Dual Targeting of Adenoviral Vectors at the Levels of Transduction and Transcription Enhances the Specificity of Gene Expression in Cancer Cells
Authors: Brian G. Barnett, Bryan W. Tillman, David T. Curiel, Joanne T. Douglas
Journal: Molecular Therapy (2002)

Adenoviral (Ad) vector-mediated strategies for cancer gene therapy mandate a vector that is capable of efficient expression of the therapeutic gene specifically within the target tumor cells. In one approach to the development of cancer cell-specific vectors, Ad vectors have been targeted at the level of transduction to achieve the selective delivery of the therapeutic gene. In an alternative approach to the derivation of cancer cell-specific vectors, Ad vectors have been targeted at the level of transcription by placing the therapeutic gene under the control of transcriptional regulatory sequences that are activated in tumor cells, but not in normal cells, and therefore target expression selectively to the tumor cell. In this report, we demonstrate that a higher degree of specificity for cancer cells can be achieved by combining the complementary approaches of transductional and transcriptional targeting, each of which is imperfect or "leaky" by itself. Adenoviral (Ad) vector-mediated strategies for cancer gene therapy mandate a vector that is capable of efficient expression of the therapeutic gene specifically within the target tumor cells. In one approach to the development of cancer cell-specific vectors, Ad vectors have been targeted at the level of transduction to achieve the selective delivery of the therapeutic gene. In an alternative approach to the derivation of cancer cell-specific vectors, Ad vectors have been targeted at the level of transcription by placing the therapeutic gene under the control of transcriptional regulatory sequences that are activated in tumor cells, but not in normal cells, and therefore target expression selectively to the tumor cell. In this report, we demonstrate that a higher degree of specificity for cancer cells can be achieved by combining the complementary approaches of transductional and transcriptional targeting, each of which is imperfect or "leaky" by itself. IntroductionAdenoviral (Ad) vectors have been used in several clinical studies of cancer gene therapy [1Roth J.A. Cristiano R.J. Gene therapy for cancer: what have we done and where are we going?.J. Natl. Cancer Inst. 1997; 89: 21-39Crossref PubMed Scopus (611) Google Scholar]. To this end, Ad vectors possess the important characteristic of in vivo stability, as well as the ability to be produced to high titers [2Zhang W.W. Development and application of adenoviral vectors for gene therapy of cancer.Cancer Gene Ther. 1999; 6: 113-138Crossref PubMed Scopus (157) Google Scholar]. However, the Ad vectors in current use possess a broad host range and can therefore infect normal cells in addition to the tumor cells [3Russell W.C. Update on adenovirus and its vectors.J. Gen. Virol. 2000; 81: 2573-2604Crossref PubMed Scopus (417) Google Scholar]. This lack of specificity limits the utility of Ad vectors for cancer gene therapy, because the therapy can result in toxicity to normal cells, particularly when expression of the therapeutic gene is designed to lead to eradication of the cell. Furthermore, although Ad is stable in the bloodstream, it has not yet proven possible to exploit the potential of Ad vectors in the treatment of metastatic disease. In this regard, sequestration of systemically administered Ad vectors by the liver reduces the number of vector particles available for delivery to the target cancer cells [4Herz J. Gerard R.D. Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice.Proc. Natl. Acad. Sci. USA. 1993; 90: 2812-2816Crossref PubMed Scopus (502) Google Scholar, 5Kass-Eisler A. 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Therefore, it is recognized that Ad-mediated in vivo cancer gene therapy strategies mandate a vector that is capable of efficient expression of the therapeutic gene specifically within the target tumor cells [9Gomez-Navarro J. Curiel D.T. Douglas J.T. Gene therapy for cancer.Eur. J. Cancer. 1999; 35: 2039-2057Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar].In one approach to the development of cancer cell-specific vectors, Ad has been targeted at the level of transduction to achieve the selective delivery of the therapeutic gene [10Krasnykh, V. N., Douglas, J. T., and van Beusechem, V. W.2000. Genetic targeting of adenoviral vectors. Mol. Ther.1: 391–405,Google Scholar, 11Wickham T.J. Targeting adenovirus.Gene Ther. 2000; 7: 110-114Crossref PubMed Scopus (269) Google Scholar]. The promiscuous tropism of Ad is due to the widespread distribution of the native primary cellular receptor, the coxsackievirus and adenovirus receptor (CAR) [12Bergelson J.M. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5.Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2605) Google Scholar, 13Tomko R.P. Xu R. Philipson L. HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses.Proc. Natl. Acad. Sci. USA. 1997; 94: 3352-3356Crossref PubMed Scopus (1025) Google Scholar]. Therefore, transductional targeting of Ad is accomplished by redirecting binding of the knob domain of the fiber capsid protein [14Henry L.J. Xia D. Wilke M.E. Deisenhofer J. Gerard R.D. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.J. Virol. 1994; 68: 5239-5246PubMed Google Scholar, 15Louis N. Fender P. Barge A. Kitts P. Chroboczek J. Cell-binding domain of adenovirus serotype 2 fiber.J. Virol. 1994; 68: 4104-4106PubMed Google Scholar] away from CAR to an alternative, cell-selective receptor, with subsequent internalization being mediated by the interaction of the capsid penton base protein with the cellular integrins αvβ3 and αvβ5 [16Wickham T.J. Mathias P. Cheresh D.A. Nemerow G.R. Integrins αvβ3 and αvβ5 promote adenovirus internalization but not virus attachment.Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1944) Google Scholar, 17Bai M. Campisi L. Freimuth P. Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2.J. Virol. 1994; 68: 5925-5932PubMed Google Scholar]. In this regard, the observation that primary tumor cells are refractory to Ad infection due to a deficiency of CAR [18Dmitriev I. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism.J. Virol. 1998; 72: 9706-9713Crossref PubMed Google Scholar, 19Hemmi S. Geertsen R. Mezzacasa A. Peter I. Dummer R. The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures.Hum. Gene Ther. 1998; 9: 2363-2373Crossref PubMed Scopus (208) Google Scholar, 20Miller C.R. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer.Cancer Res. 1998; 58: 5738-5748PubMed Google Scholar, 21Li Y. Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy.Cancer Res. 1999; 59: 325-330PubMed Google Scholar, 22Okegawa T. The dual impact of coxsackie and adenovirus receptor expression on human prostate cancer gene therapy.Cancer Res. 2000; 60: 5031-5036PubMed Google Scholar] provides a strong rationale for the use of transductionally targeted Ad vectors capable of CAR-independent gene delivery to cancer cells to enhance the efficiency of infection [23Krasnykh V. Advanced generation adenoviral vectors possess augmented gene transfer efficiency based upon coxsackie adenovirus receptor-independent cellular entry capacity.Cancer Res. 2000; 60: 6784-6787PubMed Google Scholar].In an alternative approach to the derivation of cancer cell-specific vectors, Ad has been targeted at the level of transcription. This is accomplished by placing the therapeutic gene under the control of tissue- or tumor-specific transcriptional regulatory sequences that are activated in tumor cells but not in normal cells and therefore target expression selectively to the tumor cell [24Nettelbeck D.M. Jerome V. Muller R. Gene therapy: designer promoters for tumour targeting.Trends Genet. 2000; 16: 174-181Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar].Reynolds et al. have recently reported that the use of an endothelial-specific promoter combined with transductional targeting of a systemically administered Ad vector to a pulmonary endothelial marker resulted in a marked improvement in the selectivity of transgene expression in the lung versus the liver [25Reynolds P.N. Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo.Nat. Biotechnol. 2001; 19: 838-842Crossref PubMed Scopus (212) Google Scholar]. Their study was facilitated by the existence of a truly tissue-specific cell surface marker and promoter. However, the strategy of transductional targeting to tumor cells is limited by the existence of few truly cancer-cell-specific receptors, because many candidate target receptors are also expressed at low levels by normal tissues. Similarly, transcriptional targeting of tumor cells is limited by "leakiness" of candidate promoters, thereby allowing gene expression in non-cancerous cells [26Douglas J.T. Curiel D.T. Targeted gene therapy.Tumor Target. 1995; 1: 67-84Google Scholar, 27Vile R.G. Sunassee K. Diaz R.M. Strategies for achieving multiple layers of selectivity in gene therapy.Mol. Med. Today. 1998; 4: 84-92Abstract Full Text PDF PubMed Scopus (29) Google Scholar].We hypothesized that a higher degree of specificity for cancer cells could be achieved by combining the complementary approaches of transductional and transcriptional targeting, each of which might be imperfect by itself. This is represented schematically in Fig. 1. To test this concept, we chose to target the epidermal growth factor receptor (EGFR), which is overexpressed on many tumor cells [28Modjtahedi H. Dean C. The receptor for EGF and its ligands: Expression, prognostic value and target for therapy in cancer.Int. J. Oncol. 1994; 4: 277-296PubMed Google Scholar]. To this end, we used a bispecific antibody conjugate, with specificity for both the fiber knob domain and EGFR [20Miller C.R. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer.Cancer Res. 1998; 58: 5738-5748PubMed Google Scholar]. Additionally, we placed the luciferase reporter gene under the transcriptional control of the osteocalcin gene 2 (OC) promoter [29Ducy P. Karsenty G. Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.Mol. Cell. Biol. 1995; 15: 1858-1869Crossref PubMed Scopus (518) Google Scholar], which has specificity for osteoblasts and osteoblastic metastatic lesions. In this report, we demonstrate that a dually targeted Ad vector offers a higher level of specificity for cancer cells than a transductionally or transcriptionally targeted Ad vector alone.ResultsTo evaluate the hypothesis that a higher degree of specificity for cancer cells could be achieved by combining the complementary approaches of transductional and transcriptional targeting, we chose to target EGFR, which is overexpressed on many human tumor cells [28Modjtahedi H. Dean C. The receptor for EGF and its ligands: Expression, prognostic value and target for therapy in cancer.Int. J. Oncol. 1994; 4: 277-296PubMed Google Scholar], as our approach to transductional targeting.With the aim of targeting Ad vectors to EGFR by using a conjugate-based strategy, we conjugated the Fab fragment of the neutralizing anti-knob monoclonal antibody 1D6.14 [30Douglas J.T. Targeted gene delivery by tropism-modified adenoviral vectors.Nat. Biotechnol. 1996; 14: 1574-1578Crossref PubMed Scopus (397) Google Scholar] to the Fab fragment of monoclonal antibody 425, which is specific for human EGFR, to form the conjugate Fab-425. To determine the specificity of the conjugate Fab-425 for EGFR, as well as the concentration of Fab-425 at which gene transfer is optimal, we examined Ad-mediated gene transfer to a pair of CAR-negative, αv integrin-positive cell lines differing only in EGFR expression (Fig. 2). We have previously shown that the optimal amount of conjugate determined in this system is also optimal for targeting the same number of viral particles to CAR-positive, EGFR-positive human cells [20Miller C.R. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer.Cancer Res. 1998; 58: 5738-5748PubMed Google Scholar]. AdEasy-CMV-Luc, an E1-deleted Ad vector expressing the luciferase gene under the control of the constitutive cytomegalovirus (CMV) promoter, was preincubated with increasing concentrations of Fab-425 and used to infect EGFR-negative murine Swiss 3T3-derived NR6 fibroblasts or EGFR-positive NR6WT cells. Luciferase activity was assayed 24 hours postinfection. Ad infection of the EGFR-negative NR6 cells was inefficient and did not change with increasing doses of Fab-425 (Fig. 3A). However, preincubating AdEasy-CMV-Luc with increasing concentrations of Fab-425 led to a dose-dependent enhancement of gene transfer to the EGFR-positive NR6WT cells. The maximum level of EGFR-targeted gene transfer occurred at a dose of 10 ng Fab-425 per 1.2 × 106 plaque-forming units (pfu) of AdEasy-CMV-Luc. At this optimal conjugate:virus ratio, gene transfer to EGFR-positive NR6WT cells was 154-fold greater than to EGFR-negative NR6 cells. Increasing the conjugate:virus ratio further proved inhibitory to gene transfer, presumably due to competition for EGFR binding by excess Fab-425 conjugate. Therefore, these results indicate that, as previously reported [20Miller C.R. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer.Cancer Res. 1998; 58: 5738-5748PubMed Google Scholar], Fab-425 can target Ad infection at the level of transduction in an EGFR-specific manner.FIG. 2Flow cytometry analysis for presence of CAR or EGFR on a panel of cell lines. Light line is isotype control antibody plus FITC-labeled secondary antibody. Dark line is either anti-human CAR or anti-EGFR primary antibody plus FITC-labeled secondary antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIG. 3Transductional targeting of AdEasy-CMV-Luc to EGFR by way of Fab-425 conjugate. (A) We infected 12,000 EGFR-positive NR6WT cells (filled circle) or EGFR-negative NR6 cells (filled square) with AdEasy-CMV-Luc (MOI of 100 pfu per cell) preincubated with varying concentrations of Fab-425. Luciferase activity was determined 24 hours postinfection. The optimal dose of Fab-425 was 10 ng per 1.2 × 106 pfu. Data from a representative of three separate experiments are shown as the mean RLU per 1.2 × 103 cells ± SD of triplicate determinations. (B) We infected 12,000 cells with AdEasy-CMV-Luc (MOI of 100 pfu per cell) preincubated with or without Fab-425 (10 ng per 1.2 × 106 pfu). Luciferase activity was determined 24 hours postinfection. Data from a representative of three separate experiments are shown as the mean RLU per 1.2 × 103 cells of triplicate determinations. Bars, SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further evaluate the ability to target an Ad vector to EGFR, AdEasy-CMV-Luc was premixed with either Dulbecco's phosphate-buffered saline without Ca++ and Mg++ (PBS) or with the optimal concentration of Fab-425 and used to infect a panel of cell lines whose expression of CAR and EGFR had been determined by flow cytometry (Fig. 2). Luciferase activity was assayed 24 hours postinfection. Figure 3B reveals that transductional targeting of Ad to EGFR enhanced gene transfer to CAR-negative, EGFR-positive cells. In the cell lines tested, the augmentation of gene transfer ranged from 4-fold in the 143B osteosarcoma cells (P = 0.0197) to 28-fold in the U-118 MG glioma cells (P = 0.0054). In contrast, the untargeted vector mediated more efficient gene transfer to cells that were CAR-positive and EGFR-negative. In this case, redirection of the vector to EGFR resulted in a decrease in luciferase expression compared with that of the untargeted vector, ranging from a 66% decrease in human LS 174T colon adenocarcinoma cells (P = 0.0354), to a 76% decrease in human HepG2 hepatocytes (P = 0.0092), and to an 85% decrease in human U-2 OS osteosarcoma cells (P = 0.0289). Therefore, these results show the ablation of native Ad tropism by the bispecific Fab-425 conjugate, which simultaneously provided novel tropism for EGFR.Having validated the transductional targeting component of our combinational targeting approach, we sought to confirm the ability to target Ad at the level of transcription. For this purpose, we chose to use the tissue-specific murine OC promoter, which has specificity for osteoblasts and osteoblastic metastatic lesions.We first performed a preliminary experiment to validate the activity of the OC promoter in a panel of cell lines. To this end, cells were transfected with plasmid pOC-Luc, in which expression of the luciferase gene was placed under the control of the OC promoter. As a control for the transfection efficiency of the cells, we used the isogenic plasmid pCMV-Luc, in which the constitutive CMV promoter directs expression of the luciferase gene. A promoterless plasmid, pGL3-Basic, served as a control for the basal luciferase activity in the transfected cells. The cells were lysed and the luciferase activity was determined 48 hours post-transfection. This indicated that the OC promoter was active in rat osteosarcoma (ROS) cells, weakly active in human 143B osteosarcoma cells, and inactive in U-118 MG glioma cells (Fig. 4). However, the OC promoter was also weakly active in human HepG2 hepatocytes and human LS 174T colon adenocarcinoma cells. This illustrates that the OC promoter does exhibit some nonspecific or leaky expression as previously reported.FIG. 4Determination of OC activity. Cells were transfected with plasmid pOC-Luc, pCMV-Luc, or the promoterless pGL3-Basic vector. Luciferase expression was determined 48 hours post-transfection. Results were controlled for background luciferase activity by subtracting the luciferase expression achieved by pGL3-Basic from the expression mediated by the pOC-Luc and pCMV-Luc plasmids. Data from a representative of three separate experiments are shown as the mean RLU per 1.5 × 104 cells of triplicate determinations. Bars, SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then constructed a transcriptionally targeted Ad vector, AdEasy-OC-Luc, an E1-deleted adenovirus with the luciferase gene under the control of the OC promoter. The panel of cancer cell lines was infected with AdEasy-OC-Luc or AdEasy-CMV-Luc as a control for the efficiency of infection. The luciferase activity was assayed 24 hours postinfection. The transcriptional specificity of the OC promoter in the context of the Ad vector recapitulated its activity in the context of the plasmid pOC-Luc (Fig. 5). Thus, when considered as a percentage of the luciferase activity directed by the CMV promoter, the luciferase activity directed by the OC promoter was greatest in the ROS cells. AdEasy-OC-Luc also showed moderate activity in the 143B cells but showed negligible activity in the U-118 MG and LS 174T cells. When calculated as a percentage of expression mediated by AdEasy-CMV-Luc, expression in HepG2 cells infected by AdEasy-OC-Luc was only about 3% of the level observed in 143B cells. However, the greater susceptibility of HepG2 cells to Ad infection meant that the absolute level of luciferase expression after infection by AdEasy-OC-Luc was approximately 40% of that detected in 143B cells.FIG. 5Transcriptional targeting of Ad by way of the tissue-specific OC promoter. We infected 120,000 cells with either AdEasy-OC-Luc or AdEasy-CMV-Luc (MOI of 33 pfu per cell). Luciferase expression was determined 24 hours postinfection. Data from a representative of three separate experiments are shown as the mean RLU per mg cellular protein of triplicate determinations. Bars, SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We therefore sought to determine whether a greater degree of specificity could be achieved by targeting Ad vectors at the level of both transduction and transcription. To this end, human 143B osteosarcoma cells and human HepG2 hepatocytes were infected with the untargeted vector, AdEasy-CMV-Luc; with the transductionally targeted vector, AdEasy-CMV-Luc + Fab-425; with the transcriptionally targeted vector, AdEasy-OC-Luc; or with the dually targeted vector, AdEasy-OC-Luc + Fab-425. Luciferase activity was assayed 24 hours postinfection (Figs. 6A and 6B). The relative selectivity of each vector was determined by the ratio of the relative light units (RLU) per mg protein of the 143B cells to the HepG2 cells (Fig. 6C). This revealed that the untargeted vector had a relative selectivity that was 15.2 times greater for the HepG2 cells than for the 143B cells (P = 0.0296). In contrast, the transductionally targeted vector had a relative selectivity that was 2.0 times greater for the EGFR-positive 143B cells than for the HepG2 cells, and the transcriptionally targeted vector had a relative selectivity that was 2.5 times greater for the 143B cells than for the HepG2 cells. Importantly, the relative selectivity of the dually targeted vector was 43.8 times greater for the 143B cells than for the HepG2 cells (P = 0.0055). Furthermore, the targeting index, defined as the ratio of the relative selectivity for 143B cells of the dually targeted vector to the untargeted vector, was 665. These results demonstrate that combining the strategies of transductional and transcriptional targeting into one dual targeting system increased the specificity of the Ad vectors for the target 143B cancer cells.FIG. 6Combined targeting of Ad at the levels of transduction and transcription. (A) 143B cells were infected at an MOI of 33 pfu per cell with untargeted Ad (AdEasy-CMV-Luc), transductionally targeted Ad (AdEasy-CMV-Luc + Fab-425), transcriptionally targeted Ad (AdEasy-OC-Luc), or dually targeted Ad (AdEasy-OC-Luc + Fab-425). Luciferase activity was determined 24 hours postinfection. Data from a representative of three separate experiments are shown as the mean RLU per mg cellular protein of triplicate determinations. Bars, SD. (B) HepG2 cells were infected at an MOI of 33 pfu per cell with untargeted Ad (AdEasy-CMV-Luc), transductionally targeted Ad (AdEasy-CMV-Luc + Fab-425), transcriptionally targeted Ad (AdEasy-OC-Luc), or dually targeted Ad (AdEasy-OC-Luc + Fab-425). Luciferase activity was determined 24 hours postinfection. Data from a representative of three separate experiments are shown as the mean RLU per mg cellular protein of triplicate determinations. Bars, SD. (C) The relative selectivity of each vector was determined by the ratio of the RLU per mg cellular protein of the 143B cells to the HepG2 cellsView Large Image Figure ViewerDownload Hi-res image Download (PPT)We then sought to determine whether the combination of transductional and transcriptional targeting would confer increased specificity for target 143B cells in a mixed population. To this end, we first stably transfected 143B cells with the enhanced yellow fluorescent protein (EYFP) reporter gene, which created the 143B[EYFP] cell line. Flow cytometry analysis demonstrated that stable transfection of 143B cells with EYFP did not affect the expression levels of CAR, EGFR, or αv integrins (data not shown). In addition, transfection with the plasmid pOC-Luc showed that activity of the OC promoter was the same in 143B and 143B[EYFP] cells (data not shown). We mixed 143B[EYFP] and HepG2 cells at a ratio of 1 to 3 and infected with the untargeted vector, AdEasy-CMV-Luc; the transductionally targeted vector, AdEasy-CMV-Luc + Fab-425; the transcriptionally targeted vector, AdEasy-OC-Luc; or the dually targeted vector, AdEasy-OC-Luc + Fab-425. Cells were sorted by flow cytometry to separate the fluorescent 143B[EYFP] cells from the nonfluorescent HepG2 cells 24 hours postinfection. The luciferase activity was determined in the two populations (Figs. 7A and 7B). The relative selectivity of each vector was determined by the ratio of the RLU per mg protein of the 143B[EYFP] cells to the HepG2 cells (Fig. 7C). This revealed that the untargeted vector had a relative selectivity that was 4.1 times greater for the HepG2 cells than for the 143B[EYFP] cells. In contrast, the transductionally targeted vector had a relative selectivity that was 3.3 times greater for the EGFR-positive 143B[EYFP] cells than for the HepG2 cells, and the transcriptionally targeted vector had a relative selectivity that was 8.8 times greater for the 143B[EYFP] cells than for the HepG2 cells. However, the relative selectivity of the dually targeted vector was 59.4 times greater for the 143B[EYFP] cells than for the HepG2 cells, once again showing the increased specificity of the dual targeting approach. Also, the targeting index, defined as the ratio of the relative selectivity for 143B[EYFP] cells of the dually targeted vector to the untargeted vector, was 243, further demonstrating that the overall specificity of these Ad vectors was increased by combining the strategies of transductional and transcriptional targeting into one dual targeting system.FIG. 7Combined targeting of Ad at the levels of transduction and transcription to a mixed population of cells. 143B[EYFP] and HepG2 cells were mixed at a ratio of 1 to 3 (1.2 × 105 and 3.6 × 105 cells, respectively) and infected at an MOI of 33 pfu per cell with untargeted Ad (AdEasy-CMV-Luc), transductionally targeted Ad (AdEasy-CMV-Luc + Fab-425), transcriptionally targeted Ad (AdEasy-OC-Luc), or dually targeted Ad (AdEasy-OC-Luc + Fab-425). Cells were collected 24 hours postinfection and sorted by flow cytometry into fluorescent 143B[EYFP] and nonfluorescent HepG2 populations. (A) Luciferase activity of sorted 143B[EYFP] cells was determined 24 hours postinfection. Data from a representative of three separate experiments are shown as the mean RLU per mg cellular protein of triplicate determinations. Bars, SD. (B) Luciferase activity of sorted HepG2 cells determined 24 hours postinfection. Data from a representative of three separate experiments are shown as the mean RLU per mg cellular protein of triplicate determinations. Bars, SD. (C) The relative selectivity of each vector was determined by the ratio of the RLU per mg cellular protein of the 143B[EYFP] cells to the HepG2 cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionA major limitation of the current generation of Ad vectors is the lack of specificity for cancer cells. It is therefore recognized that the successful clinical application of Ad-mediated cancer gene therapy mandates a vector that is capable of efficient expression of the therapeutic gene selectively within the target tumor cells.In this study, we have investigated the hypothesis that the specificity of Ad vectors for cancer cells could be improved by combining the complementary approaches of transductional and transcriptional targeting. To address this issue, we used a bispecific antibody conjugate to target an Ad vector at the level of transduction to EGFR, which is overexpressed on many tumor cells. The redirected vector specifically infected target EGFR-positive cells. Moreover, transductional targeting of Ad to EGFR enhanced the efficiency of infection of cancer cells expressing