Dirk M. Nettelbeck

Gene therapy: designer promoters for tumour targeting

One of the biggest challenges facing cancer therapy is to generate tumour-specific treatment strategies. Gene therapy hopes to achieve this by directing the activity of therapeutic genes specifically to the sites of disease. Of paramount importance for the success of this approach is the availability of tumour-specific delivery systems: both the transductional targeting of the vector vehicle and the restriction of transgene expression to the tumour are promising strategies towards this goal. This review will focus on the recent achievements in the field of transcriptional targeting and the different strategies to improve or design promoters with the desired specificities.

CTLA-4 and PD-L1 Checkpoint Blockade Enhances Oncolytic Measles Virus Therapy

We hypothesized that the combination of oncolytic virotherapy with immune checkpoint modulators would reduce tumor burden by direct cell lysis and stimulate antitumor immunity. In this study, we have generated attenuated Measles virus (MV) vectors encoding antibodies against CTLA-4 and PD-L1 (MV-aCTLA-4 and MV-aPD-L1). We characterized the vectors in terms of growth kinetics, antibody expression, and cytotoxicity in vitro. Immunotherapeutic effects were assessed in a newly established, fully immunocompetent murine model of malignant melanoma, B16-CD20. Analyses of tumor-infiltrating lymphocytes and restimulation experiments indicated a favorable immune profile after MV-mediated checkpoint modulation. Therapeutic benefits in terms of delayed tumor progression and prolonged median overall survival were observed for animals treated with vectors encoding anti-CTLA-4 and anti-PD-L1, respectively. Combining systemic administration of antibodies with MV treatment also improved therapeutic outcome. In vivo oncolytic efficacy against human tumors was studied in melanoma xenografts. MV-aCTLA-4 and MV-aPD-L1 were equally efficient as parental MV in this model, with high rates of complete tumor remission (> 80%). Furthermore, we could demonstrate lysis of tumor cells and transgene expression in primary tissue from melanoma patients. The current results suggest rapid translation of combining immune checkpoint modulation with oncolytic viruses into clinical application.

Cell cycle‐independent induction of apoptosis by the anti‐tumor drug flavopiridol in endothelial cells

The anti‐tumor drug Flavopiridol is a potent inhibitor of cyclin‐dependent kinases (cdks). As a consequence, Flavopiridol‐treated cells arrest in both G1 and G2, but Flavopiridol has also been shown to be cytotoxic for some tumor cell lines. The underlying molecular events are, however, unclear. We now show that Flavopiridol induces apoptosis in human umbilical vein endothelial cells (HUVECs), as judged by the occurrence of classical apoptotic markers, including chromatin condensation, internucleosomal cleavage, DNA fragmentation (TUNEL assay), annexin V binding and poly(ADP‐ribose) polymerase (PARP)‐cleavage. Such induction of apoptosis occurs with equal efficiency in both proliferating and G0/G1‐arrested cells. Because growth‐arrested HUVECs lack cdk2 activity and contain high levels of the cdk inhibitor p27, our observations suggest that cell cycle regulated cdks may not be the only critical target for Flavopiridol‐induced apoptosis. Surprisingly, A549 lung carcinoma cells were clearly dependent on cell proliferation for the induction of cell death, pointing to cell type‐related differences in the mechanism of Flavopiridol action. Int. J. Cancer 77:146–152, 1998.© 1998 Wiley‐Liss, Inc.

Transcriptional targeting of tumors with a novel tumor-specific survivin promoter

It has been demonstrated that survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, is expressed in human cancers but is undetectable in normal differentiated tissues. We employed a recombinant adenoviral vector (reAdGL3BSurvivin) in which a tumor-specific survivin promoter and a luciferase reporter gene were inserted into the E1-deleted region of adenovirus vector. Luciferase activity was measured in both multiple tumor cell lines and two primary melanoma cells infected with reAdGL3BSurvivin. Human fibroblast and mammary epithelial cell lines were used as negative controls. A reAdGL3CMV, containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity generated by the survivin promoter. Our data revealed that the survivin promoter showed high activity in both established tumor cell lines and the primary melanoma cells. In contrast, the in vivo studies indicated that the activities of survivin promoter were extremely low in the major mouse organs. The survivin promoter appears to be a promising tumor-specific promoter exhibiting a “tumor on” and “liver off” profile, and therefore, it may prove to be a good candidate for transcriptional targeting of cancer gene therapy in a wide variety of tumors.

Recombinant bispecific antibodies for the targeting of adenoviruses to CEA-expressing tumour cells: a comparative analysis of bacterially expressed single-chain diabody and tandem scFv.

We have generated two distinct recombinant bispecific antibody molecules for the retargeting of adenoviral vectors to CEA‐expressing tumour cells. These antibody molecules were produced by combining the antigen‐binding sites of a neutralising anti‐fibre knob scFv (S11) and an anti‐CEA antibody either in a single‐chain diabody format (scDb CEA‐S11) or a tandem scFv format (taFv CEA‐S11). In order to facilitate expression of taFv CEA‐S11 in bacteria we selected from a phage display library taFv molecules with an optimised linker that connects the two scFv fragments. ScDb CEA‐S11 and taFv CEA‐S11 were expressed and purified in soluble form from the bacterial periplasm with yields of approximately 100 µg per litre of bacterial culture. In vitro, both bispecific molecules mediated selective and enhanced transduction of CEA‐expressing tumour cells by recombinant adenoviruses. These assays did not reveal any differences in efficiency of adenoviral transduction by the two antibody formats. However, compared with taFv CEA‐S11, scDb CEA‐S11 exhibited a 2‐ to 3‐fold increased stability in human plasma at 37 °C. In summary, we could demonstrate that both formats are suitable for adenovirus targeting to tumour cells with similar biological activity in vitro. Copyright

Retargeting of adenoviral infection to melanoma: combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA.

Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus‐mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single‐chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma‐associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti‐HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50‐fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367‐fold increase), CAR‐independent and HMWMAA‐mediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wild‐type fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54‐fold increased adenoviral gene transfer to CAR‐negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA‐targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma.

A strategy for enhancing the transcriptional activity of weak cell type-specific promoters

Cell type- and tissue-specific promoters play an important role in the development of site-selective vectors for gene therapy. A large number of highly specific promoters has been described, but their applicability is often hampered by their inefficient transcriptional activity. In this study, we describe a new strategy for enhancing the activity of weak promoters without loss of specificity. The basic principle of this strategy is to establish a positive feedback loop which is initiated by transcription from a cell type-specific promoter. This was achieved by using a cell type-specific pro- moter to drive the simultaneous expression of the desired effector/reporter gene product and a strong artificial transcriptional activator which stimulates transcription through appropriate binding sites in the promoter. Using a VP16-LexA chimeric transcription factor, we show that this approach leads to a 14- to >100-fold enhancement of both the endothelial cell-specific von Willebrand factor promoter and the gastrointestinal-specific sucrase-isomaltase promoter while maintaining approximately 30- to >100-fold cell type specificity.

Enhanced adenovirus infection of melanoma cells by fiber-modification: Incorporation of RGD peptide or Ad5/3 chimerism

The incidence of malignant melanoma has been increasing. Unfortunately, advanced melanomas are rarely curable with standard therapy; therefore, new forms of treatment such as gene therapy are needed. The success of gene delivery or oncolysis depends on the nature of the vector. Adenoviral vectors are advantageous for several reasons; however, they are dependent of CAR (coxsackie and adenovirus receptor) which is deficient or heterogeneously expressed on melanoma cells in situ. Correspondingly, transduction of freshly purified melanoma cells has been show to be minimal or variable. In order to overcome this shortcoming, it is necessary to construct tropism modified adenoviral vectors. With this goal in mine, we generated two tropism modified vectors, Ad5lucRGD which has an RGD motif incorporated into the HI loop of the fiber knob and Ad5/3luc1 which contains the tail and shaft domain of Ad5 and the knob domain of Ad3. Herein we demonstrate that Ad5/3luc1 infects cells 1128 times better than Ad5luc1 and 34 times better than Ad5lucRGD. Furthermore we show that Ad5/3luc1 and Ad5lucRGD infect via a CAR independent route by blocking the CAR receptor. In addition, we show that the infectivity of the cells correlates with the expression of CAR and Ad3 receptors determined by FACS analysis. Therefore, Ad5/3 is very attractive as a potential therapeutic vector for malignant melanoma.

Transcriptional targeting of adenoviral vector through the CXCR4 tumor-specific promoter

Adenoviral vectors are considered to be good gene delivery vectors for cancer gene therapy due to their wide host tissue range and cell cycle-independent infectivity. However, the disadvantages include the lack of specificity for cancer cells and the high liver accumulation in vivo. The human CXCR4 gene is expressed at high levels in many types of cancers, but is repressed in the liver. We explored the CXCR4 promoter as a candidate to restrict adenoviral transgene expression to tumor cells with a low expression in host tissues. The luciferase activities in multiple cancer cell lines infected with recombinant adenovirus reAdGL3BCXCR4 or the control vector reAdGL3BCMV revealed that the CXCR4 promoter exhibited relatively high transcriptional activity in a breast cancer cell line, MDA-MB-361, and two ovarian cancer cell lines, OVCAR-3 and SKOV3. ip1, 65% (P=0.0087), 16.7% (P=0.1) and 20% (P=0.0079) compared to that of the CMV promoter, respectively, and low expression, 4.9 and 0.1%, respectively, in both normal cell lines HFBC and HMEC. In addition, CXCR4 had a low expression of luciferase (0.32%) compared to that of the CMV promoter in mouse liver in vivo. The data also revealed that the CXCR4 promoter was a stronger tumor-specific promoter (TSP) than the Cox-2M promoter in primary melanomas obtained from two patients. The CXCR4 promoter is shown to have a ‘tumor-on’ and ‘liver-off’ status in vitro and in vivo, and CXCR4 may prove to be a good candidate TSP for cancer gene therapy approaches for melanoma and breast cancers.

Combining high selectivity of replication with fiber chimerism for effective adenoviral oncolysis of CAR-negative melanoma cells.

Oncolytic adenoviruses constitute a new and promising tool for cancer treatment that has been rapidly translated into clinical trials. However, minimal or absent expression of the adenovirus serotype 5 (Ad5) receptor CAR (coxsackievirus and adenovirus receptor) on cancer cells represents a major limitation for Ad5-based oncolysis. Here, we report on the resistance of CAR-negative primary melanoma cells to cell killing by wild-type Ad5 (Ad5wt) even after high titer infection, thus underlining the need for tropism-modification of oncolytic adenoviruses. We engineered a new generation of oncolytic adenoviruses that exhibit both efficient target cell infection by swapping Ad5 fiber domains with those of Ad serotype 3, which binds to a receptor distinct from CAR, and targeted virus replication. Fiber chimerism resulted in efficient cytopathicity to primary melanoma cells, which was at least 104-fold increased relative to Ad5wt. Since viral infectivity mediated by such modified viral capsids was not cell type-specific, it was pivotal to carefully restrict adenoviral replication to target cells. Towards this end, we replaced both E1A and E4 promoters of fiber chimeric viruses by tyrosinase enhancer/promoter constructs. The resulting viruses showed melanoma-specific expression of E1A and E4 and combined efficient virus replication and cell killing in melanoma cell lines and primary melanoma cells with a remarkable specificity profile that implements strong attenuation in nonmelanoma cells, including normal fibroblasts and keratinocytes.