transduced to express a control transgene (EGFP), or transduced to express the immunomodulator CD80. Two days after transduction, irradiated (3200 cGy) primary ALL cells were coincubated with sorted autologous T cells in a primary MLR (small- and large-scale MLR). Five days later, the cells incubated in the small-scale MLR were labeled with tritium-thymidine and harvested a day later. The incorporated radioactivity was then measured.The primary MLR showed that ALL-CD80 cells (but not ALL-mock or
ALL-EGFP cells) stimulated significant T-cell proliferation (Figure
10A). The larger primary MLR cultures
were then restimulated with ALL-mock, ALL-EGFP, or ALL-CD80 cells. Five
days after the secondary MLR, the effector cells were labeled with
tritium-thymidine and harvested the next day. T cells stimulated with
ALL-CD80 in the first MLR and restimulated with ALL-mock or ALL-EGFP
cells maintained their proliferative potential, whereas T cells
reprimed with ALL-CD80 cells showed a pronounced increase in their
proliferation (Figure 10B).
In another experiment, which used paired autologous ALL and T cells from a second patient, we evaluated the function of CD80 by performing the MLR in the presence of anti-CD80 blocking antibody or the fusion protein CTLA4-Ig (which binds to CD80, thereby preventing its binding to CD28 on T cells). The findings of the previous MLR were reproduced: autologous T-cell proliferation was promoted by CD80+ ALL cells but not by nontransduced ALL cells (Figure 10C). This stimulatory effect was dramatically blocked by the anti-CD80 antibody or the CTLA4-Ig fusion protein (Figure 10C).
In a murine model, we previously showed that coexpression of CD80 and GM-CSF by a leukemia cell vaccine elicits potent immunoreactivity against pre-established leukemia.9 To translate this finding to a clinical approach, we evaluated various vector systems for reporter gene delivery into human leukemia cells, and we found lentiviral vectors to be the best candidates among those studied.10 Here, we report the use of lentiviral vectors for the efficient delivery of 2 therapeutic genes, CD80 and GM-CSF, into human leukemia cell lines and primary leukemic blasts. As shown in Figure 1, VSV-G-pseudotyped lentiviral vectors expressing CD80 (pHR-CD80) or GM-CSF (pHR-GM-CSF) and a bicistronic vector coexpressing CD80 and GM-CSF (pHR-GM/CD) were efficiently produced by transient cotransfection of 293T cells (106-107 infective particles/mL). After transduction, CD80 gene delivery was followed by immunostaining and flow cytometry. ALL cells showed stable expression of CD80 for at least 10 days after transduction (Figure 2). Transduction of ALL and AML cell lines with different concentrations of pHR-GM/CD vector showed that the expression levels of CD80 and GM-CSF were dose dependent (Table 1). Seven human leukemia cell lines (Nalm-6, Reh, Sup-B15, ML-1, U937, AML-5, and K562) were transduced with the 3 lentiviral vectors, and CD80 expression and GM-CSF expression were assessed by flow cytometry and ELISA, respectively. We observed comparable frequencies of CD80+ cells after transduction with the pHR-CD80 and pHR-GM/CD vectors (70%-95%; Figure 3). However, when we compared GM-CSF production from pHR-GM-CSF-transduced cells with that from pHR-GM/CD-transduced cells, we observed approximately 10-fold lower levels of GM-CSF production by the bicistronic vector (Figure 4). To study this discrepancy, we investigated the integration pattern of the CD80 and GM-CSF cDNAs after transduction of Nalm-6 cells with the different vectors. Southern blot analysis showed that the full-length integrated cDNAs were detectable for all 3 vectors (Figure 5A), excluding the possibility of vector rearrangement. On the other hand, Northern blot analysis of the RNA transcripts expressed by the pHR-GM/CD vector showed lower amounts of the mRNA derived from the internal CMV promoter than of the mRNA derived from the LTR promoter (Figure 5B). This indicated that the bicistronic mRNA transcribed from the CMV promoter was less stable or less efficiently processed and transported to the cytoplasm. The finding of low levels of GM-CSF production by leukemia cells transduced with the bicistronic pHR-GM/CD vector led us to evaluate whether cotransduction with 2 separate vectors, pHR-CD80 and pHR-GM-CSF, would lead to higher GM-CSF levels. This approach showed that the GM-CSF produced by the cotransduced cells reached moderate levels (120-4000 pg/106 cells per 24 hours) that were, on average, 6 times higher than the levels obtained after transduction with the bicistronic vector. The frequency of CD80+ cells and the levels of CD80 expression after the cotransduction approach were comparable or slightly superior than those after transduction with the pHR-GM/CD vector at equal MOIs. It is questionable whether the amount of GM-CSF produced by the cotransduction approach would be sufficient to elicit an antileukemia effect in vivo. To address this issue, a systematic study of GM-CSF dosage as a variable in tumor cell vaccination in a murine melanoma M-3 model was conducted.26 Interestingly, immunity against tumor development was already high at moderate secretion levels (500 pg/106 cells per 24 hours to 10 ng/106 cells per 24 hours) and reached a plateau at the highest obtainable GM-CSF production level (100-2000 ng/106 cells per 24 hours). These results indicate that high doses (> 10 ng/106 cells per 24 hours) of GM-CSF are not critical for generation of antitumor immune responses. On the other hand, dilution experiments that mixed varying numbers of transduced and nontransduced B16-F10 tumor cells showed that GM-CSF secretion below 36 ng/106 cells per 24 hours failed to generate the potent antitumor immunity observed at levels of secretion above this threshold.27 Dilution experiments with our murine BM185-CD80-GM-CSF leukemia model (expressing GM-CSF at the level of 80 ng/106 cells per 24 hours) also showed dose dependence on the ratio of transduced to nontransduced cells used for vaccination.9 Despite the lack of definitive information on this issue, the expression of GM-CSF or GM-CSF in combination with CD80 by the leukemia cells could possibly be optimized by using third-generation self-inactivating vectors (driving mRNA transcription solely from an internal promoter)28 that contain the woodchuck hepatitis virus post-transcriptional regulatory element to enhance RNA stability.29 Despite the low GM-CSF expression observed, we found that the lentiviral vectors consistently led to efficient gene transfer to primary ALL and AML cells without cytotoxic effects (Figures 6-8). These results are in agreement with findings of studies in which lentiviral vectors expressing the reporter gene EGFP were used to transduce ALL leukemia lines and primary ALL cells.10 Our initial reasons for evaluating lentiviral vectors were based on their previous success in transducing nonreplicating hematopoietic progenitor cells.15 Because primary leukemia cells replicate poorly in vitro, they are good candidates for lentiviral transduction. However, as we previously showed, other factors may also be involved, because leukemia cell lines that actively replicate in vitro are also preferentially transduced by lentiviral vectors rather than retroviral vectors.10,11 Vectors derived from herpes simplex viruses have shown a high efficiency of LacZ gene transfer into primary AML and ALL blasts (80%-100%).12 However, these vectors caused some degree of cytotoxicity, and expression declined 1 to 2 days after transduction.12 Transfection of AML blasts with a nonintegrating plasmid vector encoding CD80 showed only transient expression of CD80 on a small fraction of AML cells.14 Thus, lentiviral vectors provide various advantages for production of a leukemia cell vaccine, including (1) consistent gene-delivery efficiency; (2) high levels of transgene expression (as shown here for CD80); (3) persistent expression; (4) no cytotoxic effects; and (5) ease of use (only one transduction cycle is required). Studies in different animal models showed that the induction of CD80 expression on tumor cells is an efficient mechanism to increase their immunogenicity, leading to the rejection of established tumors and eventually conferring protection against tumor rechallenge.30-40 Here, we found that CD80-transduced leukemia cells induced proliferation of autologous leukemia-reactive T cells, which are not activated by nontransduced leukemia cells. The blockade of stimulation by either an anti-CD80 antibody or the fusion protein CTLA4-Ig showed clearly that this effect is mediated by CD80. Clinical protocols for phase I studies of vaccination with autologous irradiated tumor cells engineered to secrete human GM-CSF have been developed.41-44 A phase I clinical trial using vaccination with melanoma cells expressing GM-CSF showed an intense infiltration of T lymphocytes, dendritic cells, macrophages, and eosinophils at the vaccination site and dense infiltration of T lymphocytes and plasma cells in metastatic lesions, associated with cytotoxic T cells and antibody responses.44 Protocols aimed at evaluating the antitumor responses generated from vaccination with autologous tumor cells engineered to express CD80 have also been described.45 To advance to clinical application of lentiviral vectors, more efficient and safer vectors will be needed.20,46-47 Zufferey and colleagues28 produced a self-inactivating lentivirus vector with a 400-nucleotide deletion in the 3' LTR that abolishes the LTR promoter activity and hampers recombination with wild-type HIV-1 in an infected host. This self-inactivating vector diminishes the concern regarding oncogenesis caused by promoter insertion and substantially alleviates the risk of vector mobilization with the wild-type virus, 2 important clinical considerations. In conclusion, lentiviral vectors may be useful tools for immunotherapy of hematologic malignant diseases. Studies to optimize the expression of these vectors, improve their biosafety, and investigate the occurrence of replication-competent lentivirus after transduction will determine their potential use in future clinical trials.
We thank Dr Luigi Naldini, Dr Romain Zufferey, Dr Didier Trono, and Dr Inder M. Verma for providing us with the lentiviral vector system and useful technical advice and Lora W. Barsky and Monika Smogorzewska for technical assistance.
Submitted July 2, 1999; accepted April 17, 2000.
Supported by a translational grant from the Leukemia & Lymphoma Society to D.B.K. (LSA 6211-98) and a grant from the National Institutes of Health (PO1-CA68484) to L.M.N. D.B.K. is a recipient of an Elizabeth Glaser Scientist Award from the Pediatric AIDS Foundation. R.S. is a recipient of a career development fellowship from Childrens Hospital Los Angeles and of a Special Fellow Award from the Leukemia & Lymphoma Society.
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: Donald B. Kohn, Childrens Hospital Los Angeles, Mailstop 62, 4650 Sunset Blvd, Los Angeles CA 90027; e-mail: dkohn{at}chla.usc.edu.
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Top
Abstract
Introduction
70°C.
human hematopoietic cells by HIV-1-based lentiviral vectors.
Proc Natl Acad Sci U S A.
1999;96:2988-2993