Wing-Shing Cheng

Ex vivo stimulation of cytomegalovirus (CMV)‐specific T cells using CMV pp65‐modified dendritic cells as stimulators

Summary. Cytomegalovirus (CMV) infection is a dangerous complication in immunosuppressed individuals such as allogeneic stem cell transplant patients. CMV disease can be prevented by the early post‐transplant transfer of donor‐derived, CMV‐directed, T cells. Fast and cost efficient methods to generate CMV‐specific T cells are, therefore, warranted. The current study utilized peptide‐pulsed and adenovirus‐transduced dendritic cells (DC) to generate CMV‐restricted T cells. After one stimulation with CMV pp65495−503 peptide‐pulsed DC and three re‐stimulations with peptide‐pulsed monocytes, virtually all T cells were CD8+, expressed the relevant T cell receptor and exhibited high peptide‐specific lytic activity. After only one stimulation, pp65495−503‐restricted T cells could be sorted to a purity of higher than 95% and expanded up to 1000‐fold in 2 weeks. This technique may prove useful for the rapid generation of large quantities of specific cytolytic T lymphocytes (CTL) for cell therapy. DC transduced with an adenoviral vector encoding the full‐length pp65 protein (Adpp65) were able to simultaneously expand CTL against multiple epitopes of pp65. In addition, they activated CMV‐specific CD4+ T‐helper cells. This approach would stimulate multiple‐epitope populations of pp65‐specific T cells and could be made available to patients of any human leucocyte antigen (HLA) haplotype. DC transduced with adenoviral vectors to express full‐length antigens may prove to be potent vaccines against viral pathogens and cancer.

An oncolytic conditionally replicating adenovirus for hormone-dependent and hormone-independent prostate cancer

The use of conditionally replicating adenoviruses offers an attractive complementary treatment strategy for localized prostate cancer. We have produced a replicating adenovirus, Ad[I/PPT-E1A], where E1A gene expression is controlled by a recombinant regulatory sequence designated PPT. The PPT sequence comprises a PSA enhancer, a PSMA enhancer and a T-cell receptor γ-chain alternate reading frame protein promoter, and it is shielded from transcriptional interference from adenoviral backbone sequences by an H19 insulator. Ad[I/PPT-E1A] yields prostate-specific E1A protein expression, viral replication and cytolysis in vitro. Furthermore, Ad[I/PPT-E1A] considerably regresses the growth of subcutaneous LNCaP prostate cancer tumors in nude mice. Importantly, the viral replication and cytolytic effect of Ad[I/PPT-E1A] are independent of the testosterone levels in the prostate cancer cells. This may be beneficial in a clinical setting since many prostate cancer patients are treated with androgen withdrawal. In conclusion, Ad[I/PPT-E1A] may prove to be useful in the treatment of localized prostate cancer.

Two-step amplification of the human PPT sequence provides specific gene expression in an immunocompetent murine prostate cancer model

The recombinant prostate-specific PPT sequence comprises a prostate-specific antigen enhancer, a PSMA enhancer and a TARP promoter. It is transcriptionally active in human prostate cancer cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer, it has no detectable transcriptional activity. Herein, we describe that the PPT sequence in combination with a two-step transcriptional amplification (TSTA) system becomes active also in murine prostate cancer cells. An adenovirus with TSTA-amplified PPT-controlled expression of the luciferase reporter gene, Ad[PPT/TSTA-Luc], has up to 100-fold higher prostate-specific transcriptional activity than a non-amplified PPT-based adenovirus, Ad[PPT-Luc], in human cells. In addition, Ad[PPT/TSTA-Luc] confers prostate-specific transgene expression in murine cells, with an activity that is approximately 23% of Ad[CMV-Luc] in the transgenic adenocarcinoma of the mouse prostate (TRAMP)-C2 cells. Moreover, to visualize luciferase expression in living mice a charge-coupled device camera was used. Ad[PPT/TSTA-Luc] yielded approximately 30-fold higher transgene expression than Ad[PPT-Luc] in LNCaP tumor xenografts. Importantly, Ad[PPT/TSTA-Luc] also showed activity in murine TRAMP-C2 tumors, whereas Ad[PPT-Luc] activity was undetectable. These results highlight that the recombinant PPT sequence is active in murine prostate cancer cells when augmented by a TSTA system. This finding opens up for preclinical studies with prostate-specific therapeutic gene expression in immunocompetent mice.

1024. An Oncolytic Conditionally Replicating Adenovirus for Hormone-Dependent and Hormone-Independent Prostate Cancer

The use of conditionally replicating adenoviruses offers an attractive complementary treatment strategy for localized prostate cancer. We have produced a replicating adenovirus, Ad[I/PPT-E1A], where E1A gene expression is controlled by a recombinant regulatory sequence designated PPT. The PPT sequence comprises a PSA enhancer, a PSMA enhancer and a TARP promoter and it is shielded from transcriptional interference from adenoviral backbone sequences by an H19 insulator. Ad[I/PPT-E1A] yields prostate-specific E1A protein expression, viral replication and cytolysis in vitro. Furthermore, Ad[I/PPT-E1A] considerably regresses the growth of subcutaneous LNCaP prostate tumors in nude mice. Importantly, viral replication and cytolytic effect of Ad[I/PPT-E1A] are independent on the testosterone levels in the prostate cancer cell. This may be beneficial in a clinical setting since many prostate cancer patients are treated with androgen withdrawal. In conclusion, Ad[I/PPT-E1A] may prove to be useful in the treatment of localized prostate cancer.

626. A Novel Prostate-Specific Adenovirus for Gene Expression in Hormone-Sensitive and Hormone-Refractory Prostate Cancer Cells

To develop a transcriptional regulatory sequence for prostate-specific transgene expression that is more potent then the ones currently used in experimental and clinical prostate cancer gene therapy, we constructed various recombinant expression control sequences comprising the TARP promoter (TARPp) and the PSA enhancer (PSAe) and/or the PSMA enhancer (PSMAe). Transcriptional activities of the expression control sequences were examined by luciferase reporter gene assays on transiently transfected cells. We found that gene expression controlled by a chimeric regulatory sequences comprising PSAe linked to PSMAe, in turn linked to TARPp (PPT) is highly prostate-specific and active in prostate cancer cells both in the presence and absence of testosterone. Since prostate cancer patients are often treated by androgen withdrawal, it may be beneficial to the patient to use a regulatory sequence that results in high transgene expression also in the absence of testosterone. Recombinant adenoviruses with the PPT sequence in the E1 position of an E1/E3-deleted adenovirus serotype 5 vectors were constructed. A DNA insulator from the mouse H19 imprinting control region was inserted directly upstream of the expression cassette to protect the transcriptional integrity of PPT from interfering adenoviral sequences. The produced viruses confer prostate-specific transgene expression when screened on cell lines of various origins. We believe that an adenovirus with E1A and/or other therapeutic gene expression, controlled by the H19 insulator-shielded PPT regulatory sequence, is a promising candidate for prostate cancer gene therapy.