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. 2013 Apr;87(7):3792-800.
doi: 10.1128/JVI.02394-12. Epub 2013 Jan 23.

Prostate-specific Antigen-Retargeted Recombinant Newcastle Disease Virus for Prostate Cancer Virotherapy

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Free PMC article

Prostate-specific Antigen-Retargeted Recombinant Newcastle Disease Virus for Prostate Cancer Virotherapy

Raghunath Shobana et al. J Virol. .
Free PMC article

Abstract

Oncolytic virus (OV) therapies of cancer are based on the use of replication-competent, tumor-selective viruses with limited toxicity. Newcastle disease virus (NDV), an avian paramyxovirus, is a promising OV and is inherently tumor selective and cytotoxic only to tumor cells. Replication is restricted in normal cells. Despite encouraging phase I/II clinical trials with NDV, further refinements for tumor-specific targeting are needed to enhance its therapeutic index. Systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity, and the extracellular matrix. Overcoming these hurdles is paramount to realizing the exceptional oncolytic efficacy of NDV. We engineered the F protein of NDV and generated a recombinant NDV (rNDV) whose F protein is cleavable exclusively by prostate-specific antigen (PSA). The rNDV replicated efficiently and specifically in prostate cancer (CaP) cells and 3-dimensional prostaspheres but failed to replicate in the absence of PSA. Induction of intracellular PSA production by a synthetic androgen analog (R1881) enhanced fusogenicity in androgen-responsive CaP cells. Further, PSA-cleavable rNDV caused specific lysis of androgen-independent and androgen-responsive/nonresponsive CaP cells and prostaspheres, with a half-maximal effective concentration (EC50) ranging from a multiplicity of infection of 0.01 to 0.1. PSA-retargeted NDV efficiently lysed prostasphere tumor mimics, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating no pathogenicity for chickens. Prostate-specific antigen targeting is likely to enhance the therapeutic index of rNDV owing to tumor-restricted replication and enhanced fusogenicity.

Figures

Fig 1
Fig 1
Characterization of fusion protein mutants by using cell-based assays. (A) Schematic of the NDV fusion protein and the PSA activation mutants. (B) Fold change in cell surface expression, measured quantitatively by flow cytometry. The percentages of fluorescent cells were determined by setting the gates using mock-transfected cells as a negative control and wild-type F as a positive control. (C) Immunofluorescent staining of Vero cells cotransfected with NDV F and HN plasmids, using an antibody against F protein (magnification, ×10). (D) Activation of mutant F proteins with an exogenous PSA overlay (100 ng/ml) (at 12, 24, and 48 h posttransfection) compared to the activation of wild-type F protein with no overlay. Lysates collected after 48 h posttransfection were used to detect the F1 peptide using a polyclonal anti-NDV chicken serum. (E) Determination of the optimal PSA overlay concentration. The exogenous PSA overlay concentration was calculated based on the percentage of cell viability and the fusion index in PC-3 cells. Cotransfection of expression plasmids (mutant pCAGGS-KLQL and wt pCAGGS-HN) in PC-3 cells, followed by an exogenous PSA overlay (1 ng/ml to 400 ng/ml), resulted in variable fusion indices and cell viabilities. Results are means ± standard deviations (SD) obtained from five independent experiments. (F) Syncytium formation in BSR-T7/5 cells 72 h posttransfection (blue arrows).
Fig 2
Fig 2
Specificity of recombinant NDV for PSA. (A) Virus rescue and sequential passage of the BC-HSSKL-GFP and BC-KLQL-GFP mutant viruses in BSR-T7/5 cells. Magnification, ×20. Note the absence of multicycle replication in the HSSKL virus. (B and C) Replication kinetics of the BC-KLQL-GFP virus (MOI, 1) in Vero and WPE-int cells with or without a PSA overlay or with an overlay of PSA supplemented with 10% FBS. (D) BC-KLQL-GFP was passaged serially in WPE-int cells. The sequence of the KLQL mutation in the virus was confirmed after 10 serial passages at an MOI of 1. (E) Reverse transcription-PCR gel confirming the absence of viral RNA in the allantoic fluid of BC-KLQL-GFP-infected specific-pathogen-free chicken embryos. Lanes: a, DNA marker; b, allantoic fluid from an embryo infected with Lasota-EGFP; c, cell culture supernatant infected with BC-KLQL-GFP; d and e, allantoic fluid from embryos infected with BC-KLQL-GFP.
Fig 3
Fig 3
Virus yields in prostate cancer cells and spheres. (A) The virus yield in DU145 cells was significantly higher than those in other cells. Spheres are designated DU145S, PC-3S, WPE-intS, and C4-2BS. (B) PSA induction using the synthetic androgen analog R1881 significantly increased virus replication in AR+ cells (C4-2B and WPE-int) and decreased virus yield in AR+ spheres (C4-2BS and WPE-intS), *, P < 0.0001; n = 8. (C) Multistep growth kinetics of BC-KLQL-GFP, at an MOI of 1, after PSA induction with R1881 (1 nm/ml).
Fig 4
Fig 4
Fusogenicity of PSA-cleavable rNDV in CaP cells and spheres. AR+ and AR CaP cells and spheres were infected with recombinant BC-KLQL-GFP virus at an MOI of 1, with or without R1881 induction, and fusogenicity was examined 72 h postinfection. Note the enhanced fusogenicity in androgen-responsive C4-2B and WPE-int cells after R1881 induction. Spheres disintegrate after treatment with R1881.
Fig 5
Fig 5
Cytotoxicity of PSA-cleavable rNDV in CaP cells and spheres. Cell viability was assessed by the trypan blue dye exclusion assay at 120 h postinfection. The EC50 in AR+ and AR cells/spheres was calculated by the sigmoidal dose response-versus-inhibitor analysis with four parameters, using GraphPad Prism, version 5 (GraphPad Software). (A) AR cells and spheres with an exogenous PSA overlay (100 ng/ml). (B) AR+ cells (treated with R1881 at 1 ng/ml) and AR+ spheres (with PSA overlaid at 100 ng/ml). (C) AR+ cells (WPE-int and C4-2B) responded to PSA or R1881 supplementation. The viability of WPE-int cells treated with R1881 was significantly lower than that with the PSA overlay at 120 h postinfection. MOI, 1. *, P < 0.0001; **, P ≤ 0.0002; n = 6.

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