Previous Article | Next Article 
Journal of Virology, October 2000, p. 9540-9545, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A TVA-Single-Chain Antibody Fusion Protein Mediates Specific
Targeting of a Subgroup A Avian Leukosis Virus Vector to Cells
Expressing a Tumor-Specific Form of Epidermal Growth Factor
Receptor
Sophie
Snitkovsky,1,2
Thomas M. J.
Niederman,3
Bob S.
Carter,4
Richard C.
Mulligan,3 and
John A. T.
Young2,5,*
Committee on
Virology,1 Department of Microbiology
and Molecular Genetics,2 Howard Hughes
Medical Institute at the Children's
Hospital,3 and Harvard Institute of
Human Genetics,4 Harvard Medical School,
Boston, Massachusetts 02115, and Department of Oncology,
McArdle Laboratory for Cancer Research, University of Wisconsin at
Madison, Madison, Wisconsin 537065
Received 12 April 2000/Accepted 28 July 2000
 |
ABSTRACT |
We have previously described an approach that employs retroviral
receptor-ligand bridge proteins to target retroviral vectors to
specific cell types. To determine whether targeted retroviral entry can
also be achieved using a retroviral receptor-single-chain antibody
bridge protein, the TVA-MR1 fusion protein was generated. TVA-MR1 is
comprised of the extracellular domain of the TVA receptor for subgroup
A avian leukosis viruses (ALV-A), fused to the MR1 single-chain
antibody that binds specifically to EGFRvIII, a tumor-specific form of
the epidermal growth factor receptor. We show that TVA-MR1 binds
specifically to a murine version of EGFRvIII and promotes ALV-A entry
selectively into cells that express this cell surface marker. These
studies demonstrate that it is possible to target retroviral vectors to
specific cell types through the use of a retroviral
receptor-single-chain antibody fusion protein.
| |
INTRODUCTION |
Several different strategies have
been developed for targeting infection of specific cell types by
retroviral vectors. The most common approach has employed recombinant
viral envelope (Env) proteins that contain either cell type-specific
ligands or single-chain antibodies that bind to specific cell surface
molecules (1, 4, 7-12, 15, 18-20, 24-28, 31-35, 38, 39, 41,
42, 44, 46, 48, 51-54, 57). This approach requires that the
specific alterations made to Env do not affect the biosynthesis, virion assembly, or fusogenic function of the viral glycoprotein (12, 54,
57).
An alternative approach for targeting retroviral entry that employs
soluble retroviral receptor-ligand bridge proteins was recently
developed (5, 47). These bridge proteins are bifunctional reagents: the ligand moiety binds to specific cell surface receptors and the retroviral receptor moiety binds to Env, activating viral entry. This technique does not require making alterations to the viral
glycoprotein but instead relies on "wild-type" Env-receptor interactions to target viral entry.
To demonstrate the feasibility of this approach, the TVA-EGF and
TVB-EGF fusion proteins were generated. These bridge proteins contained
human epidermal growth factor (EGF) fused to the extracellular domains
of either the TVA receptor for subgroup A avian leukosis viruses
(ALV-A) or the TVB receptor for ALV-B and ALV-D, respectively. Each of
these bridge proteins promoted specific retroviral entry into cells
that express the EGF receptor (EGFR) when bound to cell surfaces before
viral challenge (5, 47). Furthermore, murine leukemia virus
(MLV) pseudotypes bearing ALV-B Env (EnvB) and preloaded with TVB-EGF
were targeted specifically to cells that express EGFR (5).
These data demonstrated that retroviral vectors could be targeted to
specific cell types by binding retroviral receptor-ligand bridge
proteins to virions or to cell surfaces before viral challenge.
To extend the utility of this approach, we have now asked whether
targeted retroviral entry into cells can also be achieved using
TVA-MR1, a bridge protein that contains the extracellular domain of TVA
fused to the MR1 single-chain antibody. This antibody binds
specifically to the extracellular region of EGFRvIII, a variant form of
the EGFR that is expressed at the surface of human tumor cells,
including those derived from lung and breast carcinomas and
glioblastomas (14, 17, 30, 37, 40, 49, 55, 56). EGFRvIII
lacks a substantial portion of the extracellular domain of the
wild-type receptor as a consequence of a deletion or rearrangement that
commonly occurs when the EGFR gene is amplified during tumor biogenesis. These alterations result in constitutive,
ligand-independent activation of EGFRvIII (22), which, in
turn, confers a transformed phenotype upon various cell lines (22,
40).
The MR1 antibody binds to a novel polypeptide sequence that is formed
at the site of the deletion or rearrangement that gives rise to
EGFRvIII (29). The combination of the tumor-restricted expression of EGFRvIII and the binding specificity of the MR1 antibody
makes this an attractive model system to test whether retroviral
receptor-single-chain antibody bridge proteins can mediate targeted
viral entry into cells. In this report, we demonstrate that TVA-MR1 can
support efficient and specific ALV entry into mammalian cells that have
been engineered to express a murine form of EGFRvIII.
| |
MATERIALS AND METHODS |
Viruses and immunoadhesins.
The SUA-rIgG immunoadhesin was
described elsewhere and is comprised of the surface protein of the
Schmidt-Ruppin A strain of Rous sarcoma virus fused to a rabbit Fc
chain (58). ALV-A-specific vectors encoding the enhanced
green fluorescent protein (EGFP; Clontech) were generated by
transfection of DF1 cells (45) with the RCASBP(A)-EGFP
plasmid (provided by Mark Federspiel and Matt van Brocklin). The
transfected cells were propagated until 100% of the population
expressed EGFP as determined by fluorescence microscopy. Cells were
then seeded in 1,700-cm2 roller bottles, and virions were
harvested every 12 h in 50 ml of medium that was equilibrated with
5% CO2. This medium was pooled and filtered through
0.45-µm-pore-size filters and stored at 
80°C. Before use, the
virus-containing supernatants were thawed and subjected to
centrifugation at 109,000 × g for 1.5 h at 4°C.
The viral pellets were then resuspended overnight at 4°C in 1/100 of
the original volume of TNE buffer (5).
Replication-defective MLV vectors encoding a synthetic transmembrane
form of TVA (TVAsyn) (3) or a murine form of
EGFRvIII were generated. The MLV vector pMMP.TVAsyn was
generated by first preparing a DNA fragment that encodes TVAsyn by PCR amplification using pDW1 plasmid template DNA
(D. Wenzke and J. A. T. Young, unpublished data) and the
following two primers: 5'-GCATAGCGTACCATGGCTAGATTGCTTCCTGCATTGC-3'
and
5'-CG ATCGACATGCATCCGGAACTAATCGATCTGAGCAGCGTAATCTGG-3'. The
resultant DNA fragment was digested with NcoI and
BspEI restriction enzymes and was subcloned into the
NcoI and BspEI sites of the pMMP.EGFP plasmid (K. Bradley and J. A. T. Young, unpublished data), generating the
pMMP.TVAsyn plasmid. The MLV vector pSFG.EGFRvIII contains
a gene encoding a murine form of EGFRvIII (R. Carter and R. C. Mulligan, unpublished data) located between the NcoI and
BamHI restriction enzyme sites of the pSFG vector.
Stocks of MLV vectors pseudotyped with the VSV-G protein were prepared
from human 293T cells by using a transient transfection
system
essentially as described previously (
5) with 5 µg of
the
pMD.G plasmid encoding VSV-G, 15 µg of the pMD.old.gagpol
plasmid
encoding MLV Gag and Pol, and 15 µg of either plasmid
pSFG.EGFRvIII
or plasmid pMMP.TVA
syn. Virions were harvested at 48 and
72 h postinfection, and the
supernatants containing
MLV-EGFRvIII(VSV-G) and MLV-TVA
syn(VSV-G) viruses were
separately pooled and filtered through a
0.45-µm-pore-size filter and
then stored at
80°C.
Cell lines.
Human 293T cells were propagated in Dulbecco
modified Eagle medium containing 5% fetal bovine serum. 293T-EGFRvIII
cells were generated by transducing 293T cells with
MLV-EGFRvIII(VSV-G). Approximately 72 h after viral challenge, the
transduced cell population (107 cells) was incubated for 30 min at 4°C with 5 ml of extracellular supernatant containing TVA-MR1
and then with 5 ml of extracellular supernatant containing SUA-rIgG.
These cells were then incubated for 10 min at 4°C with a fluorescein
isothiocyanate (FITC)-conjugated swine anti-rabbit antibody (DAKO
Corp.) diluted 1:100 in medium. The cells were washed with ice-cold
medium, and those expressing the EGFRvIII protein were isolated by flow
cytometric sorting using a Coulter Epics Elite cell sorter.
293T-TVAsyn cells were generated in a similar manner,
except that 293T cells were transduced with
MLV-TVAsyn(VSV-G). Cells expressing the TVAsyn
receptor were isolated by flow cytometric sorting after binding SUA-rIgG to the cells for 30 min at 4°C, followed by binding the FITC-conjugated anti-rabbit antibody; 25% of these cells with the
highest level of cell surface TVAsyn were then isolated by
subjecting the population to an additional round of flow cytometric sorting.
Construction and expression of TVA-MR1.
A DNA fragment
encoding the MR1 single-chain antibody (29) was generated by
PCR amplification, using as template DNA the plasmid pCMMP.MR1, which
contains a gene encoding MR1 located between the XbaI and
HindIII restriction enzyme sites of the pCMMP vector (T. Niederman et al., unpublished data), and the following two
oligonucleotide primers:
5'-GAACTCCTAGGGGGACCGCAGGTACAACTCCAGCAGTCCGGGGG-3' and
5'-GAGGGGCCCTCTAGATTATAGAGCTTTTTCAAGCTTGGTGCCATCACCG-3'. The resultant DNA fragment was digested with ApaI, end repaired
with the Klenow fragment of DNA polymerase, and digested with
AvrII to generate a 758-bp DNA fragment encoding MR1. This
fragment was then ligated with plasmid pSS8 that had been cut with
Asp718, end repaired, and then digested with
AvrII. Plasmid pSS8 contains the gene encoding a
TVA-heregulin
1 fusion protein (S. Snitkovsky and J. Young,
unpublished data) located between the EcoRI and HpaI restriction enzyme sites of the pCI expression vector
(Promega). The resultant plasmid pSS11 encodes the TVA-MR1 fusion
protein, and the authenticity of this open reading frame was confirmed by DNA sequence analysis (performed by the core DNA sequencing facility
in the Department of Microbiology and Molecular Genetics at Harvard
Medical School).
To generate the TVA-MR1 fusion protein, plasmid pSS11 was transfected
into human 293 cells as described previously (
47).
Aliquots
of 40 µl of extracellular supernatant taken from transfected
and
nontransfected human 293 cells were subjected to electrophoresis
on
10% polyacrylamide gel containing sodium dodecyl sulfate under
nonreducing conditions. The proteins were then transferred to
a
nitrocellulose membrane, and TVA-MR1 was detected by immunoblotting
with 10 ml of extracellular supernatant containing SUA-rIgG and
then
with a horseradish peroxidase-conjugated antibody specific
for rabbit
immunoglobulins (Amersham), followed by enhanced
chemiluminescence.
TVA-MR1 binding studies.
A suspension of 3.5 × 105 293T-EGFRvIII cells was incubated for 1 h at 4°C
with increasing amounts of TVA-MR1-containing extracellular supernatant. These samples were made up to a total volume of 500 µl
with 293 cell-conditioned medium (medium that was obtained from a
confluent monolayer of human 293 cells). A suspension of 3.5 × 105 293T cells was incubated for 1 h at 4°C with 500 µl of extracellular supernatant that either lacked or contained
TVA-MR1. Both cell populations were then washed with 2 ml of ice-cold
phosphate-buffered saline (PBS) and incubated for 1 h at 4°C
with 500 µl of extracellular supernatant containing SUA-rIgG. The
cells were washed again with ice-cold PBS and incubated for 30 min at
4°C with the FITC-conjugated swine anti-rabbit antibody (diluted
1:100) in medium. The cells were then washed again and resuspended in
500 µl of ice-cold PBS containing 1% formaldehyde, and the bound
TVA-MR1 fusion proteins were detected by flow cytometry using a Coulter
Epics Ex-L instrument.
The peptide competition binding experiments were performed by
incubating 500 µl of extracellular supernatant containing TVA-MR1
for
1 h at 4°C with a final 100 nM concentration of either the
MR1
epitope-containing peptide (LEEKKGNYVVTDH) (
29) or a
scrambled
(control) version of this peptide (YKELGVEVDNKHT). These
samples
were then incubated with 293T-EGFRvIII cells for 1 h at
4°C. TVA-MR1
proteins that were bound to the cells were then detected
using
SUA-rIgG and the FITC-conjugated antibody as described above.
For
control purposes, 500 µl of 293 cell supernatants containing
a 100 nM
concentration of either the MR1 epitope-containing peptide
or of the
control peptide was placed on ice for 1 h and then incubated
with
293T-TVA
syn cells for an additional hour. The cells were
then washed with
ice-cold PBS, and cell surface TVA
syn
proteins were detected by flow cytometry as described
above.
TVA-MR1-mediated infection.
Each of the following steps was
performed at 4°C, with a suspension of approximately 5 × 105 cells of each cell type, and each experiment was
performed in triplicate. 293T cells were rocked together for 1 h
with 500 µl of 293 cell-conditioned medium that either lacked or
contained TVA-MR1. 293T-EGFRvIII cells were similarly incubated with
500 µl of extracellular supernatant containing TVA-MR1.
293T-TVAsyn cells were incubated with 500 µl of 293 cell-conditioned medium for 1 h.
Following each of these incubations, the cells were washed and
resuspended in 500 µl of medium with or without 5 µl of
100-fold-concentrated
RCASBP(A)-EGFP (0.5 µl of 100-fold-concentrated
virus for infection
of 293T-TVA
syn cells). The cells were
then rocked with virus for 1 h at 4°C
before plating and
incubation at 37°C. The medium was replaced
20 h later; 72 h after viral challenge, the cells were resuspended
in
Ca
2+- and Mg
2+-free PBS containing 1 mM EDTA
and 7 µM propidium iodide and were
analyzed for EGFP expression using
a Coulter Epics Ex-L flow cytometer.
Dead cells that had taken up
propidium iodide were excluded from
this analysis by electronic gating.
The viral titer was then measured
by first calculating the multiplicity
of infection (MOI) observed
from the input virus: MOI =
ln[1
(number of EGFP fluorescent
cells/total number of cells
analyzed)]. The actual titers (per
microliter of 100-fold-concentrated
virus) were then calculated
using the following equation: (MOI × total number of cells that
were challenged/5 (or divided by 0.5 in the
case of 293T-TVA
syn cells). The number of fluorescent cells
seen in the absence of
virus was subtracted from each of the
calculations.
Peptide competition experiments were performed by incubating 500 µl
of extracellular supernatant containing TVA-MR1 at 4°C
for 1 h
with a 100 nM concentration of either the MR1 epitope-containing
peptide or of the control peptide. These samples were then incubated
with 293T-EGFRvIII cells for another hour at 4°C. The cells were
then
washed and incubated for 1 h with 500 µl of medium containing
5 µl of 100-fold-concentrated RCASBP(A)-EGFP before plating and
incubation at 37°C for 18 h. The medium was then replaced, and
72 h after viral challenge, the cells were analyzed by flow
cytometry
as described
above.
| |
RESULTS |
Production of the TVA-MR1 protein.
A gene that encodes the
recombinant TVA-MR1 bridge protein was constructed in plasmid pSS11
(described in Materials and Methods). TVA-MR1 is comprised of the
extracellular portion of the TVA receptor (2) fused to the N
terminus of the MR1 single-chain antibody, which recognizes a unique
sequence in the extracellular domain of EGFRvIII (29) (Fig.
1A). A proline-rich polypeptide linker that was described previously (47) was inserted between each of the two domains of TVA-MR1 (Fig. 1A) in order to maximize the probability that each domain would fold and function independently. TVA-MR1 was produced as a secreted protein, approximately 54 kDa in
size, in the extracellular supernatants taken from human 293 cells
transfected with plasmid pSS11 (Fig. 1B, compare lanes 1 and 2).
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 1.
Production of TVA-MR1. (A) TVA-MR1 was comprised of the
extracellular domain of the TVA receptor for ALV-A fused, via a
proline-rich linker region, to the N-terminal end of the MR1
single-chain antibody that binds specifically to EGFRvIII. (B)
Extracellular supernatants collected from transfected 293 cells that
expressed TVA-MR1 (lane 2) or did not express the bridge protein (lane
1) were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis under nonreducing conditions. The protein samples were
then subjected to immunoblotting with SUA-rIgG (58) and a
horseradish peroxidase-conjugated secondary antibody, and the TVA-MR1
protein was detected by enhanced chemiluminescence.
|
|
TVA-MR1 specifically binds to cells expressing an EGFRvIII
protein.
To determine whether the Env-binding and antigen-binding
domains of TVA-MR1 can each function independently, we investigated whether this bridge protein can bind both to the ALV-A surface envelope
(ALV-A SU) and to a cell surface EGFRvIII protein. Transfected human
293T cells expressing a murine form of EGFRvIII (293T-EGFRvIII cells)
were incubated with increasing amounts of an extracellular supernatant
containing TVA-MR1. For control purposes, the parental 293T cells were
also incubated with TVA-MR1-containing supernatant. The bridge proteins
that were bound to these cells were then detected by flow cytometry
after the cells had been incubated with SUA-rIgG, an immunoadhesin
composed of the ALV-A surface envelope fused to a rabbit Fc chain
(58), and with an FITC-conjugated secondary antibody.
TVA-MR1 bound specifically and in a dose-dependent manner to
293T-EGFRvIII cells (Fig. 2A), indicating
that this bridge protein can bind simultaneously to both ALV-A SU and
to cell surface EGFRvIII.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 2.
TVA-MR1 binds specifically to the EGFRvIII protein. (A)
293T cells and 293T-EGFRvIII cells were incubated with extracellular
supernatants that either lacked or contained TVA-MR1. The bound TVA-MR1
proteins were then detected by flow cytometry using SUA-rIgG and an
FITC-conjugated secondary antibody. (B) Extracellular supernatants
containing TVA-MR1 were preincubated with either no peptide (none), the
MR1 epitope-containing peptide, or a scrambled version of this peptide.
These samples were then incubated with 293T-EGFRvIII cells, and the
bound TVA-MR1 proteins were then detected by flow cytometry as shown in
panel A. The results obtained were compared with the background levels
of fluorescence obtained with cells incubated in the absence of
TVA-MR1. (C) 293T-TVAsyn cells were incubated without
SUA-rIgG or with SUA-rIgG and with either no peptide (none), the MR1
epitope-containing peptide, or the scrambled peptide. The bound
immunoadhesin was then detected by flow cytometry using an
FITC-conjugated secondary antibody. Results of a representative
experiment are shown (A through C).
|
|
To obtain direct evidence that TVA-MR1 binds to the EGFRvIII protein,
we examined whether the interaction of this bridge protein
with
293T-EGFRvIII cells could be blocked by a synthetic peptide
that
contains the target epitope for the MR1 antibody (
29).
Indeed, this peptide blocked the binding of TVA-MR1 to these cells
(Fig.
2B). By contrast, a control peptide (with the same amino
acids
scrambled in a different order) did not affect TVA-MR1 binding
to these
cells (Fig.
2B). To rule out the possibility that the
MR1
epitope-containing peptide interfered nonspecifically with
ALV-A
SU-TVA interactions, this peptide was also tested for its
effect on
the binding of SUA-rIgG to a stably transfected line
of human 293T
cells (designated as 293T-TVA
syn) that express a
transmembrane form of the TVA receptor. The MR1
epitope-containing
peptide did not block the binding of SUA-rIgG
to these cells (Fig.
2C),
confirming that this peptide specifically
blocks the interaction
between TVA-MR1 and the EGFRvIII
protein.
TVA-MR1 mediates specific ALV-A entry into 293T-EGFRvIII
cells.
To determine whether TVA-MR1 can facilitate ALV-A infection
of 293T-EGFRvIII cells, these cells and, for control purposes, 293T
cells, were incubated with this bridge protein and then challenged with
an ALV-A vector [RCASBP(A)-EGFP], encoding EGFP. The addition of
TVA-MR1 led to a significant enhancement of ALV-A entry into 293T-EGFRvIII cells (Table 1) (Fig.
3, compare panels B and C). Indeed,
TVA-MR1 acted in a dose-dependent manner to increase the susceptibility
of these cells to ALV-A infection (data not shown). In these
experiments, the level of TVA-MR1-mediated viral entry into
293T-EGFRvIII cells was 8.5% of the level seen with
293T-TVAsyn cells, and titers of approximately
108 infectious units/ml of 100-fold-concentrated virus were
achieved (Table 1). In contrast, the addition of this bridge protein
had no effect upon the susceptibility of the parental 293T cells to viral infection (Table 1) (Fig. 3, compare panels E and F).
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 3.
TVA-MR1 promotes ALV-A infection of 293T-EGFRvIII cells.
293T-EGFRvIII cells (A through C) and 293T cells (D through F) were
incubated with extracellular supernatant that contained (+) or lacked
() TVA-MR1 as indicated. These cells were incubated in the absence or
presence of the ALV-A vector RCASBP(A)-EGFP, and then aliquots of each
cell type (5 × 104 293T-EGFRvIII cells and
105 293T cells) were analyzed by flow cytometry.
|
|
To confirm that TVA-MR1 had to be bound to the EGFRvIII protein in
order to mediate enhanced levels of viral entry into 293T-EGFRvIII
cells, we determined whether this effect could be blocked by the
MR1
epitope-containing peptide. The MR1 epitope-containing peptide,
but not
the control peptide, blocked this bridge protein-dependent
viral
infection (Fig.
4). Therefore, TVA-MR1 is
capable of facilitating
targeted ALV-A entry into mammalian cells when
bound to the EGFRvIII
protein.
View larger version (58K):
[in this window]
[in a new window]
|
FIG. 4.
The TVA-MR1-EGFRvIII interaction is required for
targeted viral entry. Extracellular supernatant containing TVA-MR1 was
incubated with no peptide (none), the MR1 epitope-containing peptide,
or the scrambled peptide before addition to 293T-EGFRvIII cells. The
cells were then challenged with RCASBP(A)-EGFP and analyzed by flow
cytometry 72 h later. For control purposes, viral infection of
293T-EGFRvIII cells was also assessed in the absence of any TVA-MR1.
|
|
| |
DISCUSSION |
In this report we have demonstrated the feasibility of using a
soluble retroviral receptor-single-chain antibody bridge protein for
targeting retroviral infection to a specific cell type. Cell-binding studies and peptide competition experiments confirmed that TVA-MR1 is a
bifunctional reagent that can bind to a cell surface EGFRvIII protein
expressed on 293T cells and to ALV-A SU. Furthermore, the binding of
TVA-MR1 to the EGFRvIII protein allowed specific viral entry at a level
that was approximately 7% (ranging from 1.8 to 14% in separate
experiments) of that found in ALV-A infection of
293T-TVAsyn cells (Table 1 and data not shown). In
contrast, TVA-MR1 did not bind to or promote infection of the parental
293T cells, which are known to express cell surface EGFRs
(50). Indeed, based upon previous results that demonstrated
the unique specificity of the MR1 antibody for EGFRvIII, we fully
expected that TVA-MR1 would allow only ALV-A infection of cells
expressing mutant, but not wild-type, EGFRs. For example, Lorimer et
al. (29) have already shown that this single-chain antibody,
engineered in the context of an MR1-toxin fusion protein, directs the
potent killing of cells that express 400,000 EGFRvIII molecules (at a
concentration of 7 to 10 ng/ml), whereas this recombinant toxin is
unable to kill cells expressing 200,000 wild-type EGFR molecules even
when added at concentrations as high as 1,000 ng/ml. Taken together, our studies have demonstrated that TVA-MR1 can be an efficient and
specific facilitator of viral entry into cells when bound to EGFRvIII.
During these experiments we found that ALV-A is capable of infecting
human 293T cells and 293T-EGFRvIII cells at a low "background" level that was 1/2,000 to 1/5,000 of the level seen with
293T-TVAsyn cells (Table 1 and data not shown). The
addition of TVA-MR1 led to an 180-fold to 200-fold increase in the
susceptibility of 293T-EGFRvIII cells to ALV-A infection but did not
affect the "background" level of infection seen with 293T cells
(Table 1). The "background" level of ALV-A infection seen with
human 293T cells is similar to that seen with some other human cell
lines, e.g., U250 glioma cells, but it is approximately 100-fold higher than that seen with other mammalian cell lines (6). It is
important to understand why certain mammalian cell types are more
susceptible to ALV infection than are others, since this information
may help us eliminate such infections and thus optimize the use of
retroviral receptor-ligand and retroviral receptor-single-chain
antibody bridge proteins for viral targeting.
The results presented in this report suggest that it might be possible
both in vivo as well as in vitro, to use retroviral receptor-single-chain antibody bridge proteins as tools to deliver retroviral vectors to specific cell types that express cognate cell
surface antigens. With regard to in vivo applications, it is not yet
clear whether the background level of ALV-A infection that was observed
in cultured human 293T cells will represent a significant hurdle for
cell type-specific viral targeting: viral targeting studies, performed
with transgenic lines of mice that express a transmembrane form of TVA
in specific cell types, indicate that the "background" level of
ALV-A infection seen with cells that lack this viral receptor is
extremely low (16). Furthermore, in considering the
potential utility of this system for delivering viral vectors to tumor
cells, the use of ALV-based or MLV-based vectors for gene delivery
affords another level of specificity since these viruses only establish
proviral DNA in dividing cell types (36, 43). Indeed,
several groups have already shown that it is possible to use this
feature of MLV vectors to deliver genes specifically to tumor cells,
even in the absence of a selective Env-targeting system (13,
23). However, since the retroviral vectors used in these studies
have a broad host range, there exists the potential for infecting other
dividing cell types in addition to the target tumor cells. The use of
retroviral receptor-ligand and retroviral receptor-single-chain
antibody bridge proteins for viral delivery may lead to more specific
viral targeting in vivo. Indeed, it will be interesting to determine
the efficiency and specificity of in vivo viral targeting that can be
achieved with TVA-MR1 in mouse models of human cancer that employ tumor cells expressing EGFRvIII (21). An added advantage of using retroviral receptor-single-chain antibody fusion proteins is that such
bridge proteins might be useful for targeting retroviral vector
infection to cells expressing a number of different cell surface
factors, including those with no known ligands.
| |
ACKNOWLEDGMENTS |
We acknowledge John Daly at the Dana-Farber Cancer Institute for
expert assistance with flow cytometry and members of the core DNA
sequencing facility in the Department of Microbiology and Molecular
Genetics at Harvard Medical School for help with DNA sequencing. We
thank members of the Young laboratory for comments, suggestions, and
helpful discussions and Nathan Astrof and Walther Mothes for critical
reading of the manuscript. We also thank John Naughton for help
preparing the final figures and Mark Federspiel and Matt von Brocklin
for kindly providing viral vectors.
This work was supported by NIH grant CA 70810 from the National Cancer
Institute and by grant DAMD17-98-1-8488 from the Department of the Army.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: McArdle
Laboratory for Cancer Research, University of Wisconsin at Madison,
1400 University Ave., Madison, WI 53706. Phone: (608) 265-5151. Fax:
(608) 262-2824. E-mail: young{at}oncology.wisc.edu.
| |
REFERENCES |
| 1.
|
Ager, S.,
B. H. Nilson,
F. J. Morling,
K. W. Peng,
F. L. Cosset, and S. J. Russell.
1996.
Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity.
Hum. Gene Ther.
7:2157-2164[Medline].
|
| 2.
|
Bates, P.,
J. A. T. Young, and H. E. Varmus.
1993.
A receptor for subgroup A Rous sarcoma virus is related to the low density lipoprotein receptor.
Cell
74:1043-1051[CrossRef][Medline].
|
| 3.
|
Bélanger, C.,
K. Zingler, and J. A. T. Young.
1995.
Importance of cysteines in the LDLR-related domain of the subgroup A avian leukosis and sarcoma virus receptor for viral entry.
J. Virol.
69:1019-1024[Abstract].
|
| 4.
|
Benedict, C. A.,
R. Y. Tun,
D. B. Rubinstein,
T. Guillaume,
P. M. Cannon, and W. F. Anderson.
1999.
Targeting retroviral vectors to CD34-expressing cells: binding to CD34 does not catalyze virus-cell fusion.
Hum. Gene Ther.
10:545-557[CrossRef][Medline].
|
| 5.
|
Boerger, A. L.,
S. Snitkovsky, and J. A. T. Young.
1999.
Retroviral vectors preloaded with a viral receptor-ligand bridge protein are targeted to specific cell types.
Proc. Natl. Acad. Sci. USA
96:9867-9872[Abstract/Free Full Text].
|
| 6.
|
Bova-Hill, C.,
J. C. Olsen, and R. Swanstrom.
1991.
Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85.
J. Virol.
65:2073-2080[Abstract/Free Full Text].
|
| 7.
|
Chadwick, M. P.,
F. J. Morling,
F. L. Cosset, and S. J. Russell.
1999.
Modification of retroviral tropism by display of IGF-I.
J. Mol. Biol.
285:485-494[CrossRef][Medline].
|
| 8.
|
Chu, T. H., and R. Dornburg.
1995.
Retroviral vector particles displaying the antigen-binding site of an antibody enable cell-type-specific gene transfer.
J. Virol.
69:2659-2663[Abstract].
|
| 9.
|
Chu, T. H., and R. Dornburg.
1997.
Toward highly efficient cell-type-specific gene transfer with retroviral vectors displaying single-chain antibodies.
J. Virol.
71:720-725[Abstract].
|
| 10.
|
Chu, T. H.,
I. Martinez,
W. C. Sheay, and R. Dornburg.
1994.
Cell targeting with retroviral vector particles containing antibody-envelope fusion proteins.
Gene Ther.
1:292-299[Medline].
|
| 11.
|
Cosset, F. L.,
F. J. Morling,
Y. Takeuchi,
R. A. Weiss,
M. K. Collins, and S. J. Russell.
1995.
Retroviral retargeting by envelopes expressing an N-terminal binding domain.
J. Virol.
69:6314-6322[Abstract].
|
| 12.
|
Cosset, F. L., and S. J. Russell.
1996.
Targeting retrovirus entry.
Gene Ther.
3:946-956[Medline].
|
| 13.
|
Culver, K. W.,
Z. Ram,
S. Wallbridge,
H. Ishii,
E. H. Oldfield, and R. M. Blaese.
1992.
In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors.
Science
256:1550-1552[Abstract/Free Full Text].
|
| 14.
|
Ekstrand, A. J.,
N. Sugawa,
C. D. James, and V. P. Collins.
1992.
Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails.
Proc. Natl. Acad. Sci. USA
89:4309-4313[Abstract/Free Full Text].
|
| 15.
|
Fielding, A. K.,
M. Maurice,
F. J. Morling,
F. L. Cosset, and S. J. Russell.
1998.
Inverse targeting of retroviral vectors: selective gene transfer in a mixed population of hematopoietic and nonhematopoietic cells.
Blood
91:1802-1809[Abstract/Free Full Text].
|
| 16.
|
Fisher, G. H.,
S. Orsulic,
E. Holland,
W. P. Hively,
Y. Li,
B. C. Lewis,
B. O. Williams, and H. E. Varmus.
1999.
Development of a flexible and specific gene delivery system for production of murine tumor models.
Oncogene
18:5253-5260[CrossRef][Medline].
|
| 17.
|
Garcia de Palazzo, I. E.,
G. P. Adams,
P. Sundareshan,
A. J. Wong,
J. R. Testa,
D. D. Bigner, and L. M. Weiner.
1993.
Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas.
Cancer Res.
53:3217-3220[Abstract/Free Full Text].
|
| 18.
|
Hall, F. L.,
E. M. Gordon,
L. Wu,
N. L. Zhu,
M. J. Skotzko,
V. A. Starnes, and W. F. Anderson.
1997.
Targeting retroviral vectors to vascular lesions by genetic engineering of the MoMLV gp70 envelope protein.
Hum. Gene Ther.
8:2183-2192[Medline].
|
| 19.
|
Han, X.,
N. Kasahara, and Y. W. Kan.
1995.
Ligand-directed retroviral targeting of human breast cancer cells.
Proc. Natl. Acad. Sci. USA
92:9747-9751[Abstract/Free Full Text].
|
| 20.
|
Hatziioannou, T.,
E. Delahaye,
F. Martin,
S. J. Russell, and F. L. Cosset.
1999.
Retroviral display of functional binding domains fused to the amino terminus of influenza hemagglutinin.
Hum. Gene Ther.
10:1533-1544[CrossRef][Medline].
|
| 21.
|
Holland, E. C.,
W. P. Hively,
R. A. DePinho, and H. E. Varmus.
1998.
A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice.
Genes Dev.
12:3675-3685[Abstract/Free Full Text].
|
| 22.
|
Huang, H. S.,
M. Nagane,
C. K. Klingbeil,
H. Lin,
R. Nishikawa,
X. D. Ji,
C. M. Huang,
G. N. Gill,
H. S. Wiley, and W. K. Cavenee.
1997.
The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling.
J. Biol. Chem.
272:2927-2935[Abstract/Free Full Text].
|
| 23.
|
Hurford, R. K.,
G. Dranoff,
R. C. Mulligan, and R. I. Tepper.
1995.
Gene therapy of metastatic cancer by in vivo retroviral gene targeting.
Nat. Genet.
10:430-435[CrossRef][Medline].
|
| 24.
|
Jiang, A.,
T. H. Chu,
F. Nocken,
K. Cichutek, and R. Dornburg.
1998.
Cell-type-specific gene transfer into human cells with retroviral vectors that display single-chain antibodies.
J. Virol.
72:10148-10156[Abstract/Free Full Text].
|
| 25.
|
Jiang, A., and R. Dornburg.
1999.
In vivo cell type-specific gene delivery with retroviral vectors that display single chain antibodies.
Gene Ther.
6:1982-1987[CrossRef][Medline].
|
| 26.
|
Jiang, A.,
H. Fisher,
R. J. Pomerantz, and R. Dornburg.
1999.
A genetically engineered spleen necrosis virus-derived retroviral vector that displays the HIV type 1 glycoprotein 120 envelope peptide.
Hum. Gene Ther.
10:2627-2636[CrossRef][Medline].
|
| 27.
|
Kasahara, N.,
A. M. Dozy, and Y. W. Kan.
1994.
Tissue-specific targeting of retroviral vectors through ligand-receptor interactions.
Science
266:1373-1376[Abstract/Free Full Text].
|
| 28.
|
Konishi, H.,
T. Ochiya,
K. A. Chester,
R. H. Begent,
T. Muto,
T. Sugimura,
M. Terada, and R. H. Begent.
1998.
Targeting strategy for gene delivery to carcinoembryonic antigen-producing cancer cells by retrovirus displaying a single-chain variable fragment antibody.
Hum. Gene Ther.
9:235-248[Medline].
|
| 29.
|
Lorimer, I. A.,
A. Keppler-Hafkemeyer,
R. A. Beers,
C. N. Pegram,
D. D. Bigner, and I. Pastan.
1996.
Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display.
Proc. Natl. Acad. Sci. USA
93:14815-14820[Abstract/Free Full Text].
|
| 30.
|
Malden, L. T.,
U. Novak,
A. H. Kaye, and A. W. Burgess.
1988.
Selective amplification of the cytoplasmic domain of the epidermal growth factor receptor gene in glioblastoma multiforme.
Cancer Res.
48:2711-2714[Abstract/Free Full Text].
|
| 31.
|
Marin, M.,
D. Noel,
S. Valsesia-Wittman,
F. Brockly,
M. Etienne-Julan,
S. Russell,
F. L. Cosset, and M. Piechaczyk.
1996.
Targeted infection of human cells via major histocompatibility complex class I molecules by Moloney murine leukemia virus-derived viruses displaying single-chain antibody fragment-envelope fusion proteins.
J. Virol.
70:2957-2962[Abstract].
|
| 32.
|
Martin, F.,
J. Kupsch,
Y. Takeuchi,
S. Russell,
F. L. Cosset, and M. Collins.
1998.
Retroviral vector targeting to melanoma cells by single-chain antibody incorporation in envelope.
Hum. Gene Ther.
9:737-746[Medline].
|
| 33.
|
Martin, F.,
S. Neil,
J. Kupsch,
M. Maurice,
F. L. Cosset, and M. Collins.
1999.
Retrovirus targeting by tropism restriction to melanoma cells.
J. Virol.
73:6923-6929[Abstract/Free Full Text].
|
| 34.
|
Matano, T.,
T. Odawara,
A. Iwamoto, and H. Yoshikura.
1995.
Targeted infection of a retrovirus bearing a CD4-Env chimera into human cells expressing human immunodeficiency virus type 1.
J. Gen. Virol.
76:3165-3169[Abstract/Free Full Text].
|
| 35.
|
Maurice, M.,
S. Mazur,
F. J. Bullough,
A. Salvetti,
M. K. Collins,
S. J. Russell, and F. L. Cosset.
1999.
Efficient gene delivery to quiescent interleukin-2 (IL-2)-dependent cells by murine leukemia virus-derived vectors harboring IL-2 chimeric envelope glycoproteins.
Blood
94:401-410[Abstract/Free Full Text].
|
| 36.
|
Miller, D. G.,
M. A. Adam, and A. D. Miller.
1990.
Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.
Mol. Cell. Biol.
10:4239-4242[Abstract/Free Full Text].
|
| 37.
|
Moscatello, D. K.,
M. Holgado-Madruga,
A. K. Godwin,
G. Ramirez,
G. Gunn,
P. W. Zoltick,
J. A. Biegel,
R. L. Hayes, and A. J. Wong.
1995.
Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors.
Cancer Res.
55:5536-5539[Abstract/Free Full Text].
|
| 38.
|
Nguyen, T. H.,
J. C. Pages,
D. Farge,
P. Briand, and A. Weber.
1998.
Amphotropic retroviral vectors displaying hepatocyte growth factor-envelope fusion proteins improve transduction efficiency of primary hepatocytes.
Hum. Gene Ther.
9:2469-2479[CrossRef][Medline].
|
| 39.
|
Nilson, B. H.,
F. J. Morling,
F. L. Cosset, and S. J. Russell.
1996.
Targeting of retroviral vectors through protease-substrate interactions.
Gene Ther.
3:280-286[Medline].
|
| 40.
|
Nishikawa, R.,
X. D. Ji,
R. C. Harmon,
C. S. Lazar,
G. N. Gill,
W. K. Cavenee, and H. J. Huang.
1994.
A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity.
Proc. Natl. Acad. Sci. USA
91:7727-7731[Abstract/Free Full Text].
|
| 41.
|
Peng, K. W.,
F. J. Morling,
F. L. Cosset,
G. Murphy, and S. J. Russell.
1997.
A gene delivery system activatable by disease-associated matrix metalloproteinases.
Hum. Gene Ther.
8:729-738[Medline].
|
| 42.
|
Peng, K. W.,
R. G. Vile,
F. L. Cosset, and S. J. Russell.
1999.
Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors.
Gene Ther.
6:1552-1557[CrossRef][Medline].
|
| 43.
|
Roe, T.,
T. C. Reynolds,
G. Yu, and P. O. Brown.
1993.
Integration of murine leukemia virus DNA depends on mitosis.
EMBO J.
12:2099-2108[Medline].
|
| 44.
|
Russell, S. J.,
R. E. Hawkins, and G. Winter.
1993.
Retroviral vectors displaying functional antibody fragments.
Nucleic Acids Res.
21:1081-1085[Abstract/Free Full Text].
|
| 45.
|
Schaefer-Klein, J.,
I. Givol,
E. V. Barsov,
J. M. Whitcomb,
M. Van Brocklin,
D. N. Foster,
M. J. Federspiel, and S. H. Hughes.
1998.
The EV-O-derived cell line DF-1 supports the efficient replication of avian leukosis-sarcoma viruses and vectors.
Virology
248:305-311[CrossRef][Medline].
|
| 46.
|
Schnierle, B. S.,
D. Moritz,
M. Jeschke, and B. Groner.
1996.
Expression of chimeric envelope proteins in helper cell lines and integration into Moloney murine leukemia virus particles.
Gene Ther.
3:334-342[Medline].
|
| 47.
|
Snitkovsky, S., and J. A. T. Young.
1998.
Cell-specific viral targeting mediated by a soluble retroviral receptor-ligand fusion protein.
Proc. Natl. Acad. Sci. USA
95:7063-7068[Abstract/Free Full Text].
|
| 48.
|
Somia, N. V.,
M. Zoppe, and I. M. Verma.
1995.
Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery.
Proc. Natl. Acad. Sci. USA
92:7570-7574[Abstract/Free Full Text].
|
| 49.
|
Sugawa, N.,
A. J. Ekstrand,
C. D. James, and V. P. Collins.
1990.
Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas.
Proc. Natl. Acad. Sci. USA
87:8602-8606[Abstract/Free Full Text].
|
| 50.
|
Thomas, D., and R. A. Bradshaw.
1997.
Differential utilization of SHCA tyrosine residues and functional domains in the transduction of epidermal growth factor-induced mitigen-activated protein kinase activation in 293T cells and nerve growth factor-induced neurite outgrowth in PC12 cells identification of a new GRB2-center-DOT-SOS1 binding site.
J. Biol. Chem.
272:22293-22299[Abstract/Free Full Text].
|
| 51.
|
Valsesia-Wittmann, S.,
A. Drynda,
G. Deleage,
M. Aumailley,
J. M. Heard,
O. Danos,
G. Verdier, and F. L. Cosset.
1994.
Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors.
J. Virol.
68:4609-4619[Abstract/Free Full Text].
|
| 52.
|
Valsesia-Wittmann, S.,
F. J. Morling,
T. Hatziioannou,
S. J. Russell, and F. L. Cosset.
1997.
Receptor co-operation in retrovirus entry: recruitment of an auxiliary entry mechanism after retargeted binding.
EMBO J.
16:1214-1223[CrossRef][Medline].
|
| 53.
|
Valsesia-Wittmann, S.,
F. J. Morling,
B. H. Nilson,
Y. Takeuchi,
S. J. Russell, and F. L. Cosset.
1996.
Improvement of retroviral retargeting by using amino acid spacers between an additional binding domain and the N terminus of Moloney murine leukemia virus SU.
J. Virol.
70:2059-2064[Abstract].
|
| 54.
|
Verma, I. M., and N. Somia.
1997.
Gene therapypromises, problems and prospects.
Nature
389:239-242[CrossRef][Medline].
|
| 55.
|
Wong, A. J.,
J. M. Ruppert,
S. H. Bigner,
C. H. Grzeschik,
P. A. Humphrey,
D. S. Bigner, and B. Vogelstein.
1992.
Structural alterations of the epidermal growth factor receptor gene in human gliomas.
Proc. Natl. Acad. Sci. USA
89:2965-2969[Abstract/Free Full Text].
|
| 56.
|
Yamazaki, H.,
Y. Fukui,
Y. Ueyama,
N. Tamaoki,
T. Kawamoto,
S. Taniguchi, and M. Shibuya.
1988.
Amplification of the structurally and functionally altered epidermal growth factor receptor gene (c-erbB) in human brain tumors.
Mol. Cell. Biol.
8:1816-1820[Abstract/Free Full Text].
|
| 57.
|
Zhao, Y.,
L. Zhu,
S. Lee,
L. Li,
E. Chang,
N. W. Soong,
D. Douer, and W. F. Anderson.
1999.
Identification of the block in targeted retroviral-mediated gene transfer.
Proc. Natl. Acad. Sci. USA
96:4005-4010[Abstract/Free Full Text].
|
| 58.
|
Zingler, K., and J. A. T. Young.
1996.
Residue Trp-48 of Tva is critical for viral entry but not for high-affinity binding to the SU glycoprotein of subgroup A avian leukosis and sarcoma viruses.
J. Virol.
70:7510-7516[Abstract].
|
Journal of Virology, October 2000, p. 9540-9545, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Melikyan, G. B., Barnard, R. J. O., Abrahamyan, L. G., Mothes, W., Young, J. A. T.
(2005). Imaging individual retroviral fusion events: From hemifusion to pore formation and growth. Proc. Natl. Acad. Sci. USA
102: 8728-8733
[Abstract]
[Full Text]
-
Narayan, S., Young, J. A. T.
(2004). Reconstitution of retroviral fusion and uncoating in a cell-free system. Proc. Natl. Acad. Sci. USA
101: 7721-7726
[Abstract]
[Full Text]
-
Melikyan, G. B., Barnard, R. J. O., Markosyan, R. M., Young, J. A. T., Cohen, F. S.
(2004). Low pH Is Required for Avian Sarcoma and Leukosis Virus Env-Induced Hemifusion and Fusion Pore Formation but Not for Pore Growth. J. Virol.
78: 3753-3762
[Abstract]
[Full Text]
-
Narayan, S., Barnard, R. J. O., Young, J. A. T.
(2003). Two Retroviral Entry Pathways Distinguished by Lipid Raft Association of the Viral Receptor and Differences in Viral Infectivity. J. Virol.
77: 1977-1983
[Abstract]
[Full Text]
-
Jones, K. S., Nath, M., Petrow-Sadowski, C., Baines, A. C., Dambach, M., Huang, Y., Ruscetti, F. W.
(2002). Similar Regulation of Cell Surface Human T-Cell Leukemia Virus Type 1 (HTLV-1) Surface Binding Proteins in Cells Highly and Poorly Transduced by HTLV-1-Pseudotyped Virions. J. Virol.
76: 12723-12734
[Abstract]
[Full Text]
-
Knauss, D. J., Young, J. A. T.
(2002). A Fifteen-Amino-Acid TVB Peptide Serves as a Minimal Soluble Receptor for Subgroup B Avian Leukosis and Sarcoma Viruses. J. Virol.
76: 5404-5410
[Abstract]
[Full Text]
-
Snitkovsky, S., Niederman, T. M. J., Mulligan, R. C., Young, J. A. T.
(2001). Targeting Avian Leukosis Virus Subgroup A Vectors by Using a TVA-VEGF Bridge Protein. J. Virol.
75: 1571-1575
[Abstract]
[Full Text]
Top
Abstract
Introduction
