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Experimental Therapeutics |
Selective Genetics, Inc., San Diego, California 92121 [D. G., A. M. G., M. A. P., J. D., W. Y., M. D., D. K. H., B. A. S., A. B., S. E. A., G. F. P.], and Gene Therapy Program, University of Alabama at Birmingham, Birmingham, Alabama 35294 [D. T. C., J. T. D.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In rodent models, the majority of Ad vectors delivered i.v. is cleared rapidly (within the first 24 h) through the liver (19
, 20)
. Concomitantly, there is considerable transduction of hepatocytes and associated transgene expression (8, 9, 10)
. This is in part due to a high concentration of the Ad cellular receptor, CAR, in the rodent liver (21)
. Ad transgene expression rapidly declines over the first 7 days after Ad vector administration, but Ad transduction of hepatocytes is associated with significant liver toxicity as manifest by increased serum transaminases, hepatocellular necrosis, and inflammation (8
, 10
, 19)
. Retargeting of Ad away from its native tropism for CAR may abrogate this liver toxicity. We have developed a broadly useful method that retargets Ad by using a neutralizing Fab to the knob domain of the Ad fiber protein (12
, 22)
. The fiber protein is used by Ad for binding to its receptor, CAR. By attaching FGF2 as a targeting ligand to this Fab, this bifunctional molecule targets and redirects Ad cellular entry via high-affinity FGFRs and, additionally, blocks uptake through CAR (23). FGF2 binds FGFRs with extraordinarily high affinity (Kd, 
10-12 M) compared with most other ligand-receptor interactions. The increased affinity is due to the initial binding of the FGF ligand to cell surface heparan sulfate proteoglycans (low-affinity receptors: Kd, 10-9 M), followed by binding to, and dimerization of, the high-affinity tyrosine kinase receptors. In contrast, most other ligands used for targeting, including antibodies, bind at 3–4 logs lower affinity to their receptors.
Importantly, FGFRs are up-regulated in a number of diseases characterized by unwanted cellular proliferation, and many human malignancies contain elevated levels of one or more of the four recognized FGFRs (24, 25, 26, 27, 28, 29) . Although there are about 20 FGF ligands identified, most have specificity for specific splice variants of a subset of FGFRs (30) . FGF2, in contrast, is an injury-response molecule and can bind most splice variants of the FGFRs (30) , making it a more useful ligand for targeting FGFR-bearing malignant or injured cells. We have previously established that FGF2 targets condensed DNA in vitro (31) and have observed a greater than 10-fold increase in gene expression when FGF2-retargeted Ad was compared with Ad in delivering reporter genes or the TK gene to human Kaposi’s sarcoma, pancreatic cancer, and ovarian cancer cell lines in vitro (22 , 32) . We have also demonstrated enhanced antitumor activity when FGF-retargeted Ad was locally delivered to ovarian carcinoma in the peritoneal cavity (32) . Because this unexpectedly enhanced in vitro and in vivo potency implied greater selectivity and therapeutic benefit, we assessed whether the altered tropism of FGF2-Ad would show diminished toxicity in vivo. To establish whether the increased potency observed in vitro would translate into in vivo therapeutic benefit, FGF2-redirected Ad encoding the TK gene (AdTK) was also evaluated in mice challenged with tumor cells resistant to native Ad infection.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E1/E3-deleted type-5 adenoviral vectors encoding ßgal (Adßgal), green fluorescence protein (AdGFP), or thymidine kinase (AdTK) under control of cytomegalovirus immediate early promoter/enhancer were obtained from Molecular Medicines LCC (San Diego, CA), Quantum Biologicals (Montreal, Quebec), or David Curiel (University of Alabama at Birmingham), respectively. These vectors were then prepared as purified lots of known pfu and particle numbers using standard techniques (33) . The murine melanoma cell line B16F10 was obtained from the National Cancer Institute Tumor Cell Repository (Frederick, MD) and cultured in DMEM containing 10% FBS. B16F0 was obtained from Lou Weiner (Fox Chase Cancer Center, Philadelphia, PA).
In Vitro Transduction Studies.
Adenoviral vectors were incubated with FGF2-Fab' for 30 min and then diluted in DMEM containing 2% FBS for in vitro use. The total amount of virus needed was determined based on the desired MOI. To determine transduction efficiencies, rapidly proliferating cultures of B16F10 cells were treated for 60 min at 37°C with either nontargeted AdGFP or FGF2-conjugated AdGFP (FGF2-AdGFP) at various MOI in minimal volume. Cultures were then rinsed twice and incubated for 24 h with complete medium, at which point they were harvested with trypsin/EDTA, fixed with 1% paraformaldehyde in PBS, and examined using a Becton Dickinson FACScan analyzer. Data are presented as the percent of fluorescent cells (corrected for background controls) and their FI, in arbitrary units.
To determine transgene activity, FGF2-conjugated AdTK (FGF2-AdTK) were first constructed at various FGF2-Fab':knob monomer ratios. Rapidly proliferating cultures of B16F0 cells were then treated for 3 h at 37°C with either nontargeted AdTK or FGF2-AdTK at a MOI of 100. After a 24-h culture period in complete medium, cultures were treated with DMEM containing 10% FBS with or without 50 µm of GCV. After an additional culture period of 48 h, cell numbers were determined using a commercial assay kit based on the enzymatic conversion of the tetrazolium salt MTS (Promega, Madison, WI), and data were converted to percent of control (untreated cells).
In Vivo Targeting Studies.
All studies described herein have been approved by the Institutional Animal Care and Use Committee at Selective Genetics Incorporated and conform to all of the guidelines as set forth in the Guide for the Care and Use of Laboratory Animals (National Research Council; Washington D.C.). In vivo targeting of FGF2-Adßgal and Adßgal was assessed in female C57BL/6 mice. For preparation of FGF2-Adßgal or Adßgal, 77 µg of FGF2-Fab' or an equivalent volume of 0.9% NaCl was incubated for 30 min at room temperature with 2 x 1010 pfu of Adßgal to establish a 50:1 molar ratio of FGF2-Fab':Ad fiber monomers on the total number of Ad particles present. On day 0, 2 x 1010 pfu of either Adßgal or FGF2-Adßgal were injected i.v. via the lateral tail vein over a 30-s period in a final volume of 0.32 ml. Control mice received 0.32 ml of excipient. On days 1, 2, 4, 7, and 12 postinjection, three to six mice per group were sacrificed. Serum was collected by cardiac puncture for the analysis of transaminases (ALT, AST) and Alk Phos (BTS Laboratory, San Diego, CA.). The liver was removed, immediately snap-frozen in liquid nitrogen, stored at -80°C, and then processed for quantitative analysis of ß-Gal activity. A portion of liver was either (a) fixed for 4 h at 4°C in 10% neutral buffered formalin and then embedded in paraffin or (b) snap-frozen in OCT using isopentane precooled with dry ice and stored at -80. Statistical analyses were performed using one-way ANOVA and Fisher’s PLSD post hoc analysis.
To assess ex vivo antitumor activity, B16F0 cells were treated in suspension for 60 min at room temperature with either AdTK or FGF2-AdTK (33:1 FGF2-Fab': fiber monomer ratio) at a MOI of 50. Female BDF1 mice (n = 8/group) received 2 x 106 treated B16F0 cells implanted i.p. on day 0. Mice were then given GCV (CYTOVENE, Roche) or saline i.p. beginning on day 1, every day for 14 days, at a dose of 100 mg/kg.
For studies investigating in vivo therapeutic treatment, 1 x 106 of untreated B16F0 tumor cells were implanted i.p. on day 0 (n = 8 mice/group). On days 1 and 8, mice received 3 x 109 pfu of FGF2-AdTK or excipient. Mice were then treated with GCV or saline (i.p.) beginning on day 2, every day for 21 days, and followed for survival. Statistical analyses were performed using Kaplan-Meier survival analysis and a log-rank (Mantel-Cox) post hoc test.
Systemic administration of FGF2-AdTK was tested in a B16F10 model of experimental metastases. Briefly, 2 x 105 B16F10 untreated cells were injected i.v. on day 0 by lateral tail vein. Twenty-four h later, PBS or 8 x 109 pfu of FGF2-AdTK were delivered i.v. into the lateral tail vein. GCV (or saline), 100 mg/kg/day, was delivered once daily from days 2–10. Mice were necropsied on day 11 and the lungs were fixed in Bouin’s (Sigma, St. Louis, MO). Pulmonary metastases were enumerated with the aid of a dissecting scope.
Histological Determination of ß-Gal Activity.
Cryostat sections (8-µm) were fixed in 2% paraformaldehyde, 0.5% glutaraldehyde in PBS (pH 7.4) for 30 min. at room temperature. Tissue sections were then rinsed in PBS containing 0.03% NP40 and 2 mM MgCl2 and incubated for 16 h at 37°C in 1 mg/ml X-Gal (Fisher, Pittsburg, PA), 5 mM K3Fe(CN)6, and 5 mM K4Fe(CN)6 in PBS (pH 7.4) containing 2 mM MgCl2 and 0.03% NP40. Slides were rinsed in PBS, postfixed in 10% buffered formalin, counterstained for 15 s with Nuclear Fast Red, dehydrated, and mounted. For histopathology, routine H&E staining was performed on paraffin-embedded tissues.
Quantitation of ß-Gal Activity.
ß-Gal activity was quantitated in mouse liver homogenates according to standard techniques. Briefly, frozen liver tissue was minced and homogenized on ice in cold lysis buffer [1 ml of 0.2% Triton X-100 and 100 mM potassium phosphate (pH 7.8) per 100 mg tissue] using a glass tissue grinder. Homogenates were clarified by two centrifugation steps of 20 min each at 4°C in a microfuge at 12,000 x g. Supernatants were treated with 0.25 x volume of Chelex-100 resin (Bio-Rad, Hercules, CA; Ref. 33
). Homogenates were then vortexed briefly, incubated at room temperature for 2–5 min, and centrifuged for 30 s in a microfuge at 12,000 x g. A commercial chemiluminescent assay (Clontech, Palo Alto, CA) was used to quantify ß-Gal activity in homogenates using a luminometer (Dynatech Laboratories, Chantilly, VA). The activity of each sample was determined by extrapolation from a standard curve of ß-Gal enzyme supplied with the Clontech kit and is expressed in milliunits/g organ weight. Statistical analysis of the data was performed using an unpaired two-tailed Student’s t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. No X-Gal+ hepatocytes were observed in control mice (data not shown). Quantitation of ß-Gal activity in the liver paralleled the histochemical results and demonstrated 7- to 20-fold less ß-Gal activity in the livers of FGF2-Adßgal-treated mice than in the livers of Adßgal-treated mice on days 1, 2, 4, 7, and 12 (Table 1)
. By day 12, a modest level of ß-Gal was still present in the liver tissue of Adßgal-treated mice (mean, 259 milliunits/g) but was below the limit of detection in the livers of FGF2-Ad- ßgal-treated mice. These data suggest that FGF2-targeted adenoviral vectors have a reduced liver tropism as compared with untargeted vectors. We next addressed whether FGF2-retargeting could also reduce Ad-associated liver toxicity. On day 7 postadministration, serum transaminase levels were elevated 8- to 16-fold above normal in the Adßgal treated group but only 3- to 5-fold in the FGF2-Adßgal-treated group (Fig. 2)
. Serum Alk Phos was also elevated in Adßgal-treated mice but was within normal limits in FGF2-Adßgal-treated mice. Liver histopathology on day 7 revealed evidence of severe hepatocellular necrosis with a marked inflammatory infiltrate in mice treated with Adßgal, but histopathology analysis of liver from FGF2-Adßgal-treated mice revealed that the hepatocellular necrosis was almost completely absent; however, a minimal-to-moderate inflammatory infiltrate was observed (Fig. 3)
. Therefore, FGF2-retargeting serves to decrease both hepatic transduction and toxicity.
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, at any given MOI, FGF2-retargeting increased both the number of transduced cells as well as their level of transgene expression. Furthermore, FGF2-AdGFP was able to successfully transduce cells at MOI 
100, whereas nontargeted vectors were relatively ineffective at these levels.
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, nontargeted virus did not influence cell numbers in either the presence or the absence of GCV. By contrast, FGF2-AdTK did confer GCV sensitivity, and this effect positively correlated with increasing FGF2-Fab':knob ratios. Just as Table 2
demonstrated that B16F0 cells were transduced at the MOI used in these experiments (MOI = 100), albeit to a low level, these data demonstrate the value of enhancing transduction efficiency through the use of FGF2-retargeting.
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). In striking contrast, mice that received B16 melanoma cells treated with FGF2-AdTK demonstrated a 260% increase in median survival compared with the control groups. One of eight mice treated with FGF2-AdTK was a long-term survivor and had no evidence of disease when killed on day 126 postimplantation. Importantly, the effect was entirely GCV-dependent, ruling out nonspecific tumor cell killing due to FGF2-AdTK infection alone. A second experiment in which virus and cells were preincubated for only 15 min before implantation yielded similar results (250% increase in life span compared with control with 20% long-term survivors). These in vitro and ex vivo studies conclusively show that FGF2-retargeting can successfully be used to overcome tumor resistance to adenoviral gene therapy and enhance its therapeutic value.
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. FGF2-AdTK treatment without GCV therapy did not increase survival compared with control tumor-bearing mice, as expected.
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. In the FGF2-AdTK and GCV group, numbers of metastatic nodules were significantly reduced, although it should be noted that these animals suffered significant weight loss (average of 27%). Reduction in the number of tumor nodules was GCV-dependent.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To confirm that native Ad tropism can be redirected to cells bearing FGFRs, we have shown that an Ad-resistant tumor line (murine B16 melanoma) can be made sensitive to FGF2-AdTK transduction in vitro and ex vivo. In addition, in vitro studies established that FGF2-retargeting served to increase both the number of transduced cells and their level of transgene expression. This enhanced transduction translates into enhanced therapeutic efficacy as shown by in vitro cytotoxicity studies. Mice challenged with FGF2-AdTK-treated B16 melanoma cells have a greatly prolonged survival of 2.5 times that of mice bearing control or AdTK-treated B16 melanoma cells. Untargeted AdTK treatment is completely ineffective at the same dosage level. To demonstrate the advantage of this therapeutic system in vivo, mice were challenged with B16 melanoma cells and then treated i.p. with FGF2-AdTK. This resulted in prolonged survival (nearly 2-fold) when the mice also received the prodrug GCV. Mice treated with the retargeted Ad vector at the same dose but without GCV had no change in survival compared with untreated tumor-bearing mice. Interestingly, some evidence of systemic targeting was established using the B16F10 metastatic lung model. Although the untargeted vector was lethal at the dose tested, it should be recognized that the targeted vector was toxic, although not lethal, to the animals. The vector dose level tested in this experiment resulted in an LD65/7 for AdTK plus GCV and an LD0/7 for FGF2-AdTK plus GCV. Thus, the results show the potential of systemic dosing, but further work is required to validate this observation. Clearly, this targeting approach, which decreases the toxicity of the virus and increases the specificity of targeting and the potency of transgene delivery, results in a modified vector with an increased therapeutic window.
Many groups have attempted to target Ad to particular cell types to increase the specificity of viral delivery or cell entry. The earliest work in this regard used Ad to bind transferrin-polylysine-DNA complexes (36)
. These multiplexes had the capacity to transfect DNA into cells via cellular uptake through either the Ad receptor or the transferrin receptor. Present targeting methods block Ad uptake through its receptor and confer specificity of uptake using ligands with specificities directed toward tumors. Several physical retargeting methods for Ad vectors have been used. One targeting strategy is to block the Ad fiber protein with an antibody or antibody fragment that is coupled to a targeting molecule such as a receptor ligand or an antibody fragment that recognizes a unique cell surface molecule (12
, 14
, 15
, 17)
. This approach of using a bispecific retargeting molecule is identical to the one described here. A second strategy has also been investigated in which the knob region of the Ad fiber protein has been genetically modified with the addition of a short peptide (e.g., RGD) which can target cell surface molecules such as 
vß3 and vß5 integrins (7
, 13
, 16)
. Some retargeted vectors, such as Folate-Ad, are less than or at most equipotent to native Ad vectors (12)
, whereas several demonstrate increased potency or transduction efficiency when compared with native Ad in vitro (14
, 15
, 17)
. Certain targeting approaches, although amenable to positive gene therapy by transduction of multiple cell types, may not be specific enough (i.e., targeting cell-surface heparan sulfates) to be used in a cytotoxic gene therapy setting in vivo (7
, 15
, 16)
. The present studies are among the first to demonstrate redirected tropism in vivo.
The results with adenoviral vectors in clinical oncology trials have been disappointing. Because Ad can transduce normal cells and lead to toxicity, clinical studies have been limited to direct injection into tumors or to locoregional delivery to a compartment that contains tumor cells (1, 2, 3, 4, 5, 6) . To obviate some of the problems associated with clinical usage of these Ad vectors, new generation vectors will require increased specificity and increased potency to decrease the viral dosage required in patients. Although FGF2 targeting offers substantial benefits, further evaluation before its use in humans is required, including in vivo activity against Ad-sensitive and Ad-resistant xenogeneic tumors.
FGF2 is potentially an ideal targeting ligand for in vivo use. FGF2 binds to all of the four FGFRs with a picomolar affinity. This affinity is at least 2–3 orders of magnitude greater than other ligand-receptor systems or antibody-antigen interactions. Also, most FGFRs are differentially expressed between proliferating—often diseased or injured—cells and quiescent normal cells. In fact, few adult tissues respond to exogenously administered FGF2, even at supraphysiological concentrations (37, 38, 39, 40) . We have tested the i.v. administration of up to 5 mg FGF2/kg body weight in normal mice and observed only a 2- to 3-fold increased proliferation in bile-ductule epithelial cells and the outer cortical cells of the adrenal cortex. Significantly, proliferation in all of the other tissues examined was not increased, and no morphological changes were detected.6
Up-regulation or amplification of FGFRs in numerous human adenocarcinomas and sarcomas, when compared with the normal tissue from which they are derived, is a relatively common occurrence (24, 25, 26, 27, 28, 29) . In fact, altered FGFR expression has been associated with progression to malignancy (41) . Also, interruption of ligand-mediated signaling through FGFR1 by use of antisense has proven to be therapeutically useful in arresting tumor growth and blocking intratumoral angiogenesis in a human melanoma model (42) . Although tumor neovasculature may be a target of FGF2-AdTK, we do not presently have data to support this hypothesis.
Other nonneoplastic diseases that are characterized by hyperproliferation have also been shown to contain increased FGFR content. This includes vascular restenosis, neural ischemia, and dermal wound healing (43, 44, 45, 46) . Indeed, targeted gene delivery in these settings may also be a useful therapeutic strategy. Because of the enhanced efficacy and decreased toxicity of FGF2-retargeted Ad in comparison with Ad, the therapeutic potential of this vector in vivo is enhanced. Accordingly, the FGF2 retargeting of Ad vectors offers an innovative approach to target gene delivery for diverse clinical applications in which FGF receptors are up-regulated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Selective Genetics, Inc., 11035 Roselle Street, San Diego, CA 92121. Phone: (619) 625-0100; Fax: (619) 625-0222; E-mail: dlgu{at}selectivegenetics.com 
2 Present address: Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608.
3 The abbreviations used are: Ad, adenovirus; ALT, alanine transaminase; AST, aspartate transaminase; Alk Phos, alkaline phosphatase; ß-Gal, ß-galactosidase; CAR, Coxsackie and Ad receptor; FGF, fibroblast growth factor; FGF2, basic FGF; FGFR, FGF receptor; FGFR+, FGFR positive; pfu, plaque-forming unit(s); TK, herpes simplex virus thymidine kinase; X-Gal, 5-bromo-4-chloro-3-indolylß-D-galactopyranoside; MOI, multiplicity of infection; FI, fluorescence intensity; GCV, ganciclovir.
4 A. Chen, G. F. Pierce, and M. D’Andrea, unpublished observations.
5 M. A. Printz, J. Doukas, M. Cunningham, D. Gu, A. M. Gonzalez, G. F. Pierce, and S. L. Aukerman. FGF2 retargeted adenoviral vectors exhibit a modified biolocalization pattern and display reduced toxicity relative to native adenoviral vectors, submitted for publication.
6 A. M. Gonzalez, S. L. Aukerman, and G. F. Pierce, unpublished observations.
Received 12/18/98. Accepted 4/ 5/99.
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, excipient; 
, Adßgal; 
, FGF2-Adßgal.
, excipient and GCV; •, AdTK and GCV; 
, FGF2-AdTK and H2O; 
, FGF2-AdTK and GCV.