An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism

Abstract

Recombinant adenoviruses (Ad) have become the vector system of choice for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to cells expressing marginal levels of the coxsackievirus and adenovirus receptor (CAR). In order to achieve CAR-independent gene transfer by Ad vectors in clinically important contexts, we proposed modification of viral tropism via genetic alterations to the viral fiber protein. We have shown that incorporation of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain results in the ability of the virus to utilize an alternative receptor during the cell entry process. We have also demonstrated that due to its expanded tissue tropism, this novel vector is capable of efficient transduction of primary tumor cells. An increase in gene transfer to ovarian cancer cells of 2 to 3 orders of magnitude was demonstrated by the vector, suggesting that recombinant Ad containing fibers with an incorporated RGD peptide may be of great utility for treatment of neoplasms characterized by deficiency of the primary Ad type 5 receptor.

FIG. 1

Analysis of interaction between recombinant fiber proteins and α_vβ₃ integrin. Baculovirus-expressed fiber proteins absorbed on an ELISA plate were incubated with various concentrations of purified integrin α_vβ₃. Integrin bound to fiber proteins was then detected with anti-α subunit monoclonal antibody VNR139. Each point represents a mean of three readings obtained in one experiment. Some error bars depicting standard deviations are smaller than the symbols. wt, wild type.

FIG. 2

ELISA of α_vβ₃ integrin binding to immobilized AdCMVLuc and Ad5lucRGD virions. CsCl-purified virions of AdCMVLuc and Ad5lucRGD immobilized in the wells of an ELISA plate were incubated with affinity-purified α_vβ₃ integrin, followed by incubation with monoclonal antibody VNR139. Data shown are means ± standard deviations from an experiment performed in triplicate.

FIG. 3

Flow cytometric analysis of CAR and integrin expression in 293, HUVEC, and RD cells. Cells were incubated with anti-CAR (RmcB), anti-α_vβ₃ (LM609), or anti-α_vβ₅ (P1F6) integrin monoclonal antibodies, washed with SM to remove unbound monoclonal antibodies, and incubated with secondary FITC-labeled goat anti-mouse immunoglobulin G serum as described in Materials and Methods. After removal of the FITC-labeled antibodies, aliquots of 10⁴ cells were analyzed by flow cytometry. Expression of CAR in 293 (A), HUVEC (C), and RD (E) cells and of α_vβ₃ (thin line) and α_vβ₅ (heavy line) integrins in 293 (B), HUVEC (D), and RD (F) cells is shown. The dotted line shows the results for the negative control.

FIG. 4

Ad-mediated gene transfer to various human cell lines. 293 (A), HUVEC (B), or RD (C) cells preincubated for 10 min at room temperature in either DMEM/F12 or DMEM/F12 containing recombinant Ad5 fiber knob at 100 μg/ml were then exposed for 30 min at room temperature to AdCMVLuc or Ad5lucRGD in DMEM/F12 at 1, 10, or 100 PFU/cell. The unbound virus was aspirated, and complete medium was added. After incubation at 37°C for 30 h, the cells were lysed and the luciferase activity (in relative light units) was determined. Background luciferase activities detected in mock-infected cells were 261, 223, and 163 relative light units for 293, HUVEC, and RD cells, respectively. These activities were subtracted from all readings obtained with the corresponding cell line. Each point represents the mean ± standard deviation of three determinations.

FIG. 5

Comparison of binding of ¹²⁵I-labeled adenoviruses to 293, HUVEC, or RD cells. One-hundred-microliter aliquots of cells in DMEM-Ad medium (DMEM, 20 mM HEPES, 0.5% BSA), 10⁶ cells per aliquot, were incubated for 1 h at 4°C with 50 μl of ¹²⁵I-labeled Ad (10⁵ cpm per sample). The samples were then diluted with 4 ml of phosphate-buffered saline containing 0.1% BSA, and the cells were pelleted by centrifugation. Radioactivities of cell pellets were determined with a gamma counter. Data shown are means ± standard deviations from an experiment performed in triplicate.

FIG. 6

Inhibition of binding of labeled AdCMVLuc and Ad5lucRGD to 293 and HUVEC cells. 293 (A) or HUVEC (B) cells were preincubated with DMEM-Ad or DMEM-Ad containing either Ad5 fiber knob (100 μg/ml) or Ad2 penton base (100 μg/ml) or both for 1 h at 4°C. Fifty-microliter aliquots of ¹²⁵I-labeled viruses were then added to the samples. The rest of the procedure was as described in the legend for Fig. 5.

FIG. 7

Flow cytometric analysis of human ovarian cancer cells. Expression of CAR, α_vβ₃, and α_vβ₅ integrins in SKOV3.ip1 or OV-4 cells was analyzed by flow cytometry essentially as described in the legend for Fig. 3. Expression of CAR in SKOV3.ip1 (A) and OV-4 cells (C) and of α_vβ₃ (thin line) and α_vβ₅ (heavy line) integrins in SKOV3.ip1 (B) and OV-4 (D) cells is shown. The results for the negative control are shown by the dotted line.

FIG. 8

Comparison of the gene transfer efficiencies to cultured ovarian cancer cells mediated by AdCMVLuc and Ad5lucRGD. Human ovarian cancer cells SKOV3.ip1 (A) and OV-4 (B) were transduced with AdCMVLuc or Ad5lucRGD at an MOI of 1 or 10 PFU/cell essentially as described for 293, HUVEC, and RD cells. Recombinant Ad5 fiber knob protein was added to cells prior to infection with the virus. Each datum point is the average of three independent measurements obtained in one experiment.

FIG. 9

Transduction of primary cells isolated from ascitic fluid samples obtained from ovarian cancer patients. Cells isolated from ascitic fluid samples from two (A and B) ovarian cancer patients as described in Materials and Methods were transduced with AdCMVLuc or Ad5lucRGD at an MOI of 1 or 10 in the presence or absence of blocking Ad5 fiber knob protein. The datum points represent the means ± standard deviations of three independent determinations.