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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4591-4601
Adenovirus Vector-Based Purging of Multiple Myeloma Cells
By
Gerrard Teoh,
Ling Chen,
Mitsuyoshi Urashima,
Yu-Tzu Tai,
Leo A. Celi,
Dongshu Chen,
Dharminder Chauhan,
Atsushi Ogata,
Robert W. Finberg,
Iain J. Webb,
Donald W. Kufe, and
Kenneth C. Anderson
From the Department of Adult Oncology, Dana-Farber Cancer Institute,
Boston, MA; and the Department of Haematology, Singapore General
Hospital, Singapore.
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ABSTRACT |
Adenoviruses are efficient gene delivery agents for a variety of
neoplasms. In the present study, we have investigated the use of
adenoviruses for the delivery of the thymidine kinase
(tk) gene into multiple myeloma (MM) cells. We first
demonstrated that MM cell lines and MM patient cells express both
adenovirus receptors as well as the DF3/MUC1 protein, thus providing a
rationale for using adenoviruses to selectively deliver genes under the
control of the DF3 promoter. By using an adenoviral construct
containing  -galactosidase (-gal) gene driven by
the DF3 promoter (Ad.DF3-gal), we demonstrate greater than
80% transduction efficiency in OCI-My5 and RPMI 8226 MM cell lines at
a multiplicity of infection of 1 to 100. Importantly, transduction with
the tk gene driven by the DF3 promoter (Ad.DF3-tk)
followed by treatment with 50 µmol/L ganciclovir (GCV) purged  6
log of contaminating OCI-My5 and RPMI 8226 MM cells within bone marrow
mononuclear cells. In contrast, normal human hematopoietic progenitor
cell number was unaffected under these conditions. Selectivity of
DF3/MUC1 promoter was further confirmed, because Ad.DF3-gal
or Ad.DF3-tk did not transduce MUC1-negative HeLa cervical
carcinoma cells. In addition, GCV treatment of
Ad.DF3-tk-transduced RPMI 8226 MM cells did not induce a
significant bystander effect. These findings demonstrate that transduction with Ad vectors using a tumor-selective promoter provides
a highly efficient and selective approach for the ex vivo purging of MM
cells.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
HIGH-DOSE CHEMORADIOTHERAPY followed by
transplantation of either autologous bone marrow (BM) or peripheral
blood progenitor cells (PBPCs) has achieved high (40%) complete
response (CR) rates in patients with multiple myeloma (MM), but the
median duration of these responses has unfortunately been only 24 to 36 months at best.1,2 Patients with sensitive disease and who
are less heavily pretreated have the most favorable outcomes. Most
importantly, a national French trial of 200 patients with MM has
demonstrated significantly higher response, event-free survival (EFS),
and overall survival (OS) rates for those patients treated with
high-dose treatment, compared with those receiving conventional
therapy.3 This study is encouraging and additional
randomized trials in the United States, Scandinavia, Spain, and England
are also comparing the outcome of conventional therapy versus high-dose
therapy and autografting. However, it is unlikely that any patients
with MM are cured after a single high-dose therapy and stem cell
autografting regimen.
Attempts to improve on the outcome of high-dose therapy followed by
autografting are directed, first, to improve response rates and achieve
minimal residual disease (MRD) with transplant and, second, to develop
strategies for the treatment of MRD posttransplant to achieve prolonged
disease-free survival (DFS) and potential cure. One example of an
attempt to improve outcome after autografting is the use of multiple
high-dose therapies followed by stem cell transplantation. Outcome
appears superior to historically matched controls treated with
conventional therapy,4,5 and the relative efficacy of a
single versus double transplant is being evaluated in a randomized
trial.6 Giralt et al7 have recently reported on
the use of cyclosporine to induce graft-versus-host disease (GVHD) post
autografting in an attempt to generate the associated graft-versus-myeloma effect (GVM) and achieve the second goal, namely
to effectively treat MRD posttransplant.
Mutiple efforts have also been undertaken to obtain tumor-free BM or
PBPC autografts for transplantation. These include depletion of MM
cells from autografts,8-10 selection of normal
hematopoietic progenitor cells (HPCs) within autografts by virtue of
CD34+ expression,11,12 and selection of normal
HPCs using multiparameter cell sorting.13 Monoclonal
antibody (MoAb)-purged BM transplantation can achieve depletion of 2 to
3 log of tumor cells from autografts but few, if any, long-term
disease-free survivors.1,8-10 Although up to 5 log
depletion of MM cells can be achieved using CD34+ selection
of PBPCs, nearly half of CD34+ autografts still contain
residual tumor cells and the clinical impact of CD34+
selection requires further follow-up.11,12,14-16 A
promising highly efficient depletion of tumor cells from autografts can be achieved by transducing PBPCs ex vivo using a vector expressing the
herpes simplex virus (HSV) thymidine kinase (tk) gene,
followed by treatment with ganciclovir (GCV). For example, we have
previously used replication-defective recombinant adenoviral vectors
(Ad) containing the cytomegalovirus (CMV) or DF3/MUC1 promoters to selectively transduce contaminating breast carcinoma cells within autografts with tk gene; subsequent GCV treatment eliminated 6 log of contaminating breast cancer cells.17
In the present study, we demonstrate that MM cell lines and freshly
isolated patient MM cells, but not normal BM mononuclear cells (MNCs),
express adenovirus receptors. Moreover, these MM cells also express
DF3/MUC1, suggesting that selectivity of transgene expression may be
achieved using adenoviral vectors under the control of the DF3/MUC1
promoter. By using an adenoviral construct containing
-galactosidase (-gal) gene driven by the DF3
promoter (Ad.DF3-gal), we demonstrate greater than 80%
transduction efficiency of OCI-My5 and RPMI 8226 MM cells at a
multiplicity of infection (MOI) of 1 to 100. Importantly,
transduction with the tk gene driven by the DF3 promoter
(Ad.DF3-tk) followed by treatment with 50 µmol/L GCV purged
6 log of contaminating OCI-My5 and RPMI 8226 MM cells within BM MNCs,
without adversely affecting normal HPCs. Furthermore, GCV treatment of
AD.DF3-tk-transduced MM cells did not induce a bystander
effect. Our data therefore indicate that Ad.DF3-tk may provide
a highly efficient and selective approach for the ex vivo purging of MM
cells.
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MATERIALS AND METHODS |
Cell lines and transfectants.
The following cell lines were obtained from American Type Culture
Collection (ATCC; Rockville, MD): RPMI 8226 (CCL-155), ARH-77 (CRL-1621), HS Sultan (CRL-1484), and IM-9 (CCL-159) MM cell lines; CESS (TIB-190) Epstein-Barr virus (EBV)-transformed B
cells; HeLa (CCL-2) cervical carcinoma cells; and RD rhabdomyosarcoma
(CCL-136) cells. OCI-My5 MM cells were kindly provided by Dr H.A.
Messner (Ontario Cancer Institute, Toronto, Ontario, Canada); and JY
EBV-transformed B cells by Dr J.D. Fingeroth (Dana-Farber Cancer
Institute, Boston, MA). RD CAR, RD rhabdomyosarcoma cells transfected
with CAR adenovirus receptor cDNA, and RD PCR3, RD cells
transfected with the PCR3 plasmid vector alone that contains the
neomycin resistance (NeoR) gene, were produced by
lipofectamine-mediated gene transfer (GIBCO BRL, Gaithersburg, MD) and
selected with media containing 1 mg/mL of neomycin. Patient MM1 and MM2
cells (>99% CD38+CD45RA ), normal B
splenocytes from a cadaveric donor (T-cell and monocyte depleted), and
BM MNCs from normal donors were obtained by Ficoll-Paque (Pharmacia
Biotech, Uppsala, Sweden) density gradient separation after informed
consent had been obtained.
RPMI 8226, ARH-77, HS Sultan, and IM-9 MM cells; patient MM1 and MM2
cells; normal B splenocytes; CESS and JY EBV-transformed B cells; and
normal BM MNCs were cultured in 90% RPMI-1640 with L-glutamine medium
supplemented with 25 IU/mL penicillin, 25 µg/mL streptomycin, 5 mmol/L L-glutamine (all from GIBCO BRL), and 10% fetal bovine serum
(FBS; Sigma Diagnostics, St Louis, MO). OCI-My5 MM cells and BM MNCs
were cultured in 90% Iscove's modified Dulbecco's medium (Sigma
Diagnostics), 10% FBS, 25 IU/mL penicillin, 25 µg/mL streptomycin,
and 5 mmol/L L-glutamine. HeLa cells were maintained in 90%
Dulbecco's modified Eagle's medium (DMEM) with 4,500 mg/L D-glucose
(GIBCO BRL), 10% FBS, 1 mmol/L MEM sodium pyruvate (GIBCO BRL), 25 IU/mL penicillin, 25 µg/mL streptomycin, and 5 mmol/L L-glutamine. RD
cells were cultured in 90% Eagle's minimum essential medium with
nonessential amino acids and Earle's balanced salt solution (GIBCO
BRL), 10% FBS, 25 IU/mL penicillin, 25 µg/mL streptomycin, and 5 mmol/L L-glutamine. All cells were cultured at 37°C in a humidified
5% CO2 atmosphere. BM MNCs were treated with 2.0 Gy of
 -irradiation before tumor cell contamination and purging assays.
Antibodies for indirect immunofluorescence flow cytometry.
The following antibodies were used in indirect immunofluorescence flow
cytometry: RmcB anti-CAR adenovirus receptor mouse MoAb,18
B5-IA9 anti- v5 integrin MoAb (generously
provided by Dr Martin E. Hemler, Dana-Farber Cancer
Institute),19 DF3 anti-MUC1 MoAb,20 fluorescein
isothiocyanate (FITC)-conjugated goat
(Fab )2 antimouse IgG MoAb (Immunotech, Westbrook,
ME), and FITC-conjugated mouse IgG1 (Coulter Corp, Miami,
FL). Flow cytometric analysis was performed using the Coulter Epics XL
flow cytometer (Coulter Corp).
Specific 35S-Ad2 binding assays.
To confirm specific Ad2 binding, a competitive MoAb blocking assay was
performed by preincubating OCI-My5, RPMI 8226, ARH-77, HS Sultan, and
IM-9 MM cells; patient MM1 cells; normal B splenocytes; CESS and JY
EBV-transformed B cells; normal BM MNCs; HeLa cells (known to express
CAR protein); RD CAR cells (positive control); and RD PCR3 (negative
control) cells (1 × 106) with RmcB anti-CAR blocking
MoAb (1:100 dilution) at 4°C for 1 hour. Similarly, preincubation
with 5E2B4 mouse MoAb (IgG2; 1:100 dilution), which targets
a nonsurface protein, was used as an isotype control. Cells were next
incubated in triplicate with 20,000 cpm of 35S-Ad2
radiolabeled adenovirus18 in 200 µL of Hank's Balanced Salt Solution (HBSS; GIBCO BRL) at room temperature for 1 hour. Excess
MoAb was then washed off thrice with HBSS and cells were solubilized in
500 µL of SOLVABLE (DuPont NEN, Boston, MA). Ten milliliters of
Formula 989 (DuPont NEN) was added, and 35S-Ad2 binding was
analyzed on the LS 6000SC beta-scintillation counter (Beckman
Instruments, Inc, Fullerton, CA).
Adenovirus transduction.
Ad.DF3-gal and Ad.DF3-tk are Ad vectors in which the
-gal and tk genes, respectively, are under control
of the DF3/MUC1 tumor-selective promoter.21-23
Ad.CMV-gal and Ad.CMV-tk are vectors in which the
same genes are under control of the CMV immediate-early promoter and
enhancer24 (kindly provided by Dr Robert Gerard, University
of Texas, Austin, TX). Cells were transduced with adenovirus at MOI of
1 to 100 for 2 hours, washed out for 10 hours, resuspended in fresh
media, and evaluated for the expression of the transgene at 24 to 48 hours after transduction.
-Galalactosidase activity assays.
Expression of the transgene was assessed by -gal activity using
fluorescence-activated flow cytometry, as previously
described.25 Briefly, 0.5 to 1.0 × 106
cells were suspended in 50 µL of serum-free culture media at 37°C. An equal volume of 2 mmol/L fluorescein
di--D-galactopyranoside (FDG; Molecular Probes, Eugene, OR) was
added, rapidly mixed, and incubated at 37°C for 1 minute. The cells
were next washed once with 4 mL of ice-cold phosphate-buffered saline
(PBS) and maintained in ice-cold PBS until analysis. The -gal
activity of transduced cells was also determined using a
chemiluminescence assay (Galacto-Light system; Tropix, Inc, Bedford,
MA) that detects 2 fg to 20 ng of -galactosidase26 and
by 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal; GIBCO BRL)
staining. For X-Gal staining, tumor cells were washed three times in
PBS; fixed in 0.5% glutaldehyde; washed a further three times with
PBS/2 mmol/L MgCl2; stained at 37°C overnight with 2 mg/mL X-Gal, 5 mmol/L K3Fe(CN)6 and 5 mmol/L K4 Fe(CN)6·3H2O in PBS/2 mmol/L
MgCl2; and counted by light microscopy (200 cells/sample;
n = 3).
Cell proliferation assay.
MM cell line proliferation assays were used to define residual viable
tumor cells remaining after treatment of Ad.DF3-tk- and
Ad.CMV-tk-transduced MM cells within normal BM MNCs with
varying concentrations (0 to 50 µmol/L) of GCV (CYTOVENE;
Syntex Laboratories, Inc, Palo Alto, CA). After GCV treatment,
transduced tumor cells alone or tumor cells mixed with -irradiated
normal BM MNCs were incubated in triplicate in 96-well tissue culture
plates with 1 µCi of tritiated thymidine (3H-TdR; DuPont
NEN) for 12 hours, harvested onto glass filters using the HARVESTAR 96 MACH II harvester (Tomtec Inc, Orange, CT), and analyzed on the 1205 BETAPLATE beta-counter (Wallac, Gaithersburg, MD). Viable cell density
was determined by trypan blue exclusion.
Human hematopoietic progenitor cell assays.
Colony-forming units-granulocyte-macrophage (CFU-GM) and burst-forming
units-erythroid (BFU-E) were analyzed by plating BM MNCs (2 × 105 cells/well) obtained after Ficoll-Paque density
gradient separation in Iscove's methylcellulose medium (MethoCult GF
H4434; Stem Cell Technologies, Vancouver, British Columbia, Canada)
containing recombinant human stem cell factor (rhSCF; 50 ng/mL),
recombinant human granulocyte-macrophage colony-stimulating factor
(rhGM-CSF; 10 ng/mL), recombinant human interleukin-3 (rhIL-3; 10 ng/mL), and recombinant human erythropoietin (rhEPO; 3 U/mL).27 CFU-GM (40 cells) and BFU-E (3 erythroid
clusters) colonies were enumerated and classified on the basis of
morphology at 14 days of culture using indirect microscopy.
Polymerase chain reaction (PCR) detection of Ad5 E2B gene.
PCR of Ad5 E2B gene was used to detect transduced cells. Cells
were washed three times in PBS, resuspended in 0.2 mL of TE buffer (10 mmol/L Tris pH 8.0, 1 mmol/L EDTA), lysed by freeze-thaw (×2),
and centrifuged to remove cellular DNA and debris. The supernatant containing viral DNA was incubated with 100 mmol/L Tris, pH 8.0, 0.5%
sodium dodecyl sulphate, and 200 µg/mL proteinase K (Sigma Diagnostics) at 50°C for 2 hours; and viral DNA was obtained by phenol/chloroform (GIBCO BRL) extraction followed by 100%
ethanol/sodium acetate (Sigma Diagnostics) precipitation.28
The E2B gene was amplified using the GeneAmp PCR reagent kit
and Perkin-Elmer thermal cycler (Applied Biosystems Corp, Foster City,
CA) under the following conditions: 94°C for 5 minutes, 94°C
for 30 seconds (denaturation), 56°C for 30 seconds (annealing), and
72°C for 1 minute (extension) for 25 cycles; and 72°C for 10 minutes.17,28 The primers used for Ad5 E2B gene
were 5-TCGTTTCTCAGCAGCTGTTG-3 (forward) and 5-CATCTGAACTCAAAGCGTGG-3 (reverse).28
-actin (control) gene was amplified using the following
primers: 5-CAGCCATGTACGTTGCTATCCAG-3 (forward) and
5-GTTTCGTGGATGCCACAGGAC-3 (reverse).
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RESULTS |
Expression of CAR adenovirus receptor on HeLa cells; OCI-My5, RPMI
8226, ARH-77, HS Sultan, and IM-9 MM cells; patient MM1 cells; normal B
splenocytes; CESS and JY EBV-transformed B cells; and normal BM MNCs.
To explore the feasibility of using adenoviral vector transduction of
MM cells with the tk gene followed by GCV treatment for purging
MM cells, we first characterized the expression of adenoviral receptors
on MM cells by indirect immunofluorescence flow cytometric analysis
with RmcB anti-CAR adenoviral receptor MoAb
(Fig 1).18 In this assay, RD
PCR3 rhabdomyosarcoma cells transfected with vector alone served as a
negative control (Fig 1A). RD rhabdomyosarcoma cells transduced with
CAR adenovirus receptor gene (Fig 1B) and HeLa cells (Fig 1C),
which are known to express adenoviral receptors, served as positive
controls. Reactivity was observed on 5 MM cell lines (OCI-My5, RPMI
8226, ARH-77, HS Sultan, and IM-9; Fig 1D through H) and patient MM 1cells (Fig 1I) but absent on normal B splenocytes (Fig 1J), CESS and
JY EBV-transformed B cells (Fig 1K and L), and normal BM MNCs (Fig 1M).
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| Fig 1.
Expression of CAR adenovirus receptor on HeLa cells;
OCI-My5, RPMI 8226, ARH-77, HS Sultan, and IM-9 MM cells; patient MM1
cells; normal B splenocytes; CESS and JY EBV-transformed B cells; and
normal BM MNCs. Cells (5 × 105/mL) were stained with RmcB
anti-CAR MoAb (1:100) followed by 1 µL of FITC-conjugated goat
(Fab)2 antimouse IgG MoAb, or with 5 µL of
FITC-conjugated mouse IgG1 (isotype control), and analyzed
by indirect immunofluorescence flow cytometry. Cells examined included
RD PCR3 (A; negative control) and RD CAR (B; positive control) cells;
HeLa (C) cells, known to express adenovirus receptors; OCI-My5 (D),
RPMI 8226 (E), ARH-77 (F), HS Sultan (G), and IM-9 (H) MM cells;
patient MM1 (I) cells; normal B splenocytes (J); CESS (K) and JY (L)
EBV-transformed B cells; and normal BM MNCs (M).
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Specific 35S-Ad2 binding to HeLa cells; to OCI-My5, RPMI
8226, ARH-77, HS Sultan, and IM-9 MM cells; to patient MM1 cells; to
normal B splenocytes; to CESS and JY EBV-transformed B cells; and to
normal BM MNCs.
To confirm the presence of adenovirus receptors on MM cells and to
compare the relative affinity of viral binding to MM cells versus cells
of other lineages, we performed assays of specific 35S-Ad2
binding to tumor cells relative to HeLa cells
(Fig 2). Preincubation of tumor cells with
RmcB anti-CAR adenovirus receptor blocking MoAb defined the levels of
nonspecific Ad2 binding; and preincubation with 5E2B4 mouse MoAb
(IgG2) was used as an unreactive isotype control. RD CAR
(184% specific HeLa binding) and RD PCR3 (0% specific HeLa binding)
transfectants served as positive and negative controls for total Ad2
binding, respectively. MM cell lines (OCI-My5, RPMI 8226, ARH-77, HS
Sultan, and IM-9) and patient MM1 cells demonstrated 25% to 100%
specific adenoviral binding, compared with HeLa cells (100% specific
Ad2 binding). In contrast, normal B splenocytes, CESS and JY
EBV-transformed B cells, and normal BM MNCs did not exhibit significant
binding of adenovirus.
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| Fig 2.
Specific 35S-Ad2 binding to HeLa cells; to
OCI-My5, RPMI 8226, ARH-77, HS Sultan, and IM-9 MM cells; to patient
MM1 cells; to normal B splenocytes; to CESS and JY EBV-transformed B
cells; and to normal BM MNCs. HeLa cells; OCI-My5, RPMI 8226, ARH-77,
HS Sultan, and IM-9 MM cells; patient MM1 cells; normal B splenocytes;
CESS and JY EBV-transformed B cells; and normal BM MNCs (1 × 106) were incubated with either RmcB anti-CAR MoAb (1:100)
or 5E2B4 mouse MoAb (IgG2; 1:100; isotype control),
followed by incubation with 35S-labeled Ad2, and then
washed, solubilized, and analyzed on a -counter. The percentage of
specific Ad2 binding (difference in Ad2 binding by 5E2B4-treated versus
RmcB-treated cells) relative to control HeLa cells (100% specific Ad2
binding) was determined. RD PCR3 and RD CAR transfectants served as
negative and positive controls, respectively.
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Expression of v5 integrin and DF3/MUC1
on OCI-My5 and RPMI 8226 MM cells as well as patient MM1 cells.
Because internalization of adenovirus into mammalian cells requires
viral binding to v5
integrin29,30 and all 5 integrin subunits
are associated with v subunits,19 we assayed
v5 integrin expression on CAR adenovirus
receptor-positive OCI-My5 and RPMI 8226 MM cells, as well as patient
MM1 cells, using B5-IA9 anti-v5
MoAb19 in indirect immunofluorescence flow cytometry. Both
MM cell lines and patient MM1 cells strongly expressed (85% to 98%)
v5 integrin
(Fig 3A). Because prior reports have shown that MM cell lines and patient MM cells express
DF3/MUC1,31,32 we examined our MM cell lines and patient
MM1 cells for DF3/MUC1 expression (Fig 3B). Indirect immunofluorescence
flow cytometric analysis confirmed strong (77% to 95%) DF3/MUC1
expression on MM cell lines and patient MM1 cells. Collectively, these
findings suggest that DF3/MUC1 adenoviral vector-based therapies may
offer the advantage of tumor cell selectivity.
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| Fig 3.
Expression of v5 integrin
and DF3/MUC1 on OCI-My5 and RPMI 8226 MM cells and on patient MM1
cells. OCI-My5 and RPMI 8226 MM cells and patient MM1 cells (5 × 105/mL) were stained with 1 µL of either
v5 MoAb (A) or anti-DF3/MUC1 MoAb (B),
followed by 1 µL of FITC-conjugated goat (Fab)2
antimouse IgG MoAb, or with 5 µL of FITC-conjugated mouse
IgG1 alone (isotype control). The percentage of cells
expressing these antigens was determined using indirect
immunofluorescence flow cytometric analysis.
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Efficiency of Ad vector transduction assessed by -gal reporter
gene expression in OCI-My5 and RPMI 8226 MM cells as well as RPMI 8226 MM cells within normal BM MNCs.
We next determined the efficiency of adenovirus vector-mediated
reporter gene expression in MM cells using Ad.DF3-gal vector (Fig 4A). -gal activity was observed in
80% to 100% of OCI-My5 and RPMI 8226 MM cells transduced with
Ad.DF3-gal at MOI = 1. To determine whether transduction
using Ad vectors under the control of the DF3/MUC1 promoter may augment
transduction efficiency for tumor cells, we compared the relative
transduction efficiencies using Ad.CMV-gal (nonspecific CMV
promoter) or Ad.DF3-gal (Fig 4B). Transduction efficiency,
reflected in -gal activity, was similar in OCI-My5 and RPMI 8226 MM
cells using either promoter.
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| Fig 4.
Efficiency of Ad vector transduction assessed by
-gal reporter gene expression in OCI-My5 and RPMI 8226 MM
cells, as well as RPMI 8226 MM cells within normal BM MNCs. OCI-My5 and
RPMI 8226 MM cells (5 × 105/mL) were transduced with
Ad.DF3-gal at MOI = 1 for 2 hours, incubated with FDG, and
analyzed by fluorescence flow cytometry at 24 hours (A). These tumor
cells were also transduced with Ad.DF3-gal ( ) or
Ad.CMV-gal ( ) at MOI = 10 for 2 hours and analyzed by
chemiluminescence assay at 24 hours (B). Mock-irradiated () and
-irradiated (; 2.0 Gy) normal BM MNCs (3 × 104/well) were cultured in media alone or transduced with
either Ad.CMV-gal or Ad.DF3-gal (both MOI = 100) for 2 hours and then analyzed by X-Gal staining at 24 hours (n = 3). X-Gal staining of mock-irradiated and -irradiated BM MNCs was
compared with untransduced normal BM MNCs (negative control) and RPMI
8226 MM cells (positive control; C). RPMI 8226 MM cells (0.0%,
0.0001%, 0.001%, and 0.01%) mixed with normal BM MNCs were
transduced with Ad.DF3-gal at MOI = 1 for 2 hours and
similarly analyzed using a chemiluminescence assay at 24 hours (n = 3; D).
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We next mixed Ad.DF3-gal-transduced RPMI 8226 MM cells
(0.01% to 0.0001%) within -irradiated normal BM MNCs to assess the level of detectability by chemiluminescence. Our previous studies showed that both BM and PB MNCs are not transduced by
Ad.DF3-gal.17 Moreover, -irradiation (2.0 Gy)
of BM MNCs before incubation with Ad.CMV-gal or
Ad.DF3-gal at MOI = 100 does not alter their tranducibility,
evidenced by lack of X-Gal staining (Fig 4C). As seen in Fig 4D,
0.0001% tumor cell contamination within BM MNCs is detectable by
-gal activity.
Treatment with GCV to purge Ad.DF3-tk-transduced OCI-My5 and RPMI
8226 MM cells within BM MNCs.
Having demonstrated high transduction efficiency of adenoviral vectors
in MM cells, we next examined the efficiency of purging of MM cells
within BM MNCs using tk gene transduction followed by GCV
treatment. Proliferation of both OCI-My5
(Fig 5A) and RPMI 8226 (Fig 5B) MM cells
was correlated with cell number up to 1 × 106
cells/mL. Moreover, proliferation of control (nontransduced) OCI-My5,
RPMI 8226, CESS, and JY (Fig 5C through F) cells was unaffected by
treatment with GCV alone. Therefore, proliferation was used as a
measure of residual viable tumor cells after tk transduction
and GCV treatment. Tumor cells (2 × 105) were mixed
with 2 × 106 -irradiated normal BM MNCs and
transduced with either Ad.DF3-tk (MOI = 1, 10, or
100) or with Ad.CMV-tk (MOI = 10) for 2 hours and washed out
for 10 hours, followed by GCV (0 to 50 µmol/L) treatment for 36 hours. The decrease in 3H-TdR incorporation observed after
GCV (50 µmol/L) treatment of Ad.DF3-tk-transduced (MOI = 100) OCI-My5 (Fig 5C) and RPMI 8226 (Fig 5D) MM cells indicates a 6
log depletion of these MM cell lines (Fig 5G and H, respectively). As
shown in Fig 5C and D, transduction with Ad.DF3-tk at MOI = 100 (6 to 7 log depletion) was associated with more efficient purging of MM
cells than at MOI = 1 (1 to 2 log depletion) at similar GCV
concentrations. In addition, purging efficiency of Ad.CMV-tk at
MOI = 10 was comparable to that of Ad.DF3-tk at MOI = 10. CESS
and JY (Fig 5E and F) cells were similarly mixed with BM MNCs and
treated with Ad.DF3-tk or Ad.CMV-tk, followed by GCV.
However, neither CESS nor JY EBV-transformed B cells were depleted from
BM MNCs by this treatment. These data demonstrate that
Ad.DF3-tk transduction of MM cells followed by treatment with
GCV is effective in selectively eradicating 6 to 7 log of contaminating
tumor cells.
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| Fig 5.
Treatment with GCV to purge
Ad.DF3-tk-transduced OCI-My5 and RPMI 8226 MM cells within BM
MNCs. To construct standard curves for measuring residual viable tumor
cell contamination after purging, OCI-My5 cells (A) or RPMI 8226 MM
cells (B; 0 to 1 × 106 cells) were incubated in
triplicate with 3H-TdR (1 µCi) for 12 hours, harvested,
and analyzed on a -counter. OCI-My5 (C) and RPMI 8226 MM cells (D; 2 × 105 cells) were mixed with -irradiated (2.0 Gy)
normal BM MNCs (2 × 106 cells). BM MNCs containing tumor
cells were transduced with Ad.DF3-tk at MOI = 1, MOI = 10, or MOI = 100 or with Ad.CMV-tk at MOI = 10 for 2 hours,
washed out for 10 hours, and treated with GCV at 0 (), 0.5 ( ), 5 ( ), or 50 () µmol/L for 36 hours. 3H-TdR
incorporation was measured as described above and compared with that
for nontransfected tumor cell in BM MNC controls. CESS (E) and JY (F)
EBV-transformed B cells, which do not express adenoviral receptors,
served as negative controls. All experiments were performed in
triplicate. Log depeletion of OCI-My5 (G) and RPMI 8226 (H) MM cells
with Ad.DF3-tk at MOI = 100 and 50 µmol/L GCV is shown.
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Effect of Ad.DF3-tk transduction and GCV treatment on the
growth of normal human hematopoietic progenitor cells.
To assess the effects of Ad.DF3-tk transduction and GCV
treatment on the growth of normal HPCs, BM MNCs were cultured with Ad.DF3-tk (MOI = 10) and/or GCV (50 µmol/L) and
CFU-GM and BFU-E colonies were enumerated
(Table 1). CFU-GM and BFU-E growth was unaffected relative to untreated control BM MNC cultures in media alone. Untreated RPMI 8226 MM cells (1%) mixed within normal BM MNCs
formed a monolayer and overgrew HPC colonies. In contrast, transduction
of BM MNCs containing 1% RPMI 8226 cells with Ad.DF3-tk, followed by GCV treatment, eradicated all detectable contaminating tumor cells (5 to 6 logs); importantly, this treatment does not affect
growth of normal CFU-GM and BFU-E.
View this table:
[in this window]
[in a new window]
|
Table 1.
Effect of Ad.DF3-tk Transduction and GCV
Treatment on the Growth of Normal Human Hematopoietic Progenitor
Cells
|
|
Detection of Ad5 E2B adenoviral DNA in Ad.DF3-tk
transduced RPMI 8226 MM cells, patient MM2 cells, and normal BM MNCs
before and after treatment with GCV.
To further assess the potential efficacy and selectivity of using Ad
vectors for purging MM cells within BM MNCs, we transduced RPMI 8226 MM
cells (Fig 6A) and patient MM2 cells (Fig
6B) within normal BM MNCs (1 × 104 /sample) with
Ad.DF3-tk (MOI = 100) and then treated with GCV (50 µmol/L).
The presence of E2B adenoviral DNA in each sample was assessed
by PCR, as in previous studies.17,28 Only RPMI 8226 MM
cells (Fig 6A) and patient MM2 cells (Fig 6B) transduced with
Ad.DF3-tk were positive for the E2B PCR transcript
(lanes 3 and 4), whereas untransduced MM cells (lanes 1 and 2), as well as both untransduced and transduced normal BM MNCs (lanes 9 through 12)
were E2B PCR-negative. Moreover, GCV treatment of
Ad.DF3-tk-transduced tumor cells (lane 4) was associated with
decreased PCR product, compared with Ad.DF3-tk-transduced
tumor cells that were not treated with GCV (lane 3). The pattern of
PCR-positivity in samples of RPMI 8226 MM cells (Fig 6A) and patient
MM2 cells (Fig 6B) within normal BM MNCs (lanes 5 through 8) was
similar to that observed for samples containing RPMI 8226 MM cells (Fig
6A) or patient MM2 cells (Fig 6B) alone (lanes 1 through 4), consistent
with the above-noted observation that tumor cells, but not normal BM MNCs, are transduced by Ad.DF3-tk (MOI = 100). Viral DNA
obtained from Ad.DF3-tk particles (1 × 103 to
1 × 105 plaque-forming units
[PFU]) served as a positive control for E2B PCR (lanes 13 through 15). -actin
confirmed integrity of DNA.
View larger version (41K):
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| Fig 6.
Detection of Ad5 E2B adenoviral DNA in RPMI 8226 MM cells, patient MM2 cells, and normal BM MNCS transduced with
Ad.DF3-tk and then treated with GCV. RPMI 8226 MM cells (A) or
patient MM2 cells (B; 1 × 103/sample; lanes 1 through 4);
RPMI 8226 MM cells (A) or patient MM2 cells (B; 1 × 103/sample) mixed with normal BM MNCs (1 × 104/sample; lanes 5 through 8); or normal BM MNCs (1 × 104/sample; lanes 9 through 12) were either nontransduced
(lanes 1, 2, 5, 6, 9, and 10) or transduced with Ad.DF3-tk (MOI
= 100; lanes 3, 4, 7, 8, 11, and 12) for 2 hours and then washed out
for 10 hours. Cells were next cultured with GCV (50 µmol/L; lanes 2, 4, 6, 8, 10, and 12) or without GCV (lanes 1, 3, 5, 7, 9, and 11) for
36 hours. The E2B gene was amplified by PCR, as previously
reported,17,28 from viral DNA extracted following cell
lysis. Viral DNA obtained from Ad.DF3-tk particles (1 × 103 to 1 × 105 PFU) served as a positive
control (lanes 13 through 15). PCR for -actin confirmed
integrity of DNA.
|
|
Bystander effect after GCV treatment of tk-transduced RPMI
8226 MM cells.
Although GCV-induced killing of tk-transduced cells may extend
to proximate untransduced cells (ie, a bystander effect),33 this effect has not been observed in lymphoid cells.34
Nonetheless, we next determined whether GCV treatment of
tk-transduced RPMI 8226 MM cells induced a bystander effect.
GCV treatment of tk-transduced HeLa cells was used as a
positive control for the induction of the bystander
effect.34 As can be seen in Fig
7, the percentage of RPMI 8226 MM cells killed by transduction with
Ad.CMV-tk or Ad.DF3-tk at MOI = 1 to 100 followed by
treatment with GCV cells is equivalent (P > .5) to the
percentage of RPMI 8226 MM cells staining for X-Gal after transduction
with Ad.CMV-gal or Ad.DF3-gal, suggesting the
absence of a bystander effect in these tumor cells. Moreover, there was
no significant difference (P > .5) in X-Gal staining or
killing of RPMI 8226 MM cells transduced using CMV versus DF3
promoters. In contrast, the percentage of HeLa cells killed after
transduction with Ad.CMV-tk at MOI = 1 to 100 followed by
treatment with GCV was significantly greater (P < .001) than the percentage of HeLa cells transduced with Ad.CMV-gal,
confirming the presence of a bystander effect, as previously
reported.34
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| Fig 7.
Bystander effect after GCV treatment of
tk-transduced RPMI 8226 MM cells. RPMI 8226 MM cells (3 × 104/well) were transduced with Ad.CMV-gal or
Ad.CMV-tk ( ) or with Ad.DF3-gal or
Ad.DF3-tk ( ) at MOI = 0, 1, 5, 10, 50, and 100 for 2 hours. Cells were washed out for 10 hours, and tk-transduced
tumor cells were then treated with GCV (50 µmol/L) for 36 hours. The
bystander effect was determined by comparing the percentage of
transduced cells after Ad.CMV-gal and Ad.DF3-gal
transduction, assessed by X-Gal staining, with the percentage of cells
killed after GCV treatment of Ad.CMV-tk- and
Ad.DF3-tk-transduced cells, assessed by trypan blue exclusion.
HeLa cervical carcinoma cells ( ), known to demonstrate the bystander
effect,34 were similarly transduced with
Ad.CMV-gal or Ad.CMV-tk and served as positive
controls.
|
|
Selectivity of the DF3 promoter in RPMI 8226 MM cells.
Because RPMI 8226 MM cells express DF3/MUC1 and HeLa cervical carcinoma
cells lack expression of the major polymorphic MUC1/REP protein,35 we next examined the pattern of transduction of
these tumor cells using the CMV and DF3 promoters. There was no
significant difference (P > .5) in X-Gal staining of RPMI
8226 MM cells transduced with Ad.CMV-gal versus
Ad.DF3-gal at MOI = 1 to 100 (Fig 8A). In contrast, a highly significant
(P < .0001) difference in X-Gal staining was observed in HeLa
cells transduced with CMV versus DF3 promoters at MOI = 1 to 100 (Fig
8B). Importantly, no increase in X-Gal staining was observed in HeLa
cells transduced with Ad.DF3-gal at MOI = 1 to 100 (Fig 8B).
View larger version (32K):
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| Fig 8.
Specificity of the DF3 promoter for RPMI 8226 MM cells.
DF3-positive RPMI 8226 MM and DF3-negative HeLa cervical carcinoma
cells (3 × 104/well) were transduced with
Ad.CMV-gal (A and B; ) and Ad.CMV-tk (C and D,
) or with Ad.DF3-gal (A and B, ) and Ad.DF3-tk
(C and D, ) at MOI = 0, 1, 5, 10, 50, and 100 for 2 hours. Cells
were washed out for 10 hours and tk-transduced tumor cells were
then treated with GCV (50 µmol/L) for 36 hours. Comparison of the
transduction efficiency (ie, X-Gal staining after transduction with
Ad.CMV-gal or Ad.DF3-gal) and GCV killing of
tk-transduced tumor cells (ie, viable cells by trypan blue
exclusion after transduction with Ad.CMV-tk and
Ad.DF3-tk followed by GCV treatment) was compared for Ad.CMV
versus Ad.DF3 promoters.
|
|
There was also no significant difference (P > .5) in the
percentage of killing of RPMI 8226 MM cells transduced with
Ad.CMV-tk and Ad.DF3-tk at MOI = 1 to 100, followed by
treatment with GCV (Fig 8C). In contrast, killing after GCV treatment
of Ad.CMV-tk-transduced HeLa cells was significantly
greater (P < .0001) than observed after GCV treatment of
Ad.DF3-tk-transduced HeLa cells (Fig 8D). Indeed, GCV
treatment resulted in equivalent low-level killing of untransduced HeLa
cells versus HeLa cells transduced with Ad.DF3-tk at MOI = 1 to
100 (Fig 8D), further supporting selectivity of the DF3 promoter for
DF3-positive target cells.
| |
DISCUSSION |
Multiple studies have used transfection or transduction of the
tk gene into target cells followed by treatment with GCV as a
strategy for their selective depletion. In the context of allogeneic transplantation, for example, tk gene transduction into T cells or T lymphocyte subsets offers the possibility of abrogating GVHD in
murine models as well as in humans.34,36 In MM, Dilber et al37 have used a replication deficient retroviral vector,
containing the bacterial NeoR and HSV-tk fusion gene,
to transduce ARH-77 and RPMI 8226 MM cells. Transgene expression after
2 weeks of G418 selection was only 13% and 9% for ARH-77 and RPMI
8226 MM cells; nonetheless, these investigators confirmed the utility of GCV treatment to eradicate tk-gene-transfected MM cells in a SCID mouse model. In the present study, we examined the feasibility of Ad vector-based strategies for delivering the tk gene,
followed by treatment with GCV, for the depletion of contaminating MM
cells within autologous stem cell grafts before transplantation.
Although early studies suggested that transduction efficiency of Ad
vectors in hematologic malignancies is low,38 more recent
reports demonstrating successful Ad transduction of genes encoding
-gal or murine CD80 into human chronic lymphocytic leukemia
B cells39 suggest the potential utility of Ad gene transfer
into human MM cells. In the present study, we demonstrate the presence
of Ad receptors on human MM cells and that adenoviral vectors under the
control of tumor selective promoters (Ad.DF3) have high transduction
efficiency (80% to 100%) in human MM cells, but not in normal BM
MNCs. Importantly, Ad.DF3-tk transduction followed by GCV
treatment can selectively deplete 6 logs of MM cells, without either
bystander effect or altering normal HPCs. These studies therefore
suggest that tk gene transduction using Ad vectors with a
tumor-selective promoter, followed by treatment with GCV, may provide a
highly efficient and selective approach for the ex vivo purging of MM
cells.
To test the feasibility of Ad vector-based purging of MM cells, we
first needed to demonstrate the presence of functional Ad receptors on
tumor cells. We showed that both MM cell lines and freshly isolated
patient MM cells express the CAR adenovirus receptor, evidenced by
reactivity with RmcB MoAb using indirect immunofluorescence flow
cytometric analysis.18 In contrast, normal B splenocytes,
CESS and JY EBV-transformed B cells, and normal BM MNCs lacked CAR
expression; these observations are consistent with Ad receptor
positivity being restricted to MM cells. Second, our specific
radiolabeled viral binding studies confirmed this pattern of receptor
expression. Third, because Ad infection is a two-step process involving
attachment of virus followed by internalization mediated by
v3 and v5
integrins,29,30 we next assayed for v5 expression on tumor cells and found it
to be present on both MM cell lines and freshly isolated tumor cells.
These observations confirmed the presence of Ad receptors on human MM
cells.
To improve the selectivity of Ad vectors for human MM cells, we next
used Ad vectors under the control of tumor selective promoters. In
previous studies, we had shown that Ad.DF3 could be used to selectively
transduce DF3-positive breast cancer cells.17 Because prior
studies by others31 and by our laboratory32 have demonstrated that DF3/MUC1 may also be expressed on MM cells but
not normal BM plasma cells, we assayed for DF3 cell surface expression
on the human MM cell lines and freshly isolated patient MM cells used
in this study. Indirect flow cytometric analysis confirmed that the
majority of cells expressed DF3/MUC1, suggesting the potential utility
of Ad.DF3 for selectively targeting MM cells. In our study we achieved
transfection using Ad.DF3-gal equivalent to that observed
with Ad vector under the control of the CMV vector (Ad.CMV-gal), as assessed by X-Gal staining. The lack of
transduction of MUC1-negative HeLa cells using Ad.DF3-gal,
even at MOI = 100, confirmed selectivity.
To directly test our purging strategy, we performed mixing experiments
in which contaminating MM cells mixed with normal BM MNCs were
transduced with Ad.DF3-tk and then treated ex vivo with GCV.
Importantly, this strategy depleted 6 to 7 log of tumor cells, assayed
first by MM cell proliferation. In both MM cell line and MM patient
cells, we also used PCR to detect Ad5 E2B gene within transduced tumor cells and not normal BM MNCs and to further assess depletion of transduced cells achieved by Ad.DF3-tk
transduction and GCV treatment. These data further confirm the
effectiveness and specificity of this procedure for purging
contaminating MM cells from normal BM MNCs. We also showed that there
was no bystander effect on BM MNCs with the use of Ad.DF3-tk
and GCV purging in this way. Moreover, to probe further the feasibility
of using this technique for purging MM autografts, we showed that
normal HPCs, assessed by BFU-E and CFU-GM, were unaffected. These
characteristics suggest utility and provide the framework for testing
the efficacy and safety of this method for ex vivo purging of MM cells
within autografts.
Multiple other reports, as well as our prior studies in breast
carcinoma, suggest that tumor cells can be effectively purged using Ad
vectors without altering normal HPCs.17,40,41 However, other studies have shown that HPCs can be transduced using Ad vectors
under some culture conditions.42,43 For example, entry of
Ad may occur via passive mechanisms at the very high MOI (ie, 500) used
in these studies, including into CD34+ HPCs that lack
v, v3, or
v5 integrins.17 In the
present study, we achieved 6 log depletion of MM cells under
conditions that did not affect the in vitro growth of CFU-GM and BFU-E.
Although our study suggests that Ad purging of MM cells is unlikely to adversely affect HPCs, this methodology must be scaled up for clinical
use to evaluate not only efficacy of tumor cell purging, but also to
assure that it provides for engraftment that is at least equivalent to
that observed in similarly treated recipients of non-Ad-purged
autografts.
What then is the role for Ad purging of tumor cells in autografting for
MM? To date, a single report suggests that high-dose therapy and
autografting has a superior outcome to conventional therapy3; however, all patients treated with a single
transplant appear destined to relapse. Although double autografting can
be safely attempted and in a retrospective analysis is superior to conventional therapy,5 its utility compared with a single
autograft remains to be determined in ongoing prospective randomized
trials.6 To prolong DFS, we are attempting to increase the
likelihood of achieving MRD using autografting, setting the stage for
the use of pharmacologic and/or immunologic approaches
posttransplant to treat MRD. To address the former goal of achieving
MRD posttransplant, attempts are being made to both improve ablative
anti-MM therapy before transplant and to provide tumor-free autografts.
Selection of CD34+ cells within autologous PBPCs can
achieve a median 3.1 log reduction in tumor cell contamination, but
approximately 50% CD34+ autografts still contain residual
tumor cells.11,12,15,16 Depletion techniques to date using
MoAbs have only achieved 2 to 3 log reduction of tumor cells in MM
autografts.1,8,9 Use of Ad transduction therefore achieves
levels of depletion (6 logs) that have not been previously
attainable; it could be used alone or in combination with other purging
or selection techniques in an attempt to achieve MM-free autografts for
transplantation. Based on the present results, our plan is therefore to
test the potential utility of Ad vector based depletion of MM cells
within autografts both in terms of efficacy of ex vivo purging of tumor cells, as well as satisfactory hematologic engraftment and immunologic reconstitution in patients receiving such autografts posttransplant. Ultimately, it may be both more practical and efficient to first select
CD34+ cells and then purge MM stem cell autografts using
these Ad-based techniques.
| |
FOOTNOTES |
Submitted November 25, 1997;
accepted August 19, 1998.
Supported by National Institutes of Health Grant No. CA 78378 and the
Kraft Family Reseach Fund. G.T. was supported by the Health Manpower
Development Plan Fellowship, Ministry of Health, Singapore; the Medical
Research Council-Shaw Medical Research Fellowship, Singapore; the
Medical Research Council-Singapore Totalisator Board Medical Research
Fellowship, Singapore; and the Singapore General Hospital Medical
Research Fellowship, Singapore.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kenneth C. Anderson, MD, Department of
Adult Oncology, Dana-Farber Cancer Institute, 44 Binney St, Boston, MA
02115; e-mail: kenneth_anderson{at}dfci.harvard.edu.
| |
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Top
Abstract
Introduction