HUMAN GENE THERAPY 13:497–508 (March 1, 2002)
© Mary Ann Liebert, Inc.
Gene Therapy of Murine Solid Tumors with T Cells
Transduced with a Retroviral Vascular Endothelial
Growth Factor–Immunotoxin Target Gene
NI JIN, 1 WEI CHEN,2 BRUCE R. BLAZAR,2 S. RAMAKRISHNAN,3 and DANIEL A. VALLERA1
ABSTRACT
Solid tumor growth can be inhibited by targeting its neovasculature with vascular endothelial growth factor
(VEGF)–toxin fusion proteins (FPs), but these agents have been limited by their inability to localize at the tumor site. In this study, we devised a gene therapy approach intended to deliver VEGF–toxin directly to tumor. Antigen-specific cytotoxic T lymphocytes (CTLs) served as vehicles to deliver a retroviral VEGF–toxin
fusion protein to its specific leukemia cell target in vivo. A retroviral vector was constructed for gene therapy
with VEGF positioned downstream of its 27-amino acid leader sequence, which promoted secretion of a catalytic immunotoxin containing either truncated diphtheria toxin or Pseudomonas exotoxin A. VEGF was chosen on the basis of the expression of VEGF receptor on endothelial cells in the tumor neovasculature. The
VEGF FP was first expressed and secreted by mammalian NIH 3T3 cells. Intracellular expression of both
VEGF and toxin was verified by immunofluorescence. In vitro, supernatants collected from transfected cells
specifically inhibited the growth of VEGF receptor-expressing human umbilical vein endothelial cells (HUVECs), but not a control cell line. In vivo findings correlated with in vitro findings. A retroviral vector containing the target gene and a nerve growth factor receptor (NGFR) reporter gene was used to transiently
transduce T15, a CD81 CTL line that specifically recognizes C1498, a lethal C57BL/6 myeloid tumor. Transduced T15 cells injected intravenously significantly inhibited the growth of subcutaneous tumor, whereas nontransduced controls did not. Together, these data indicate that gene therapy of T cells with retrovirus containing a VEGF–immunotoxin target gene may be a valid means of inhibiting a broad range of solid tumors
dependent on angiogenesis.
OVERVIEW SUMMARY
Previous studies show that tumor-binding immunotoxins
can be selectively delivered to tumor targets by retroviruses,
but the approach has been limited by the narrow reactivity
of the immunotoxin produced by the target gene and the
failure of carrier cells to access tumor. Here, we addressed
these issues by targeting the tumor neovasculature, instead
of the tumor, with antiangiogenic VEGF–immunotoxin
retrovirus. Also, instead of non-antigen-specific immune
cells, an antigen-specific CTL line specifically recognizing
acute myeloid leukemia tumor C1498 was used as the virally infected carrier cell line. Data showed the selective in
vitro inhibition of HUVECs with supernatants from infected
cells and significant inhibition of myeloid tumor growth in
1Section
mice in vivo. Together, data indicate that gene therapy of T
cells with retrovirus carrying VEGF–immunotoxin target
gene may be a valid means of directing more biological
reagent to the tumor site and inhibiting a broad range of
solid tumors dependent on angiogenesis.
INTRODUCTION
T
and progression is primarily dependent on the angiogenic capability of the tumor (Folkman,
1990). Some believe that inhibition of this process may hold
the key to the future development of effective anticancer agents
(Gimbrone et al., 1972). Several reports indicate that therapies
aimed at destroying tumor vasculature can lead to rapid reUMOR DEVELOPMENT
on Experimental Cancer Immunology, Department of Therapeutic Radiology-Radiation Oncology, 2Department of Pediatrics, and
of Pharmacology, University of Minnesota Cancer Center, Minneapolis, MN 55455.
3Department
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498
gression of existing tumors (Huang et al., 1997; O’Reilly et al.,
1997; Arap et al., 1998). One of these approaches has used gene
therapy targeted to tumor cells to suppress the expression of the
angiogenic factor vascular endothelial growth factor (VEGF).
Tumor cells directly transfected with VEGF antisense (Saleh et
al., 1996; Oku et al., 1998) have demonstrated partial inhibition of tumor growth mediated through reduced neovascularization. A different approach has been to target the tumor vasculature with fusion toxins consisting of cell-binding ligands
and potent catalytic toxins (Ramakrishnan et al., 1996; Olson
et al., 1997; Wild et al., 2000). In one study, recombinant VEGF
(VEGF165 isoform) was linked to a truncated form of diphtheria toxin (DT390) that contained the catalytic fragment A and
all of the translocation domain (T domain) but lacked the
native binding domain (Ramakrishnan et al., 1996). This
VEGF–toxin selectively inhibited vascular endothelial cell proliferation and angiogenesis in vitro and inhibited tumor growth
in vivo.
For the targeted therapy of cancer, one of the most important issues to be considered is how to target only cancer cells
and to protect the surrounding normal tissues from side effects.
Generally, less than 0.001% of any injected biological ever
reaches its tumor target (Baxter et al., 1992; Shockley et al.,
1992; Sung et al., 1992), and therefore numerous laboratories
have been exploring the use of (1) specific gene therapy to more
effectively localize treatment and (2) adoptive immunotherapy
with cancer-specific T cells, which can access in vivo tumors
in a way that no biological can.
In a previous report, we demonstrated that retroviral delivery of a leukemia-specific targeted toxin was indeed possible,
using antigen-specific T cell lines in mice (Vallera et al., 2000).
Thus, a mode of therapy in which leukemia-specific immunotoxins could be combined with gene therapy by T cells showed
promise. This approach showed several advantages over conventional systemic injections of recombinant immunotoxins
(ITs), including (1) therapy localized at the tumor site, (2) the
added benefit of T cell therapy, and (3) no need for the time
and expense of recombinant protein scale-up and production
because the retroviral protein was secreted in situ.
Although our study indicated that directly targeting the overexpressed interleukin 4 (IL-4) receptor with a retroviral fusion
protein resulted in a significant anticancer effect, tumor was reduced and not cured, mandating future improvements. One potential improvement would be to target a receptor that is more
universally involved in various cancers. Therefore, in this study,
we targeted the VEGF receptor with retrovirus encoding the
VEGF–PE (Pseudomonas exotoxin) or VEGF–DT390 target
gene. The model was chosen because human VEGF is species
cross-reactive and would bind to murine VEGF receptors expressed on cells of the neovasculature of mice (Ramakrishnan
et al., 1996).
To assemble a VEGF retroviral immunotoxin (retIT), we
used the signal sequence of VEGF to direct VEGF–toxin retroviral protein to the lumen of the endoplasmic reticulum (Walter and Johnson, 1994; Rapoport et al., 1996), thereby avoiding release of the protein into the cytosolic compartment, which
would otherwise destroy the carrier cell. Downstream of the
VEGF gene, we positioned DNA fragments encoding either
truncated Pseudomonas exotoxin A (PE40) or diphtheria toxin
(DT390). We chose the DT390 truncation of diphtheria toxin be-
JIN ET AL.
cause Williams and co-workers described a series of internal
in-frame deletion mutations that established position 389 as the
optimal site for genetic fusion of DT and targeting ligands
(Williams et al., 1990). Both PE40 and DT390 have identical
ADP-ribosylating mechanisms, inactivating elongation factor 2
(EF-2) and protein synthesis at the ribosomal level (Fitzgerald
et al., 1992). Both have been used in constructing fusion toxins for phase I clinical studies (Kreitman, 2000) and both have
been previously used in the construction of retIT (Chen et al.,
1997; Vallera et al., 2000).
In this paper, the target gene is cloned into a retroviral vector and transduced into a previously characterized CD81 T cell
line called T15 (Boyer et al., 1997), which selectively recognizes the lethal myeloid leukemia C1498 and has been used as
a carrier of retIT in previous studies (Vallera et al., 2000). These
studies show for the first time that cells transfected with target
gene stain intracellularly for the presence of both human VEGF
and toxin. Supernatants from these cells selectively inhibit
human umbilical vein endothelial cells (HUVECs), but not control cell lines. Transduced T15 T cells administered intravenously inhibit growth of subcutaneous tumors in mice, providing evidence that targeting the neovasculature in this manner
may be a valid approach to cancer therapy.
MATERIALS AND METHODS
Constructions
A cDNA hybrid gene was assembled to encode amino acids
1 through 237 of the human VEGF protein, including its 27amino acid signal sequence. The sequence encoding the VEGF
polypeptide was positioned upstream of a gene encoding truncated Pseudomonas exotoxin A (PE). The structurally mature PE
peptide was included (GenBank accession number K01397), except for the first 250 amino acids, which encode the native binding site for human cells. The other domains include a potent
catalytic inhibitor of intracellular protein synthesis and a domain that facilitates the membrane translocation of the toxin
into the cytosol (Fitzgerald et al., 1992). VEGF–DT was cloned
as previously described (Vallera et al., 2000). Target genes were
cloned into the nonviral eukaryotic expression vector pcDNA3
(Invitrogen, Carlsbad, CA) to determine whether mammalian
cells could express this fusion protein without undergoing selfdestruction (Fig. 1A). The vector was used to transfect mouse
fibroblast NIH 3T3 cells from the American Type Culture Collection (ATCC, Rockville MD). A second construct was assembled for retroviral transduction, pLNCX.NGFR (generously
provided by S.-Y. Chen, Baylor University, Houston, TX). This
vector contained a DNA fragment encoding human nerve
growth factor receptor (NGFR) as a reporter gene for transduction efficiency (Fig. 1B). Both VEGF–PE and VEGF–DT
were cloned into pLNCX.NGFR, forming vectors LNCVP and
LNCVD, respectively (Fig. 1).
Cells
Human umbilical vein endothelial cells (HUVECs) were obtained from the ATCC and maintained in medium 199 (GIBCOBRL, Rockville, MD) supplemented with EGM SingleQuots
(Clonetics, Walkersville, MD) containing human epithelial
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499
FIG. 1. Construction of recombinant VEGF–PE fusion toxins used in the studies. (A) sigVEGF–PE/pcDNA3 encodes 1683bp gene fragment consisting of the VEGF leader sequence (amino acids 1–26) followed by the human VEGF165 gene (amino
acids 27–237), a flexible EASGGPE linker, and a downstream fragment of PE40 (amino acids 244–535). Relevant restriction
sites are indicated. The fusion gene was cloned into the pcDNA3 vector (Invitrogen), a eukaryotic expression vector, and then
transfected into NIH 3T3 cells. (B) LNCVP encodes the sigVEGF–PE fusion gene described in (A), cloned into the retroviral
vector LNCX.NGFR, which possesses the LNCX backbone except that the neo marker was replaced by the human NGFR gene
to provide a marker for assessing transduction frequency with flow cytometry. (C) LNCVD encodes the sigVEGF–DT fusion
gene and the sigVEGF fusion with DT390 (the first 389 amino acids of diphtheria toxin devoid of the native binding region),
cloned into the retroviral vector LNCX.NGFR.
growth factor (hEGF), hydrocortisone, GA-1000, bovine brain
extract, and fetal bovine serum (FBS). C1498 is an IL-4 receptor-positive (IL-4R1 ), spontaneously occurring C57BL/6
myeloid leukemia, which is lethal to mice in 20–30 days when
injected at doses greater than 105 cells (Durham and Stewart,
1953; Bradner and Pindell, 1966). C1498 cells were maintained
in RPMI 1640 with FCS, L -glutamine, and penicillin–streptomycin (Life Technologies, Grand Island, NY). FBL3 is a control erythroleukemia line cultured similarly (Chen and Cheever,
1997). T15 is an MHC class I-restricted CD81 cytotoxic T cell
line produced by hyperimmunizing C57BL/6 mice with an irradiated subline of C1498 cells with enhanced costimulatory
activity. Previous studies showed that T15 cells respond against
C1498 in vitro and in vivo (Boyer et al., 1997). T15 cells were
maintained in RPMI 1640 with FCS, penicillin–streptomycin,
amphotericin B (Fungizone), gentamicin sulfate, L -glutamine,
sodium pyruvate, minimal essential medium (MEM), nonessential amino acids, 50 mM 2-mercaptoethanol, and mIL-2 (100
units/ml; Cetus, Emeryville, CA), and stimulated every 2 weeks
with irradiated C1498.B7-2 as described previously (Boyer et
al., 1997). NIH 3T3 cells were obtained from the ATCC and
were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% FCS and antibiotics. PA317 packaging cells
were obtained from the ATCC and were maintained in
DMEM–10% FCS with antibiotics.
Transfection and immunofluorescence detection
NIH 3T3 cells were grown to 70% confluency in a six-well
plate (Corning, Corning, NY) at 2 3 105/well in DMEM–10%
FCS and then transfected with VEGF–PE/pcDNA3, using LipofectAMINE according to the manufacturer instructions (GIBCO).
For immunofluorescence analysis, NIH 3T3 cells were cultured
on coverslips and transfected with VEGF–PE/pcDNA3, and then
30 hr after transfection coverslips were washed with phosphatebuffered saline (PBS) and fixed with 95% ethanol–5% acetic acid
at 220°C for 5 min. Fixed cells were washed with PBS and incubated with primary polyclonal rabbit anti-PE (Sigma, Saint
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Louis, MO) and anti-VEGF (provided by S. Ramakrishnan, University of Minnesota, Minneapolis, MN) and secondary fluorescein isothiocyanate (FITC)-labeled antibody (goat anti-rabbit
IgG–FITC) (Sigma, St. Louis, MO) (Vallera et al., 2000). After
mounting with a SlowFade light antifade kit (Molecular Probes,
Eugene, OR), the cells were observed and digitally photographed
with a Nikon fluorescence microscope with Spot Advanced software (Diagnostic Instruments, Ann Arbor, MI).
Transduction and flow cytometry analysis
For retroviral supernatant production, the amphotropic packaging cell line PA317 was transfected with LNCVP or LNCVD,
using LipofectAMINE. Supernatants were harvested 48 hr after transfection and used for transduction of the targeted toxin
gene in T15 cells. The infection procedure has been previously
described (Vallera et al., 2000).
To assess the transduction frequency in T15 cells, transduced
and nontransduced cells were stained with mouse anti-NGFR
polyclonal antibody (diluted 1:1000; Roche, Indianapolis, IN)
for 15 min at room temperature, and further incubated with
FITC-labeled anti-mouse IgG secondary antibody (diluted
1:100; Chemicon, Temecula, CA) for 15 min at room temperature. The anti-NGFR-labeled cell samples were analyzed on a
FACSCalibur (Becton Dickinson, Franklin Lakes, NJ).
Selective killing of target cells by VEGF–PE
To investigate whether VEGF–PE was secreted into the supernatant and whether it could kill target cells, HUVECs and
C1498 cells were cultured with medium containing supernatants
harvested from LNCVP virus-transduced T15 cells. The cells
were plated in a 24-well plate (Corning). A mixture of 0.5 ml
of filtered supernatant from transduced T15 cells and 0.5 ml of
fresh medium was added to each well and the incubated cells
were sampled at 24, 48, and 72 hr. Viable HUVECs and C1498
cells were visually counted by trypan blue dye exclusion (0.5%
trypan blue). The cytotoxicity of supernatants from VEGF–PEtransduced T15 cells was also assessed by proliferation assay.
The HUVECs or C1498 cells were plated into 96-well plates
at 104/well in 100 ml of medium. After an overnight incubation
at 37°C, 100 ml of serial diluted supernatants from transduced
T15 cells was added to each well. After further incubation for
24, 48, or 72 hr, these cultures were pulsed with 1.0 mCi of tritiated thymidine for 24 hr. The amount of radioactivity incorporated into DNA was determined after washing the wells with
PBS to remove unincorporated tritium.
Tumor model
C1498 cells (106) suspended in 100 ml of PBS, pH 7.4, were
inoculated subcutaneously into the flanks of 8- to 10-week-old
female C57BL/6 mice. Five days after implantation, 5 3 106
T15 cells transduced or nontransduced with VEGF–PE were injected intravenously. Because the T15 cell line is dependent on
IL-2 for growth, mIL-2 (5000 units/mouse) was administered
intraperitoneally daily for five consecutive days (days 5–9). Serial measurements of subcutaneous tumor implants were made
with calipers, and tumor sizes were calculated as tumor volume 5 w2 3 l.
JIN ET AL.
Statistical analysis
Groupwise comparisons of continuous data were made by
Student’s t test.
RESULTS
Expression of the sigVEGF–PE gene
in mammalian cells
To determine the feasibility of producing VEGF–PE intracellularly, the VEGF target gene with a 27-amino acid leader
human VEGF was assembled and cloned into the mammalian
expression vector pcDNA3. Figure 1A shows the initial design
of this vector and the correct assembly was confirmed by DNA
sequencing. Thirty hours after transient transfection of NIH 3T3
cells with VEGF–PE/pcDNA3, indirect immunofluorescence
(IF) staining with anti-VEGF or anti-PE revealed the definitive
intracellular presence of both the VEGF (Fig. 2A) and toxin
moiety (Fig. 2B) of the hybrid protein. No positive staining was
observed in controls transduced with the empty pcDNA3 vector and stained with anti-PE (Fig. 2C).
Transduction of T15 cells by sigVEGF–PE gene. Antigenspecific CD81 CD42 MHC class I-restricted T15 T cells were
chosen for delivery of VEGF–PE to C1498 tumors. The T15
cell line was generated by immunizing mouse T cells with irradiated C1498 myeloid leukemia cells and creating an antigenspecific T cell line (Boyer et al., 1997). Typically, T15 cells
are monitored by a 51Cr release assay to verify that the line is
maintaining activity. When T15 activity was measured against
C1498 at effector:target (E:T) ratios of 12.5, 6.3, 3.1, and 1.6
(Table 1), the percent cytotoxicity was 33.3, 23.4, 22.2, and
12.2%, respectively. Against a control unrelated leukemia,
FBL3, at the same ratios 14.9, 6.3, 0, and 0% activity were measured. These data indicate the high degree of antigen specificity.
T15 cells were successfully used as the carrier of an IL4–diphtheria target gene construct in a separate study (Vallera
et al., 2000). Just as in that study, we used a reporter gene to
determine whether the target gene had successfully been expressed in retrovirally transduced T15 cells. Target gene was
cloned into an LNCX vector with an NGFR reporter gene (Fig.
1B). The retrovirus was used to transfect packaging cell lines
and viral supernatant was collected. Viral supernatant from
VEGF–PE/LNCX.NGFR-infected packaging cells was used to
infect T15 cells and the cells were studied for NGFR expression by flow cytometry. The histogram in Fig. 3B shows that
after 48 hr, 30.2% of the transduced cells expressed NGFR on
their surface. The background for the nontransduced controlexpressed NGFR is shown in Fig. 3A. Together, these studies
provided an indication as to whether target gene had been successfully transduced into T15 cells and, even more importantly,
provided a measure of the transduction frequency. These frequencies varied from 25 to 35% in various experiments.
Inhibition of HUVECs by supernatants of VEGF–PE-transduced T15 cells. Because two independent lines of in vitro studies confirmed that VEGF–PE was expressed in mammalian
cells, we next determined whether VEGF–PE was indeed se-
FIG. 2. Expression of recombinant VEGF–PE fusion toxin in mammalian cells. (A) Immunofluorescent staining of sigVEGF–PE/pcDNA3-transfected NIH 3T3 cells with rabbit anti-human VEGF and secondary FITC-labeled anti-rabbit IgG, showing transfectants with normal morphology expressing VEGF in their cytoplasm. (B) Immunofluorescent
staining of sigVEGF–PE/pcDNA3-transfected NIH 3T3 cells with rabbit anti-PE (Sigma) and secondary FITC-labeled anti-rabbit IgG, showing transfectants expressing PE in
their cytoplasm. (C) Immunofluorescent staining of control vector pcDNA3-transfected NIH 3T3 cells with anti-PE and secondary FITC-labeled anti-rabbit IgG. No target gene
was included in this control vector pcDNA3 and no reactivity was observed.
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TABLE 1. SPECIFIC CYTOLYTIC ACTIVITY
T15 T CELL LINE a
E:T ratio
25.0
12.5
6.3
3.1
1.6
OF
C1498
FBL3
44.7
33.3
23.4
22.2
12.2
29.2
14.9
6.3
0.0
0.0
aTo
demonstrate that the T15 cell line was specifically cytotoxic for C1498 cells, cytotoxicity was measured against C1498
and control FBL3 targets in triplicate wells. Data are plotted as
percent cytotoxicity versus the effector–target ratio. Standard
error did not exceed 11% of mean values. The cytolytic curve
measured for C1498 cells differed significantly (p , 0.05) from
the cytolytic curve measured for FBL3.
creted by retrovirus-infected cells. Supernatants that putatively
contained VEGF–PE were collected from transduced T15 cells
and then added to VEGF receptor-expressing HUVECs. Figure
4A shows that HUVECs cultured for 24 hr in the presence of
supernatant diluted with an equal volume of medium (1:1)
showed significantly (p , 0.03) reduced viability, 8000
cells/ml, compared with 20,000 cells/ml in nontransduced controls (also diluted 1:1 in medium). Viability was determined on
the basis of the ability of cells to exclude trypan blue dye as a
vital stain. The inhibitory effect of the supernatant was dose dependent because increasing dilutions of the supernatant with
medium reduced the effect.
In this same experiment, we also evaluated a different agent,
VEGF–DT. Treatment of HUVECs with supernatants from
transduced T15 cells resulted in a similar pattern of reactivity
(Fig. 4B). Supernatants from transduced cells resulted in sig-
nificant (p , 0.02), dose-dependent inhibition of HUVECs,
whereas supernatants from nontransduced cells did not.
The killing observed in the supernatant assays shown in Fig.
4A and B was modest, but was specific to the supernatants from
the transduced clones. Levels of killing might have been higher
if we had given the immunotoxin longer than 24 hr to act on
the target cells.
Figure 5 is a photograph of treated HUVECs taken while the
cells were in culture 48 hr following treatment. Figure 5 (right)
shows healthy control HUVECs that were treated with supernatants from nontransduced T15 cells. The cells are large with
healthy cytoplasm. In contrast, Fig. 5 (left) shows HUVECs after treatment with supernatants from transduced T15 cells. Cytoplasmic compartments are smaller. Extensive vacuolization
has occurred. Several cells have lysed and some of the intracellular debris is visible.
In a separate experiment, we determined whether the cell
killing observed in Fig. 4A and B was specific only to the HUVEC line. Instead of HUVECs, we used C1498 cells as a negative control target line. C1498 is a leukemic line that does not
express the VEGF receptor on its surface. Therefore we would
not expect it to be a target for VEGF–IT killing. Just as in Fig.
4B, HUVECs were treated with supernatants from T15 cells infected with virus containing the VEGF–DT target gene. Figure
6A shows they were inhibited even when diluted 1:4. In contrast, control supernatants from nontransduced T15 cells did not
inhibit HUVECs as much. The dashed line represents HUVEC
cell proliferation in the presence of medium alone. This suggests that supernatants from nontransduced cells were inhibitory
partly because of nutrient depletion and the presence of cell
waste by-products. This problem was solved by adding medium.
In contrast to our findings with HUVECs, the control C1498
cell line (Fig. 6B) was not inhibited when treated with these
identical supernatants, indicating that the inhibition observed
FIG. 3. Retroviral transduction of LNCVP virus into C1498 tumor-specific CD81 T15 clone expressing human NGFR. Transduction frequency of the NGFR reporter gene was determined by immunofluorescent staining of cell surface NGFR and analyzed. (A) Flow cytometry showed that only 4.2% of nontransduced cells were in the M1 gate and represent background; (B)
30.2% of T15 cells in the M1 gate were transduced by LNCVP virus. This agreed with the visual assessment in Fig. 2.
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FIG. 4. Cytotoxicity of VEGF–PE (A) and VEGF–DT (B) to HUVECs in vitro. About 2 3 104 HUVECs per well were cultured in 24-well plates and incubated with supernatants from VEGF–PE transductants for 24 hr. The trypan blue viability assay
showed that HUVEC growth was inhibited by supernatants of transduced T15 cells (undiluted, and diluted 1:1, 1:2, and 1:4)
compared with supernatants from nontransduced T15 cells (NT). Comparisons of data were made between the transduced and
nontransduced groups, using the Student’s t test.
with supernatants from transduced T15 cells was specific to
VEGF-expressing HUVECs. Together, these studies provided
evidence that HUVECs could be specifically inhibited by
VEGF–toxin fusion protein in vitro.
Inhibition of C1498 tumor growth in vivo
Because in vitro data showed that this approach was feasible, we constructed a murine model to determine whether T15
T cells could deliver retIT to C1498 target cells in vivo. C1498
leukemia cells were injected subcutaneously into C57BL/6 mice
to form subcutaneous tumor. Four days after tumor inoculation,
5 3 106 VEGF–PE-transduced T15 cells were intravenously
administered. Nontransduced T15 T cells or PBS was given to
groups of mice as controls. Four of five mice responded well
to the VEGF–PE-transduced T15 cells. Only one of the mice
showed a tumor growth curve that was no different from that
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FIG. 5. Effects of VEGF–DT supernatants on HUVECs in vitro. HUVECs were cultured with supernatants from VEGF–DTtransduced T15 cells (diluted 1:4) for 24 hr or with supernatants prepared identically from nontransduced cells. Left: HUVECs
treated with supernatants from transduced cells show distinctive changes in morphology and decreased numbers in culture. Right:
HUVECS treated with supernatants from nontransduced cells show no changes in morphology.
of the PBS controls. Because the results for four of the responder mice were similar, these were pooled and compared.
Figure 7 shows that tumor growth in the mice receiving transduced T cells was significantly (p , 0.05) slower than tumor
growth in mice that received nontransduced T cells. Mice receiving nontransduced T cells exhibited a slight depression in
tumor growth as compared with untreated controls, likely attributed to the antitumor effect of the cytolytic T cells. Growth
FIG. 6. Selective cytotoxicity of VEGF–DT to HUVECs in vitro. (A) HUVECs (2 3 104/well in 24-well plates) were cultured
with supernatants from VEGF–DT-transduced or nontransduced T15 cells (undiluted, and diluted 1:2 and 1:4). HUVECs were
inhibited with supernatants from transduced cells, but not nontransduced cells. The dashed line represents the level of HUVECs
that were cultured in the presence of medium only. (B) C1498 cells (105/well in 24-well plates) were cultured with supernatants
from VEGF–DT-transduced or nontransduced T15 cells (undiluted, and 1:2 and 1:4). There were no major differences. The dashed
line represents the level of C1498 cells that were cultured in the presence of medium only. Comparisons of data were made between transduced and nontransduced groups, using the Student’s t test.
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FIG. 7. The effects of VEGF–PE-transduced T15 cells on C1498 subcutaneous tumors in vivo. On day 0, 106 viable C1498
leukemia cells were administered to C57BL/6 mice. On day 4 groups of mice were intravenously given 5 3 106 VEGF–PE-transduced T15 cells. Mean tumor size (mean 6 standard deviation) is shown for the group given transduced T15 cells, nontransduced
T15 cells, and PBS only. All groups contained five mice except for the VEGF–PE-transduced T15 cells group, which contained
four mice.
rates did not differ significantly when the untreated group was
compared with the group receiving nontransduced T cells. Together, in vitro and in vivo studies indicate that a single inoculation of transiently transduced T cells significantly inhibited
tumor growth.
The above-described experiment was repeated with the C8
T cell clone, which does not recognize C1498, as a specificity
control (data not shown). C8 is a previously reported MHC class
I-restricted clone recognizing the FBL3 tumor (Chen and
Cheever, 1997). Each mouse in a group of eight was given 5 3
106 C8 cells transduced in the same way as T15 cells (see the
experiment shown in Fig. 7). The rate of tumor growth in these
mice over a 20-day interval was not significantly different from
the rate of tumor growth in control mice (n 5 8/group) given
nontransduced C8 cells or PBS. This indicated that the antitumor effect was limited to T cells that could specifically recognize leukemia cells.
DISCUSSION
The major contribution of this work is the assembly and description of a retIT that potentially can be used against any established solid tumor. All tumor growth beyond a few millimeters and metastasis are dependent on the induction of angio-
genesis mediated by the release of angiogenic factors secreted by
the tumor cells (Folkman, 1995). VEGF is one of the major factors mediating tumor angiogenesis, a complex process in which
endothelial cells (ECs) undergo activation, proliferation, and migration, a process culminating in the formation of new blood vessels (Folkman, 1995; Thomas, 1996). In this study, VEGF–toxin
fusion proteins were fashioned to undergo cotranslational protein
transport so that VEGF–DT and VEGF–PE were secreted from
transduced carrier cells, in this case T cells. Two different toxins were studied because they are not immunologically cross-reactive. Thus, in later clinical studies, if an immune response
negates the therapeutic activity of one biological in vivo, we can
switch to the other. In vitro experiments showed that supernatants
from cells containing the VEGF–retIT target genes were highly
selective for HUVECs. In vivo findings correlated with in vitro
findings because the intravenous administration of antigen-specific T cells transiently transduced with VEGF–toxin virus resulted in significant anticancer effects against a vascularized
subcutaneous myeloid tumor, whereas nontransduced control
T cells had little effect on tumor in vivo. Tumors administered
nontransduced T cells grew at a slightly slower rate than tumors
of untreated controls. Nontransduced T cells did not contribute
to an anti-C1498 effect, as they did in past studies by others
(Chen and Cheever, 1997), which could be explained by our failure to administer enough T cells or to administer them often
506
enough. Perhaps we did not include enough IL-2 to drive the
growth of IL-2-dependent CD81 T15 cells.
VEGF receptors (VEGFRs) are expressed most abundantly
in the tumor vasculature and less abundantly in the endothelium of resting blood vessels (DeVries et al., 1992; Terman et
al., 1992). Thus, high levels of VEGFR expression in the tumor vasculature provide a unique opportunity for tumor targeting. Two tyrosine kinase receptors have been cloned and
characterized to be high-affinity receptors for VEGF localized
predominantly on ECs. These are the 180-kDa Fms-like tyrosine kinase (Flt-1) and the 200-kDa kinase insert domain-containing receptor (KDR) and its murine analog, Flk-1 (Klagsbrun and D’Amore, 1996). Also, alternate splicing from a single
VEGF gene containing eight exons generates the multiple
species of VEGF mRNA. These mRNA species result in putative proteins of 121, 145, 165, 189, and 206 amino acids, which
all recognize the high-affinity receptor (Tischer et al., 1991).
We chose to assemble retIT with VEGF165 because VEGF165
has exon 7, which encodes a peptide recognized by low-affinity receptors neuropilin 1 and neuropilin 2 on the EC membrane. Thus, in addition to recognizing high-affinity VEGF receptors, VEGF165 has the ability to recognize low-affinity ones
as well. Also, Arora et al. previously synthesized recombinant
hybrid proteins consisting of VEGF165 or VEGF 121 spliced to
DT390 (Arora et al., 1999). A comparison revealed that the
VEGF 165 form was 3- to 4-fold more toxic to endothelial cells.
These studies also showed that recombinant VEGF–IT is more
potent than chemically conjugated VEGF–IT.
Several retITs have now been reported. For example, investigators have used transduced lymphokine-activated killer
(LAK) cells to deliver an sFV immunotoxin recognizing Her2/Neu expression human breast cancer in severe combined immunodeficient mice (Chen et al., 1997). In a different study,
transduced T cells were used to deliver an IL-4–IT recognizing
IL-4R-expressing myeloid leukemia cells (Vallera et al., 2000).
Targeting the VEGF receptor with retIT may be prove better because the approach is more broadly reactive with a greater range
of human cancers and therefore may have wider applicability.
The first report of retIT involved the use of LAK cells, which
are IL-2-expanded lymphokine-activated killer cells (Chen et
al., 1997). The use of antigen-specific T cell clones in our model
represents a significant improvement because these T cells
specifically recognize and home to antigen-expressing tumor
cells. There is not yet direct proof in this model that our T cells
home specifically. However, we did demonstrate that a control
T cell clone that did not recognize tumor did not show a significant antitumor effect when transduced in the same manner
as T15 cells. Although tumor growth was significantly inhibited by retIT treatment, it was only slowed and there were no
cures. We do not know why our effect was incomplete, but there
are several possible explanations. The most common problem
with cellular immune therapy is a failure of immune cells to
properly home (Chen and Cheever, 1997). Too many T cells
may be filtered into nontarget organs such as lung or liver or
the amount of IL-2 in vivo was limiting.
Future experiments designed to measure the localization of
transduced T cells in the tumor and their expansion are critical.
Currently, we are adapting a published model (Chen et al.,
1990) in which the tumor cells, host T cells, and antigen-specific T cell clone can be independently marked and the trans-
JIN ET AL.
duced T cells can be distinguished from nontransduced T cells
by using a retroviral vector with an NGFR reporter gene. These
studies will be critical for measuring T cell penetration into tumor and determining whether, when, and how many T cells
should be given.
What is the proposed mechanism of VEGF–retIT? The physiologic factors that contribute to the poor delivery of biologicals in solid tumors include heterogeneous blood supply, elevated interstitial pressure, fluid loss from the periphery, and
large distances in the interstitium (Jain, 1989). Although protein molecules are mostly unable to overcome the barriers, antigen-specific T cells may be able to do so as evidenced by the
presence of T cells within tumors. As mentioned, inhibition of
the activity of angiogenic factors such as VEGF inhibits new
blood vessel growth and, consequently, tumor growth (Kim et
al., 1993; Olson et al., 1997). VEGF receptors are expressed
on the vasculature around tumors. Therefore, VEGF–immunotoxins released from transduced cells attack new vasculature,
reducing or shutting down tumor blood flow. Retroviral delivery of immunotoxins has been theoretically limited by cell suicide expected to result from the presence of toxin in the cytosol. Although posttranslational protein transport in yeast does
indeed permit translated toxins to react with their target, most
mammalian cells undergo cotranslational protein transport, in
which the signal peptide directs the single-chain protein through
the endoplasmic reticulum (ER) membrane directly into the ER
lumen (Walter and Johnson, 1994; Rapoport et al., 1996). Data
in this report indicate that toxin separation between the intracellular ER lumen and cytosolic compartments is remarkably
conserved, so that even minute amounts of toxin are prevented
from gaining access to ribosomes. It is known that leader sequences are responsible for the passage of proteins into or
through membranes and retroviral ITs assembled in our laboratory without a leader sequence positioned upstream from the
ligand were lethal to transfected cells. The leader peptide permits recognition by the signal recognition particle (SRP) and
translocation of IT polypeptide directly into the ER lumen,
where it is sequestered (reviewed in Walter and Johnson, 1994).
Whether this separation is maintained indefinitely is unlikely,
and therefore the duration of the effect must be measured in future experiments.
These studies were performed with transiently transduced T15
cells (transduced and then injected 24 hr later) on the basis of
previous studies (Chen et al., 1997). However, higher levels of
killing may be obtained if we select for stable T cell transductants that have stably integrated the target gene. It is not known
how long the target gene could be carried before a breach in the
ER–cytosol gradient would result in cell death. Investigators have
shown that the toxin provirus is present in transduced T cells for
at least 2 weeks after transduction (Vallera et al., 2000). However, stable producers would increase the likelihood that T cells
attacking the tumor would still be producing IT.
How might this approach be translated clinically? No exact
answer can be given at this early point in time. However, patient T cells might be collected, transiently or stably transduced,
and then returned to the patient with IL-2 to promote T cell expansion. Whether it will be somehow necessary to stimulate T
cells with antigen before administration or whether the cancer
cells within the patient might provide enough antigen stimulation remains to be determined.
RETROVIRAL GENE THERAPY WITH VEGF-IMMUNOTOXIN
These studies support the feasibility of using an retIT for
therapy of solid tumors because intracellular staining showed
the presence of VEGF and toxin proteins inside cells transfected
with the target gene. These findings correlated with studies in
which supernatants from these cells selectively inhibited HUVECs in vitro. Furthermore, in vitro findings correlated with in
vivo studies in which subcutaneous tumor-bearing mice given
intravenous injections of antigen-specific T cells transduced
with VEGF–PE virus resulted in significant anticancer effects.
Thus, this form of gene therapy may prove useful for bolstering the cytolytic capabilities of antigen-specific T cells for a
variety of different cancer types.
ACKNOWLEDGMENT
We thank Dr. Scott McIvor (University of Minnesota) for
helpful comments. This work was supported in part by National Institutes of Health grants ROICA82154, ROICA72669 ,
ROICA71803, and ROICA85922.
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Address reprint requests to:
Dr. D.A. Vallera
University of Minnesota Cancer Center
Mayo mail code: 367, Harvard St. at East River Road
Minneapolis, MN 55455
E-mail: [email protected]
Received for publication June 28, 2001; accepted after revision
January 18, 2002.
Published online: February 4, 2002.