FASEB J. Pierce now sold as Thermo Scientific
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by DOUKAS, J.
Right arrow Articles by SOSNOWSKI, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by DOUKAS, J.
Right arrow Articles by SOSNOWSKI, B. A.
(The FASEB Journal. 1999;13:1459-1466.)
© 1999 FASEB

Retargeted delivery of adenoviral vectors through fibroblast growth factor receptors involves unique cellular pathways

JOHN DOUKAS1, DIANA K. HOGANSON, MICHAEL ONG, WENBIN YING, DAVID L. LACEY*, ANDREW BAIRD, GLENN F. PIERCE and BARBARA A. SOSNOWSKI

* Amgen, Inc., Thousand Oaks, California 91320, USA; and Selective Genetics, Inc., San Diego, California 92121, USA

1Correspondence: Selective Genetics, Inc., 11035Roselle St., San Diego, CA 92121, USA. E-mail: jdoukas{at}selectivegenetics.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major goal of gene therapy is to improve target specificity by delivering vectors through alternative cellular receptors. We previously reported that adenoviral vector delivery through basic fibroblast growth factor (FGF2) receptors enhances both cellular transduction and in vivo efficacy. We now present studies addressing the cellular pathways and mechanisms underlying these events. Cellular receptors for adenoviruses are not required for transduction by FGF2-retargeted vectors. Moreover, {alpha}V integrins can antagonize FGF2 retargeting, in contrast to their obligatory role in non-retargeted vector delivery. By contrast, high-affinity FGF receptors, which are overexpressed on potential tumor targets, are required for FGF2-retargeted transduction. Low-affinity heparan sulfate proteoglycan interactions, however, are not a prerequisite, in marked contrast to their obligatory role in FGF2 mitogenic signaling. By comparing receptor expression and ligand binding with transgene expression, we also demonstrate that FGF2 retargeting enhances transduction by mechanisms other than increasing the number of targeted cells. Rather, the use of alternative targeting ligands supports the conclusion that specific receptor interactions and intracellular events serve to enhance transgene expression. Together, these studies highlight the unique delivery and transduction pathways used by FGF2-retargeted adenoviruses, and help define the basis for their enhanced in vivo efficacy.—Doukas, J., Hoganson, D. K., Ong, M., Ying, W., Lacey, D. L., Baird, A., Pierce, G. F., Sosnowski, B. A. Retargeted delivery of adenoviral vectors through fibroblast growth factor receptors involves unique cellular pathways.


Key Words: gene therapy • cancer • integrin • affinity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE USE OF recombinant adenoviral vectors for gene therapy is limited by the widespread cellular expression of adenoviral receptors, which can lead to inappropriate vector targeting to the liver and other organs (1 , 2) . Conversely, some potential targets may be resistant to gene transfer due to low receptor expression (3 4 5) . Clearly, a means is needed to both ablate normal adenoviral tropism and introduce novel cellular specificity. In addition, the ability to enhance transduction efficiency would both improve therapeutic efficacy and allow for the use of reduced virus volumes, thereby reducing potential viral-associated toxicity. Several approaches have been proposed for altering adenoviral tropism (6 7 8 9 10 11 12 13) . However, many of the targeting receptors selected are ubiquitously expressed in vivo, and would therefore result in extensive nonspecific transduction. Conversely, targeting to receptors expressed by highly specific cell populations, such as the T cell antigen CD3 (12) , would greatly limit the general utility of adenoviruses for gene therapy applications.

Recently we described a successful solution to viral retargeting using a member of the fibroblast growth factor (FGF)2 family (14 , 15) . Basic FGF (FGF2) was chemically conjugated to a neutralizing anti-adenoviral antibody in order to both ablate normal viral tropism and confer FGF2 receptor specificity. In these initial studies, FGF2-retargeted vectors were observed to transduce cells at higher levels compared with non-retargeted vectors. Furthermore, when adenoviral vectors encoding therapeutic transgenes were administered to tumor-bearing animals, the clinical benefit of enhanced transduction was demonstrated as significantly improved survival rates in groups treated with FGF2-retargeted compared with non-retargeted vectors (16 , 17) .

Although these reports established the value of FGF2 retargeting for adenoviral gene therapy, they did not fully characterize the underlying receptor or cellular pathways. For example, adenoviruses normally transduce cells via interactions with both the coxsackie adenovirus receptor (CAR) (2 , 18) and the integrins {alpha}Vß3 and {alpha}Vß5 (19) . By contrast, FGF2 mitogenic signaling involves a cooperative interaction between low-affinity heparan sulfate proteoglycans (HSPG) and a family of high-affinity tyrosine kinases termed FGFR1-R4 (20) . The ability of FGF2- retargeted vectors to transduce specific cell populations is predicated on the expression pattern of these tyrosine kinases. High-affinity FGF receptors (FGFR) are down-regulated in normal adult tissues but highly up-regulated in proliferating tumors, angiogenic endothelium, and wound repair sites (21 22 23 24) . However, to better understand the mechanisms by which FGF2 retargeting enhances adenoviral transduction and in vivo efficacy, a more complete understanding of the cellular pathways that FGF2-retargeted vectors follow is required.

To characterize these pathways, we have compared non-retargeted and retargeted transduction under various experimental conditions. These studies reveal a unique and unexpected pattern of receptor involvement for FGF2-mediated adenovirus delivery. In addition, the ability of FGF2 retargeting to enhance transduction is shown not to simply reflect enhanced delivery to an abundantly expressed receptor system. Rather, a more complex pattern of receptor–ligand interactions and intracellular events appears to govern enhanced transgene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines and viruses
Tumor cell lines included A549 (human lung carcinoma, American Type Culture Collection, Manassas, Va.), B16F0 (murine melanoma, gift of Lou Weiner, Fox Chase Cancer Center, Philadelphia, Pa.), BxPC-3 (human pancreatic adenocarcinoma, ATCC), HCT116 (human colon carcinoma, ATCC), K-562 (human chronic myelogenous leukemia, ATCC), KM12 (human colorectal carcinoma, NCI), KM20L2 (human colorectal carcinoma, NCI), OVCAR-5 (human ovarian carcinoma, NCI), PANC-1 (human pancreatic epithelioid carcinoma, ATCC), SKOV3.ip1 (human ovarian carcinoma, gift of Janet Price, M.D. Anderson Cancer Center, Houston, Tex.), and RENCA (murine renal carcinoma, gift of Marston Manthorpe, Vical, Inc., San Diego, Calif.). CHO-K1-FR2 cells (murine FGFR2 and HSPG positive) and CHO-745-FR1 cells (murine FGFR1 positive and HSPG negative) were gifts of Jeff Esko (University of California at San Diego) and Avner Yayon (Weizmann Institute, Israel). Cells were grown in either RPMI medium containing 10% fetal bovine serum (FBS) (A549, BxPC-3, HCT116, K-562, KM12, KM20L2, OVCAR-5), Dulbecco's modified Eagle's medium containing 10% FBS (B16F0, PANC-1, SKOV3.ip1, RENCA), or Ham's F12 medium containing 10% FBS (CHO). E1-, E3-deleted type 5 adenoviruses encoding ß-galactosidase (AdlacZ) or green fluorescence protein (AdGFP) were obtained from Molecular Medicines LCC (San Diego, Calif.) or Quantum Biologics (Montreal, Quebec) and prepared as purified lots of known plaque-forming units and particle numbers using standard techniques.

Ligands and conjugates
Recombinant FGF2 and keratinocyte growth factor (KGF) were produced and purified as described previously (25 , 26) . The anti-knob antibody 1D6.14 (gift of David Curiel, University of Alabama at Birmingham) was prepared as Fab fragments (Fab), which were then conjugated to FGF2 to yield FGF2-Fab, as described previously (14) . Similar techniques were used to conjugate Fab to KGF or the monoclonal anti-FGFR1 antibody 11A8 in order to yield KGF-Fab or 11A8-Fab, respectively. Finally, FGF2-Fab was iodinated by the lactoperoxidase method as described previously (27) .

Cell transduction
Anchorage-dependent cells were plated into 12-well cluster plates 18 h prior to experimental use so as to yield rapidly proliferating cultures at assay. Adenoviruses were ligated to ligand-Fab conjugates by incubation at room temperature for 30 min. Virus volume was determined based on the desired multiplicity of infection (MOI), and ligand-Fab conjugate volume based on the desired ligand-Fab:virus ratio. Unless otherwise stated, experiments used 2.5 x 104 cells/well treated at 300 MOI and 10:1 Fab:knob monomer ratio. Cultures were treated for 60 min at 37°C with 250 µl volumes of either buffer, adenovirus, or adenovirus ligated to Fab conjugates (FGF2-Ad, KGF-Ad, or 11A8-Ad) in medium containing 2% FBS. After two rinses, cultures were then incubated for 24–72 h with complete medium prior to transgene analysis as described below. Alternatively, K-562 cells, which are a suspension line, were resuspended at 2 x 104/ml, treated in microcentrifuge test tubes as outlined above, and placed in 12-well cluster plates for further culture prior to analysis.

Receptor blocking experiments
To assess receptor usage, the following reagents were included with vector treatments as competitive inhibitors of ligand binding: purified recombinant knob protein (10 µg/ml, gift of David Curiel), GRGDSP peptide (100 µg/ml; Life Technologies, Grand Island, N.Y.), FGF2 (5 µg/ml), recombinant FGFR1 (10 µg/ml; Austral Biologics, San Ramon, Calif.), heparin (100 µg/ml, Sigma Chemicals, St. Louis, Mo.), and rabbit anti-FGF1 (#F5521) and -FGF2 (#F3393, Sigma Chemicals) neutralizing antibodies (1:200 final dilution). Anti-FGF2 antibodies and dilutions were selected based on their ability to neutralize FGF2-induced mitogenesis of bovine aortic endothelial cells, but not to inhibit FGF2 binding to heparin sulfate as shown by both heparin-Sepharose high-performance liquid chromatography and a capture assay using immobilized heparin-bovine serum albumin (data not shown). Alternatively, cells were pretreated for 30 min prior to viral transduction with the anti-{alpha}V integrin neutralizing antibody MAB-1970 (Chemicon, Temecula, Calif.) at a 1:25 final dilution, and in addition this antibody was included with vector treatments.

Transgene expression analysis
For analysis of AdlacZ-treated cultures, cells were rinsed with phosphate-buffered saline (PBS), followed by lysis in 20 mM phosphate buffer containing 0.6% Triton X-100 (pH 7). Protein and ß-galactosidase concentrations were then determined using commercial bicinchoninic acid (Pierce, Rockford, Ill.) and chemiluminescent assays (Clontech Labs., Palo Alto, Calif.), respectively. Data were converted to mU ß-galactosidase activity/mg protein, subtracted for background expression (buffer-treated controls), and are presented as either means ± SD (n=3) or normalized to AdlacZ values (% non-retargeted transgene expression). Statistically significant differences were determined using one-way analysis of variance and Fisher's LSD procedure (StatView, Abacus Concepts, Berkeley, Calif.).

For AdGFP-treated cultures, cells were harvested with trypsin/EDTA, rinsed with PBS, and fixed with 1% paraformaldehyde in PBS prior to analysis using a Becton-Dickinson FACScan analyzer. Data are presented as the percent fluorescent cells (corrected for background controls) and the mean fluorescence intensity of positive cells (in arbitrary units).

Receptor expression and ligand binding analyses
Flow cytometric analyses were performed as described previously (28) , using rapidly proliferating cell cultures harvested nonenzymatically in order to preserve receptor expression. Primary antibodies included normal mouse immunoglobulin G (Sigma), RmcB (anti-CAR, gift of Jeffrey Bergelson, Children's Hospital of Philadelphia), LM609 (anti-{alpha}Vß3, Chemicon), P1F6 (anti-{alpha}Vß5, Chemicon), and the anti-pan FGFR antibody Ab6 (29) . For FGFR detection, cells were pretreated for 30 min with 1% paraformaldehyde in PBS, as fixation is required in order to reveal Ab6 binding epitopes. Data are presented as described above.

For receptor binding studies, rapidly proliferating cells were harvested nonenzymatically, rinsed three times with Dulbecco's modified Eagle's medium containing 0.05% bovine serum albumin, and incubated with 125I-FGF2-Fab (0.16 µCi/5 x 105 cells/120 µl volume) for 60 min on ice. After five rinses in cold buffer, cell-associated radioactivity was determined using a gamma counter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FGF2 retargeting involves integrin but not CAR viral receptors
We first determined the role of viral receptors in adenoviral transduction using SKOV3.ip1 cells, as this line was successfully used to demonstrate FGF2 retargeting in vivo (16 , 17) . Recombinant knob protein, the viral ligand for CAR, inhibited non-retargeted AdlacZ transduction by 76% (P<0.001), but minimally influenced FGF2-retargeted transduction (Fig. 1 ). These data demonstrate that FGF2 retargeting is relatively independent of knob–CAR interactions. By contrast, a neutralizing antibody directed against {alpha}V integrins (CD51), which inhibited non-retargeted transduction by 93%, enhanced FGF2-retargeted transduction by 60% (P<0.001).



View larger version (35K):
[in this window]
[in a new window]
  		Figure 1. Influence of viral receptor antagonists on transduction. SKOV3.ip1 and A549 cells were treated for 60 min with AdlacZ either alone (Ad) or conjugated to FGF2-Fab (FGF2-Ad) in the presence or absence of free knob protein, anti-CD51 antibody, or GRGDSP peptide. After an additional 72 h culture period, cells were analyzed for ß-galactosidase expression and protein content. Data were then converted to mU ß-galactosidase/mg protein, corrected for background expression (buffer-treated cells), and after normalization to the AdlacZ group value (such that this value equals 100%), presented as means ± SD (n=3). For both cell lines, those groups with integrin blockers differ from those without blockers by P<=0.009.

To further explore this unexpected enhancement of FGF2 retargeting, we next examined the influence of integrin blockers using A549 cells, as this line is commonly used to examine adenoviral receptor interactions (3 , 19 , 30) . As expected, the integrin binding peptide GRGDSP reduced non-retargeted transduction by 44% and, in agreement with our anti-CD51 data, enhanced retargeted transduction by 35% (P<0.009, Fig. 1 ). In other experiments, a combination of anti-{alpha}Vß3 and anti-{alpha}Vß5 integrin neutralizing antibodies also enhanced FGF2-retargeted transduction of A549 cells (data not shown). We conclude that in contrast to non-retargeted adenoviruses, FGF2-retargeted vectors need not engage integrins to transduce cells, and in fact may be most effective in the absence of such interactions.

FGF2 retargeting is dependent on high-affinity but not low-affinity FGF receptors
We examined whether FGF2-retargeted transduction involves high- and/or low-affinity FGF receptors. As shown in Fig. 2 , both free FGF2 and soluble FGFR1 antagonize FGF2-retargeted transduction. In control experiments, these reagents had no influence on non-retargeted viral transduction (data not shown). However, as these reagents may compete with FGF2 for binding to either HSPG or FGFR, these data only demonstrate a role for FGF receptors in retargeted transduction without differentiating between receptor types. We therefore used anti-FGF neutralizing antibodies capable of blocking high-affinity but not low-affinity receptor interactions. As a further assurance of our blocking only high-affinity receptor interactions, we also used the FGFR+/HSPG- cell line CHO-745-FR1 in these studies (31) . We observed that a polyclonal anti-FGF2 antibody inhibited FGF2-retargeted transduction of these cells; as expected, an anti-FGF1 (acidic FGF) antibody had no effect (Fig. 2) . Similar data were obtained using SKOV3.ip1 cells and a set of mouse monoclonal anti-FGF reagents (data not shown). We therefore conclude that FGF2-Ad are specifically targeted through high-affinity FGF receptors.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 2. Influence of FGF2 receptor antagonists on transduction. SKOV3.ip1 and CHO-745-FR1 cells were treated for 60 min with AdlacZ either alone (Ad) or conjugated to FGF2-Fab (FGF2-Ad) in the presence or absence of free FGF2 (an 800-fold excess to the FGF2 present on FGF2-Ad), FGFR1, or anti-FGF antibodies. SKOV3.ip1 cells were analyzed after 24 h and CHO-745-FR1 cells after 72 h additional culture; data are presented as in Fig. 1 . For SKOV3.ip1 cells, the FGF2-Ad group differs significantly from all others by P <= 0.004. For CHO-K1-FR2 cells, the FGF2-Ad group differs significantly from all others (P<=0.01) except for the anti-FGF1 group.

As FGF2-mediated transduction was accomplished using an HSPG-negative cell line, these data also demonstrate that HSPG are not required for FGF2 retargeting, an unexpected finding that contrasts with previous studies of FGF2 mitogenic signaling (20 , 31) . To elaborate on this finding, we compared the FGF2 responsiveness of FGFR+/HSPG+ CHO-K1-FR2 cells with that of FGFR+/HSPG- CHO-745-FR1 cells. As shown in Fig. 3 , FGF2 retargeting enhanced transduction of both cell lines, confirming our previous demonstration that FGF2 retargeting can occur in the absence of low-affinity interactions. Furthermore, free heparin sulfate inhibited FGF2-AdlacZ transduction of HSPG+ cells, as would be predicted due to its competition with cellular HSPG for FGF2 binding. By contrast, free heparin enhanced retargeted transduction of CHO-745-FR1 cells, suggesting that low-affinity interactions can enhance FGF2-mediated viral delivery.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 3. FGF2-retargeted transduction of HSPG+/- cell lines. CHO-K1-FR2 cells (HSPG+) and CHO-745-FR1 cells (HSPG-) were treated for 60 min with AdlacZ either alone (Ad) or conjugated to FGF2-Fab (FGF2-Ad) in the presence or absence of heparin sulfate. After a 72 h culture period, experiments were analyzed and data are presented as in Fig. 1 . For both cell lines, the addition of heparin leads to significant differences in FGF2-Ad groups (P<=0.001) but not in non-retargeted Ad groups.

FGF2 retargeting does not correlate with extent of FGFR expression
Having defined the receptors involved in FGF2-retargeted transduction, we next addressed the mechanism(s) by which retargeting increases transgene expression. As shown in Table 1 , FGF2 retargeting enhanced transgene expression by a panel of cell lines selected to reflect various tumor types, although the extent of enhanced expression varied between 225% and 2,233% of that observed using non-retargeted AdlacZ. These results suggest that cell-specific characteristics such as receptor density may regulate transduction efficiency.


View this table:
[in this window]
[in a new window]
  		Table 1. FGF2-retargeted transduction of various tumor cell linesa

To address the contributions of receptor density to transduction efficiency, we determined the FGF and viral receptor expression of several cell lines. As shown in Table 2 , CAR, integrin, and FGFR expression vary considerably between these lines. More important, the ability of FGF2 to enhance transgene expression (as shown in Table 1 ) could not be correlated to the relative expression of any single receptor. For example, whereas FGFR expression could be ranked as A549> KM20L2> SKOV3.ip1> KM12, FGF2-enhanced transduction ranked SKOV3.ip1> A549 >= KM12 > KM20L2. Therefore, the extent of FGFR expression does not appear to be the sole determinant of FGF2-enhanced transgene expression.


View this table:
[in this window]
[in a new window]
  		Table 2. Tumor cell receptor expression and FGF2-Fab bindinga

To further explore the relationship of ligand binding to transduction, we also used iodinated FGF2-Fab in order to determine total cellular binding of this ligand to both high-affinity FGFR and low-affinity HSPG. We observed that both SKOV3.ip1 and A549 cells bind similar levels of 125I-FGF2-Fab (Table 2) . As our tumor cell survey data revealed that FGF2 retargeting enhances transgene expression much more effectively for SKOV3.ip1 than A549 cells, these data also support the conclusion that cellular mechanisms other than receptor density contribute to the extent of transgene expression.

FGF2 retargeting increases the level of transgene expression
We next used flow cytometry to further characterize the ability of FGF2 retargeting to enhance transgene expression. As shown in Table 3 , three patterns of transgene expression were seen. The predominant pattern observed, and typified by SKOV3.ip1 cells, consists of increased numbers of transduced cells with higher transgene expression levels for FGF2- retargeted vs. non-retargeted transduction. A second pattern, typified by KM20L2 cells, showed equal numbers of transduced cells but higher transgene expression levels. Finally, KM12 cells revealed a third pattern of decreased numbers of transduced cells, but higher transgene expression levels. These data further suggest that FGF2 retargeting enhances transduction not simply by increasing the number of targeted cells, but rather via other cellular mechanisms that lead to enhanced transgene expression.


View this table:
[in this window]
[in a new window]
  		Table 3. Flow cytometric analysis of FGF2-retargeted transductiona

Retargeted transduction can be achieved using other FGF ligands
To further delineate the cellular pathways underlying enhanced transgene expression, we delivered adenoviruses to cells using alternative FGF receptor ligands. These included KGF (FGF7), a potent mitogen that signals through the high-affinity receptor FGFR2b (32 , 33) , and 11A8, an anti-FGFR1 antibody that is internalized after receptor binding but does not induce mitogenesis (34) . We also used HCT116 and KM12 cells, which express appropriate receptors for these targeting ligands (35) . As shown in Fig. 4 , AdlacZ conjugation to either FGF2-Fab or KGF-Fab enhanced transgene expression by both cell lines. In contrast, conjugation to 11A8-Fab did not enhance transduction over that obtained using virus alone. These data further support the conclusion that the extent of cellular transduction observed after FGF2 retargeting does not simply result from vector binding to FGFR. Rather, the nature of ligand–receptor interaction governs the extent of transgene expression. Finally, as we also observed that free FGF2 or KGF did not enhance non-retargeted AdlacZ transduction, mitogenic signaling does not appear to be the required event in enhanced transgene expression.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 4. Comparison of alternative FGFR ligands as retargeting agents. HCT116 and KM12 cells were treated for 60 min with AdlacZ either alone (Ad) or conjugated to FGF2-Fab (FGF2-Ad), KGF-Fab (KGF-Ad), or 11A8-Fab (11A8-Ad). In some groups, free FGF2 or KGF was also added to treatments at equivalent concentrations as that present in FGF2-Ad or KGF-Ad groups (7 ng/ml). Experiments were then analyzed after a 72 h culture period; data presented as in Fig. 1 . For HCT116 cells, the FGF2-Ad, KGF-Ad, and 11A8-Ad groups differ from each other and the Ad group by P<=0.03. For KM12 cells, the FGF2-Ad and KGF-Ad groups differ from each other and the Ad group by P<=0.002.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Retargeting adenoviral vector delivery through FGF receptors allows for enhanced in vivo efficacy, in part due to an enhanced cellular transduction (14 15 16 17) . The present studies explore the mechanisms underlying these observations and reveal that FGF2-conjugated adenoviral transduction occurs via distinct pathways compared with both non-retargeted vector transduction and FGF2 mitogenic signaling. The unique characteristics of FGF2 retargeting offer several advantages regarding gene therapy applications, including the ability to preferentially target proliferating tumor cells based on their overexpression of high-affinity FGF receptors.

A primary characteristic that establishes FGF2-retargeted transduction as unique is receptor usage. Non-retargeted adenovirus transduction requires engagement of both CAR and {alpha}V integrins (2 , 18 , 19) , whereas FGF2 mitogenic signaling requires cooperative interactions between HSPG and FGFR (20) . By contrast, we observed that only FGFR interactions are prerequisite for FGF2-retargeted transduction. Independence from CAR binding was anticipated, as a neutralizing antibody directed against this receptor's ligand was used to introduce FGF2 onto viral capsids. Indeed, this is a key component of our vector delivery strategy, as it serves to limit the general transduction of nontarget organs after systemic delivery of non-retargeted virus (1 , 2) . In preliminary in vivo studies, we have confirmed this benefit of FGF2 retargeting by demonstrating a reduced liver toxicity for FGF2-retargeted adenoviruses (36) .

A second benefit of this vector delivery design is that FGF-retargeted transduction depends on high-affinity binding to FGFR rather than only low-affinity binding to HSPG. HSPG are ubiquitously expressed (20 , 31) , and solely targeting these receptors would lead to nonspecific delivery and potential toxicity. FGFR, by contrast, are down-regulated in most tissues but highly up-regulated on rapidly proliferating tumor cells and angiogenic endothelium (21 22 23 24 , 35) . In fact, continuous intravenous infusion of FGF2 does not induce mitogenesis of normal endothelium or smooth muscle cells (21) , and FGF2- retargeted adenoviruses are not efficient transducers of quiescent endothelial cells in vitro (15) . Therefore, by using high-affinity FGF receptors for transduction, FGF2-conjugated vectors should specifically transduce the primary targets of cancer gene therapy.

The ability of FGF2 retargeting to proceed in the absence of HSPG interactions also distinguishes FGF2-mediated transduction from FGF2 mitogenic signaling. FGF2 mitogenic signaling requires HSPG to promote FGFR dimerization (20 , 31 , 37 , 38) , a key event in ligand internalization. By contrast, although HSPG interactions can enhance FGF2 retargeting, they are not required. We suggest that the trimeric form of the adenoviral fiber structure (18) allows for multiple FGF2-Fab molecules to bind any single knob unit. The resulting spatial immobilization of FGF2 on viral capsids may then directly promote receptor dimerization and thus substitute for the receptor clustering activity of HSPG. This mechanism would be analogous to that observed for anti-CD3-mediated T cell signaling, in which immobilized but not soluble antibodies trigger cellular proliferation via receptor clustering (39) . The ability of heparin sulfate or HSPG, when present, to enhance these interactions may be due to either their direct influence on FGF2 or FGFR (40 , 41) .

Perhaps our most striking observation regarding receptor usage, however, is that FGF2 retargeting is most efficient in the absence of integrin interactions. This is in direct contrast to the obligatory role of {alpha}V integrins in the internalization of non-retargeted adenoviruses (19) . We conclude that FGF2 internalization pathways, which involve receptor-mediated endocytosis, lysosomal processing, and ligand trafficking into the nucleus (20 , 42 , 43) , are more efficient than integrin-mediated pathways. Part of this efficiency may be due to the extreme high affinity of FGFR (Kd ~ 2 pM) (20) , which permits avid ligand binding at limiting ligand concentrations. Our observation that competitive inhibitors such as free FGF2 or FGFR1 were unable to completely abrogate FGF2-retargeted transduction supports this conclusion. Similarly, neutralizing anti-FGF2 antibodies, which are expected to bind FGF2 with only nanomolar affinities (44) , could only partially inhibit FGF2-retargeted transduction. By contrast, CAR and integrins bind their viral ligands with >=1000-fold lower affinities (18) , and therefore may require the involvement of multiple receptor species in order to mediate efficient transduction. This conclusion is also supported by a report that epidermal growth factor (EGF), which binds its receptor with >=100-fold lower affinity than that displayed by FGF2 (45) , can only efficiently retarget adenoviruses to cells highly expressing EGF receptors (11) . By contrast, we observed efficient FGF2-retargeted transduction over a relatively wide range of FGFR expression densities.

The potential widespread utility for FGF2-mediated adenovirus delivery was demonstrated by the successful targeting of a broad panel of tumor lines. Along with receptor expression and ligand binding data, these studies also demonstrated that the ability of FGF2 retargeting to enhance transduction does not result exclusively from a greater expression of FGF receptors compared with viral receptors. Rather, flow cytometric studies demonstrate that, although FGF2 retargeting can often increase the number of cells transduced within a given population, the overriding mechanism underlying enhanced transduction is enhanced transgene expression.

Enhanced transduction was also observed when KGF was used to retarget vectors. KGF binds to a specific high-affinity FGF receptor not recognized by FGF2 (32 , 33) , suggesting that the ability of FGF ligand retargeting to enhance transduction lies in some common characteristic of FGFR. The inability of the anti-FGFR antibody 11A8 to enhance transgene expression when used as a retargeting agent may relate to the fact that antibodies generally bind their antigens with nanomolar affinities (44) , and supports our hypothesis that one key to FGF ligand-enhanced transgene expression is the picomolar affinity constants displayed by FGFR. Alternatively, as FGF2 and KGF are mitogenic and 11A8 is not, some event(s) in receptor or intracellular signaling may influence transgene expression. However, we did observe that neither free FGF2 nor KGF enhanced non-retargeted viral transduction; therefore, if FGFR-mediated signaling does influence transduction, these events must be tightly linked to vector internalization and/or intracellular trafficking. Work is currently in progress to address these questions.

In conclusion, our studies support the concept that retargeting adenoviral delivery to FGF receptors will help overcome several limitations to the general utility of these vectors. These include unwanted native viral tropism, the inability to selectively target appropriate cell populations, and the requirement for very high virus concentrations. Furthermore, by using alternative conjugation methods, we may also provide superior delivery for other gene therapy agents. In fact, we have already used FGF receptors as targeting sites for DNA (25) and bacteriophages (46) . Finally, as FGF receptors are up-regulated on most proliferating cells, the potential targets for these approaches extend beyond cancer to indications such as tissue repair.


   ACKNOWLEDGMENTS
 
We thank Emelie Amburn, Gail Fieser, and Graham Fleurbaaij for their technical assistance. We also thank Jeffrey Bergelson, David Curiel, Jeff Esko, Marston Manthorpe, Janet Price, Lou Weiner, and Avner Yayon for their gifts of cells and reagents. This publication was made possible in part by NIH grant number R43CA79294 (J.D.).


   FOOTNOTES
 
2 Abbreviations: CAR, coxsackie adenovirus receptor; EGF, epidermal growth factor; FBS, fetal bovine serum; FGF, fibroblast growth factor; FGFR, FGF receptors; HSPG, heparan sulfate proteoglycans; KGF, keratinocyte growth factor; MOI, multiplicity of infection; PBS, phosphate-buffered saline.

Received for publication January 12, 1999. Revision received March 3, 1999.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Worgall, S., Wolff, G., Falck-Pedersen, E., Crystal, R. G. (1997) Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration. Hum. Gene Ther. 8,37-44[Medline]
  2. Tomko, R. P., Xu, R., Philipson, L. (1998) HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc. Natl. Acad. Sci. USA 94,3352-3356[Abstract/Free Full Text]
  3. Freimuth, P. (1996) A human cell line selected for resistance to adenovirus infection has reduced levels of the virus receptor. J. Virol. 70,4081-4085[Abstract]
  4. Zabner, J., Freimuth, P., Puga, A., Fabrega, A., Welsh, M. J. (1997) Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection. J. Clin. Invest. 100,1144-1149[Medline]
  5. Li, Y., Pong, R. C., Bergelson, J. M., Hall, M. C., Sagalowsky, A. I., Tseng, C. P., Wang, Z., Hsieh, J. T. (1999) Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy. Cancer Res 59,325-330[Abstract/Free Full Text]
  6. Wickham, T. J., Carrion, M. E., Kovesdi, I. (1995) Targeting of adenovirus penton base to new receptors through replacement of its RGD motif with other receptor-specific peptide motifs. Gene Ther 2,750-756[Medline]
  7. Douglas, J. T., Rogers, B. E., Rosenfeld, M. E., Michael, S. I., Feng, M., Curiel, D. T. (1996) Targeted gene delivery by tropism-modified adenoviral vectors. Nat. Biotechnol. 14,1574-1578[Medline]
  8. Wickham, T. J., Segal, D. M., Roelvink, P. W., Carrion, M. E., Lizonova, A., Lee, G. M., Kovesdi, I. (1996) Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies. J. Virol. 70,6831-6838[Abstract/Free Full Text]
  9. Wickham, T. J., Roelvink, P. W., Brough, D. E., Kovesdi, I. (1996) Adenovirus targeted to heparan-containing receptors increases its gene delivery efficiency to multiple cell types. Nat. Biotechnol. 14,1570-1573[Medline]
  10. Stevenson, S. C., Rollence, M., Marshall-Neff, J., McClelland, A. (1997) Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein. J. Virol. 71,4782-4790[Abstract]
  11. Watkins, S. J., Mesyanzhinov, V. V., Kurochkina, L. P., Hawkins, R. E. (1997) The `adenobody' approach to viral targeting: specific and enhanced adenoviral gene delivery. Gene Ther 4,1004-1012[Medline]
  12. Wickham, T. J., Lee, G. M., Titus, J. A., Sconocchia, G., Bakacs, T., Kovesdi, I., Segal, D. M. (1997) Targeted adenovirus-mediated gene delivery to T cells via CD3. J. Virol. 71,7663-7669[Abstract]
  13. Wickham, T. J., Haskard, D., Segal, D., Kovesdi, I. (1997) Targeting endothelium for gene therapy via receptors up-regulated during angiogenesis and inflammation. Cancer Immunol. Immunother. 45,149-151[Medline]
  14. Goldman, C. K., Rogers, B. E., Douglas, J. T., Sosnowski, B. A., Ying, W., Siegal, G. P., Baird, A., Campain, J. A., Curiel, D. T. (1997) Targeted gene delivery to Kaposi's sarcoma cells via the fibroblast growth factor receptor. Cancer Res 57,1447-1451[Abstract/Free Full Text]
  15. Reynolds, P. N., Miller, C. R., Goldman, C. K., Doukas, J., Sosnowski, B. A., Rogers, B. E., Gomez-Navarro, J., Pierce, G. F., Curiel, D. T., Douglas, J. T. (1998) Targeting adenoviral infection with basic fibroblast growth factor enhances gene delivery to vascular endothelial and smooth muscle cells. Tumor Targeting 3,156-168
  16. Rogers, B. E., Douglas, J. T., Sosnowski, B. A., Ying, W., Pierce, G., Buchsbaum, D. J., Della Manna, D., Baird, A., Curiel, D. T. (1998) Enhanced in vivo gene delivery to human ovarian cancer xenografts utilizing a tropism-modified adenovirus vector. Tumor Targeting 3,25-31
  17. Rancourt, C., Rogers, B. E., Sosnowski, B. A., Wang, M., Piche, A., Pierce, G. F., Alvarez, R. D., Siegal, G. P., Douglas, J. T., Curiel, D. T. (1998) Basic fibroblast growth factor enhancement of adenovirus-mediated delivery of the herpes simplex virus thymidine kinase gene results in augmented therapeutic benefit in a murine model of ovarian cancer. Clin. Cancer Res. 4,2455-2461[Abstract/Free Full Text]
  18. Nemerow, G. R., Cheresh, D. A., Wickham, T. J. (1994) Adenovirus entry into host cells: a role for {alpha}v integrins. Trends Cell Biol 4,52-55[Medline]
  19. Wickham, T. J., Mathias, P., Cheresh, D. A., Nemerow, G. R. (1993) Integrins {alpha}vß3 and {alpha}vß5 promote adenovirus internalization but not virus attachment. Cell 73,309-319[Medline]
  20. Klagsburn, M., Baird, A. (1991) A dual receptor system is required for basic fibroblast growth factor activity. Cell 67,229-231[Medline]
  21. Whalen, G. F., Shing, Y., Folkman, J. (1989) The fate of intravenously administered bFGF and the effect of heparin. Growth Factors 1,157-164[Medline]
  22. Lindner, V., Reidy, M. A. (1993) Expression of basic fibroblast growth factor and its receptor by smooth muscle cells and endothelium in injured rat arteries. An en face study. Circ. Res. 73,589-595[Abstract/Free Full Text]
  23. Takanami, I., Tanaka, F., Hashizume, T., Kodaira, S. (1997) Tumor angiogenesis in pulmonary adenocarcinomas: relationship with basic fibroblast growth factor, its receptor, and survival. Neoplasma 44,295-298[Medline]
  24. Wang, Y., Becker, D. (1997) Antisense targeting of basic fibroblast growth factor and fibroblast growth factor receptor-1 in human melanomas blocks intratumoral angiogenesis and tumor growth. Nat. Med. 3,887-893[Medline]
  25. Sosnowski, B. A., Gonzalez, A. M., Chandler, L. A., Buechler, Y. J., Pierce, G. F., Baird, A. (1996) Targeting DNA to cells with basic fibroblast growth factor (FGF2). J. Biol. Chem. 271,33647-33653[Abstract/Free Full Text]
  26. Housley, R. M., Morris, C. F., Boyle, W., Ring, B., Biltz, R., Tarpley, J. E., Aukerman, S. L., Devine, P. L., Whitehead, R. H., Pierce, G. F. (1994) Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract. J. Clin. Invest. 94,1764-1777
  27. Gonzalez, A. M., Carman, L. S., Ong, M., Ray, J., Gage, F. H., Shults, C. W., Baird, A. (1994) Storage, metabolism, and processing of 125I-fibroblast growth factor-2 after intracerebral injection. Brain Res 665,285-292[Medline]
  28. Doukas, J., Majno, G., Mordes, J. P. (1996) Anti-endothelial cell autoantibodies in BB rats with spontaneous and induced IDDM. Diabetes 45,1209-1216[Abstract]
  29. Gonzalez, A. M., Logan, A., Ying, W., Lappi, D. A., Berry, M., Baird, A. (1994) Fibroblast growth factor in the hypothalamic-pituitary axis: differential expression of fibroblast growth factor-2 and a high affinity receptor. Endocrinology 134,2289-2297[Abstract]
  30. Greber, U. F., Willetts, M., Webster, P., Helenius, A. (1993) Stepwise dismantling of adenovirus 2 during entry into cells. Cell 75,477-486[Medline]
  31. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., Ornitz, D. M. (1991) Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64,841-848[Medline]
  32. Bottaro, D. P., Rubin, J. S., Ron, D., Finch, P. W., Florio, C., Aaronson, S. A. (1990) Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors. J. Biol. Chem. 265,12767-12770[Abstract/Free Full Text]
  33. Ornitz, D. M., Xu, J., Colvin, J. S., McEwen, D. G., MacArthur, C. A., Coulier, F., Gao, G., Goldfarb, M. (1996) Receptor specificity of the fibroblast growth factor family. J. Biol. Chem. 271,15292-15297[Abstract/Free Full Text]
  34. Ying, W., Martineau, D., Kiefer, M., Lappi, D. A., and Baird, A. (1993) Characterization and activities of an anti-basic fibroblast growth factor receptor immunotoxin. Proc. Am. Assoc. Cancer Res. 34, 480 (abstr.)
  35. Chandler, L. A., Sosnowski, B. A., Greenlees, L., Aukerman, S. L., Baird, A., Pierce, G. F. (1999) Prevalent expression of functional fibroblast growth factor (FGF) receptors and FGF2 in human tumor cell lines. Int. J. Cancer 81,451-458[Medline]
  36. Gu, D., Hoganson, D. K., Printz, M., Gonzalez, A. M., Ying, W., Sosnowski, B. A., Curiel, D. T., Pierce, G. F., and Aukerman, S. L. (1998) Retargeting adenovirus vectors with FGF-2 leads to enhanced efficacy and decreased toxicity. Proc. Am. Assoc. Cancer Res. 39, 512 (abstr.)
  37. Rapraeger, A. C. (1995) In the clutches of proteoglycans: how does heparan sulfate regulate FGF binding?. Chem. Biol. 2,645-649[Medline]
  38. Venkataraman, G., Sasisekharan, V., Herr, A. B., Ornitz, D. M., Waksman, G., Cooney, C. L., Langer, R., Sasisekharan, R. (1996) Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation. Proc. Natl. Acad. Sci. USA 93,845-850[Abstract/Free Full Text]
  39. Geppert, T. D., Lipsky, P. E. (1987) Accessory cell independent proliferation of human T4 cells stimulated by immobilized monoclonal antibodies to CD3. J. Immunol. 138,1660-1666[Abstract]
  40. Herr, A. B., Ornitz, D. M., Sasisekharan, R., Venkataraman, G., Waksman, G. (1997) Heparin-induced self-association of fibroblast growth factor-2. Evidence for two oligomerization processes. J. Biol. Chem. 272,16382-16389[Abstract/Free Full Text]
  41. Givol, D., Yayon, A. (1992) Complexity of FGF receptors: genetic basis for structural diversity and functional specificity. FASEB J 6,3362-3369[Abstract]
  42. Gleizes, P. E., Noaillac-Depeyre, J., Amalric, F., Gas, N. (1995) Basic fibroblast growth factor (FGF-2) internalization through the heparan sulfate proteoglycans-mediated pathway: an ultrastructural approach. Eur. J. Cell Biol. 66,47-59[Medline]
  43. Prudovsky, I., Savion, N., Zhan, X., Friesel, R., Xu, J., Hou, J., McKeehan, W. L., Maciag, T. (1994) Intact and functional fibroblast growth factor (FGF) receptor-1 trafficks near the nucleus in response to FGF-1. J. Biol. Chem. 269,31720-31724[Abstract/Free Full Text]
  44. Bachmann, M. F., Kalinke, U., Althage, A., Freer, G., Burkhart, C., Roost, H. P., Aguet, M., Hengartner, H., Zinkernagel, R. M. (1997) The role of antibody concentration and avidity in antiviral protection. Science 276,2024-2027[Abstract/Free Full Text]
  45. Kwok, T. T., Sutherland, R. M. (1991) Differences in EGF related radiosensitisation of human squamous carcinoma cells with high and low numbers of EGF receptors. Br. J. Cancer 64,251-254[Medline]
  46. Larocca, D., Witte, A., Johnson, W., Pierce, G. F., Baird, A. (1998) Targeting bacteriophage to mammalian cell surface receptors for gene delivery. Hum. Gene Ther. 9,2393-2399[Medline]



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
D. Hoffmann and O. Wildner
Restriction of adenoviral replication to the transcriptional intersection of two different promoters for colorectal and pancreatic cancer treatment.
Mol. Cancer Ther., February 1, 2006; 5(2): 374 - 381.
[Abstract] [Full Text] [PDF]

Home page
 JBJSHome page
R.E. Schreiber, K. Blease, A. Ambrosio, E. Amburn, B. Sosnowski, and T.K. Sampath
Bone Induction by AdBMP-2/Collagen Implants
J. Bone Joint Surg. Am., May 1, 2005; 87(5): 1059 - 1068.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
M. Qin, B. Escuadro, S. Sharma, and R. K. Batra
Gene Transfer Mediated by Native versus Fibroblast Growth Factor-Retargeted Adenoviral Vectors into Lung Cancer Cells
Am. J. Respir. Cell Mol. Biol., March 1, 2005; 32(3): 211 - 217.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
V. W. van Beusechem, J. Grill, D. C. J. Mastenbroek, T. J. Wickham, P. W. Roelvink, H. J. Haisma, M. L. M. Lamfers, C. M. F. Dirven, H. M. Pinedo, and W. R. Gerritsen
Efficient and Selective Gene Transfer into Primary Human Brain Tumors by Using Single-Chain Antibody-Targeted Adenoviral Vectors with Native Tropism Abolished
J. Virol., February 22, 2002; 76(6): 2753 - 2762.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
R. Wiewrodt, A. P. Thomas, L. Cipelletti, M. Christofidou-Solomidou, D. A. Weitz, S. I. Feinstein, D. Schaffer, S. M. Albelda, M. Koval, and V. R. Muzykantov
Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells
Blood, February 1, 2002; 99(3): 912 - 922.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
R. K. Batra, S. M. Dubinett, B. W. Henkle, S. Sharma, and B. K. Gardner
Adenoviral Gene Transfer Is Inhibited by Soluble Factors in Malignant Pleural Effusions
Am. J. Respir. Cell Mol. Biol., May 1, 2000; 22(5): 613 - 619.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by DOUKAS, J.
Right arrow Articles by SOSNOWSKI, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by DOUKAS, J.
Right arrow Articles by SOSNOWSKI, B. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS