Victor W. van Beusechem

Targeting of adenoviral vectors through a bispecific single-chain antibody

Recombinant adenoviral vectors are attractive in the context of cancer gene therapy because they are capable of delivering genes to a wide variety of tissues. The utility of adenoviruses is limited by their lack of specificity and by the absence of the receptor(s) for these viruses on many tumor cells. Redirecting adenoviral vectors to tissue- or tumor-specific targets can be achieved by using bispecific conjugates produced by chemical linkage of an anti-adenovirus antibody (Ab) and a ligand or Ab directed toward a specific target. To avoid the limitations of chemical conjugates, molecular conjugates of anti-fiber knob and ligand have been proposed. We present here a novel strategy that allows the production of recombinant bispecific single-chain Abs directed at cell surface molecules. A construct was made that encodes a neutralizing anti-adenovirus fiber single-chain Fv (scFv) Ab (S11) fused to a scFv Ab (425) directed against the epidermal growth factor receptor. The fusion protein markedly enhanced the infection efficiency of adenoviral vectors in epidermal growth factor receptor-expressing cell lines. The bispecific scFv could be purified and concentrated after binding of its 6His tag to a nickel column without significant loss of activity. This approach should permit the production of high quantities of active bispecific scFv for in vivo use. The universal design of the construct allows rapid screening for relevant specific scFv directed at cell surface antigens that can be incorporated into adenoviral targeting strategies.

Efficient and Selective Gene Transfer into Primary Human Brain Tumors by Using Single-Chain Antibody-Targeted Adenoviral Vectors with Native Tropism Abolished

ABSTRACT The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeting adenoviral vectors toward tumor cells may improve cancer gene therapy procedures by providing augmented tumor transduction and decreased toxicity to normal tissues. Targeting requires both the complete abolition of native tropism and the addition of a new specific binding ligand onto the viral capsid. Here we accomplished this by using doubly ablated adenoviral vectors, lacking coxsackievirus-adenovirus receptor and αv integrin binding capacities, together with bispecific single-chain antibodies targeted toward human epidermal growth factor receptor (EGFR) or the epithelial cell adhesion molecule. These vectors efficiently and selectively targeted both alternative receptors on the surface of human cancer cells. Targeted doubly ablated adenoviral vectors were also very efficient and specific with primary human tumor specimens. With primary glioma cell cultures, EGFR targeting augmented the median gene transfer efficiency of doubly ablated adenoviral vectors 123-fold. Moreover, EGFR-targeted doubly ablated vectors were selective for human brain tumors versus the surrounding normal brain tissue. They transduced organotypic glioma and meningioma spheroids with efficiencies similar to those of native adenoviral vectors, while exhibiting greater-than-10-fold-reduced background levels on normal brain explants from the same patients. As a result, EGFR-targeted doubly ablated adenoviral vectors had a 5- to 38-fold-improved tumor-to-normal brain targeting index compared to native vectors. Hence, single-chain targeted doubly ablated adenoviral vectors are promising tools for cancer gene therapy. They should provide an improved therapeutic index with efficient tumor transduction and effective protection of normal tissue.

Selective gene delivery toward gastric and esophageal adenocarcinoma cells via EpCAM-targeted adenoviral vectors

Application of recombinant adenoviral vectors for cancer gene therapy is currently limited due to lack of specificity for tumor cells. For gastric and esophageal adenocarcinoma, we present here that the relative abundant expression of the primary adenovirus receptor, coxsackie/adenovirus receptor (CAR), on normal epithelium compared to carcinoma favors the transduction of the epithelium. As such, to achieve specific transduction of cancer cells, targeting approaches are required that ablate the binding of the virus to CAR and redirect the virus to tumor-specific receptors. By immunohistochemistry and reverse transcriptase polymerase chain reaction assays, we demonstrate a marked difference in expression of the human epithelial cell adhesion molecule (EpCAM) between normal and (pre)malignant lesions of the stomach and esophagus. Based on this, we explored the feasibility of using EpCAM to achieve gastric and esophageal adenocarcinoma selective gene transfer. Adenoviral vectors redirected to EpCAM using bispecific antibodies against the adenovirus fiber-knob protein and EpCAM specifically infected gastric and esophageal cancer cell lines. Using primary human cells, an improved ratio of tumor transduction over normal epithelium transduction was accomplished by the EpCAM-targeted vectors. This study thus indicates that EpCAM-targeted adenoviral vectors may be useful for gastric and esophageal cancer–specific gene therapy in patients. Cancer Gene Therapy (2001) 8, 342–351

Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a target gene in cancer cells.

RNA interference (RNAi) is a posttranscriptional silencing mechanism triggered by double-stranded RNA that was recently shown to function in mammalian cells. Expression of cancer-associated genes was knocked down by expressing short hairpin RNAs (shRNAs) in cancer cells. By virtue of its excellent target specificity, RNAi may be used as a new therapeutic modality for cancer. The success of this approach will largely depend on efficient delivery of shRNAs to tumor cells. Tumor-selective replication competent viruses are especially suited to efficiently deliver anticancer genes to tumors. In addition, their intrinsic capacity to kill cancer cells makes these viruses promising anticancer agents per se. In this study, conditionally replicating adenoviruses were constructed encoding shRNAs targeted against firefly luciferase. These replicating viruses were shown to specifically silence the expression of the target gene in human cancer cells down to 30% relative to control virus. This finding offers the promise of using RNAi in the context of cancer gene therapy with oncolytic viruses.

Oncolytic Activity of p53-Expressing Conditionally Replicative Adenovirus AdΔ24-p53 against Human Malignant Glioma

Prognosis of malignant glioma is poor, and results of treatment remain mediocre. Conditionally replicative adenoviruses hold promise as alternative anticancer agents for the treatment of malignant glioma. Here, we evaluated the conditionally replicative adenovirus AdΔ24 and its recently developed derivative AdΔ24-p53, which expresses functional p53 tumor suppressor protein while replicating in cancer cells, for treatment of malignant glioma. In comparison to its parent AdΔ24, AdΔ24-p53 killed most malignant glioma cell lines and primary glioblastoma multiforme short-term cultures more effectively, irrespective of their p53 status. Moreover, AdΔ24-p53 caused more frequent regression and more delayed growth of IGRG121 xenografts derived from a glioblastoma multiforme in vivo. Five intratumoral injections of 107 pfu AdΔ24 gave 24 days median tumor growth delay (P < 0.01), 30% tumor regressions, and 30% animals surviving >120 days tumor-free or with a minimal tumor residual. The same dose of AdΔ24-p53 caused >113 days of median tumor growth delay (P < 0.001), 70% tumor regressions, and 60% animals surviving >120 days tumor-free or with a minimal tumor residual. Antitumor effects in vivo were associated with extensive conditionally replicative adenovirus replication, apoptosis induction, and tumor morphology changes, including dissociation, inflammatory cell infiltration, and necrosis. We conclude that conditionally replicative adenoviruses expressing p53 are promising new agents for treatment of malignant glioma.

WEE1 inhibition sensitizes osteosarcoma to radiotherapy

BackgroundThe use of radiotherapy in osteosarcoma (OS) is controversial due to its radioresistance. OS patients currently treated with radiotherapy generally are inoperable, have painful skeletal metastases, refuse surgery or have undergone an intralesional resection of the primary tumor. After irradiation-induced DNA damage, OS cells sustain a prolonged G2 cell cycle checkpoint arrest allowing DNA repair and evasion of cell death. Inhibition of WEE1 kinase leads to abrogation of the G2 arrest and could sensitize OS cells to irradiation induced cell death.MethodsWEE1 expression in OS was investigated by gene-expression data analysis and immunohistochemistry of tumor samples. WEE1 expression in OS cell lines and human osteoblasts was investigated by Western blot. The effect of WEE1 inhibition on the radiosensitivity of OS cells was assessed by cell viability and caspase activation analyses after combination treatment. The presence of DNA damage was visualized using immunofluorescence microscopy. Cell cycle effects were investigated by flow cytometry and WEE1 kinase regulation was analyzed by Western blot.ResultsWEE1 expression is found in the majority of tested OS tissue samples. Small molecule drug PD0166285 inhibits WEE1 kinase activity. In the presence of WEE1-inhibitor, irradiated cells fail to repair their damaged DNA, and show higher levels of caspase activation. The inhibition of WEE1 effectively abrogates the irradiation-induced G2 arrest in OS cells, forcing the cells into premature, catastrophic mitosis, thus enhancing cell death after irradiation treatment.ConclusionWe show that PD0166285, a small molecule WEE1 kinase inhibitor, can abrogate the G2 checkpoint in OS cells, pushing them into mitotic catastrophe and thus sensitizing OS cells to irradiation-induced cell death. This suggests that WEE1 inhibition may be a promising strategy to enhance the radiotherapy effect in patients with OS.

Conditionally Replicative Adenovirus with Tropism Expanded towards Integrins Inhibits Osteosarcoma Tumor Growth in Vitro and in Vivo

Purpose: The clinical course of osteosarcoma (OS) demands the development of new therapeutic options. Conditionally replicative adenoviruses (CRAds) represent promising agents for the treatment of solid tumors, because CRAds have an intrinsic replication capacity that allows in situ amplification and extensive tumor infection through lateral spread. The CRAd AdΔ24 has been developed to replicate selectively in cells with a defective retinoblastoma (Rb) pathway. Because genetic alterations in the Rb pathway are frequently observed in OS, AdΔ24 might be useful in the treatment of this cancer. Experimental Design: Because the lack of Coxsackie adenovirus receptor on OS cells limits the efficacy of CRAd treatment, we explored alternative target molecules on OS. Insertion of an Arg-Gly-Asp (RGD-4C) integrin-targeting motif into the adenovirus fiber knob expanded tropism toward the ανβ3 and ανβ5 integrins. The oncolytic capacity of the CRAd Ad5-Δ24RGD was tested on primary OS cells in vitro and in vivo. Results: The ανβ3and ανβ5 integrins are abundantly expressed on OS cells. RGD-mediated infection augmented adenovirus infection of primary OS cells by two orders of magnitude. Ad5-Δ24RGD was shown to be highly active in killing human OS cell lines, as well as primary cell cultures. Furthermore, intratumoral injections with Ad5-Δ24RGD into established human OS xenografts that were derived directly from a patient with OS refractory for chemotherapeutic treatment caused a significant tumor-growth delay. Furthermore, adenoviral particles could be detected in tumor cells 25 days posttumor injection. Conclusions: Targeting adenovirus toward integrins rendered OS cells more sensitive to killing by Ad5-Δ24RGD. These findings suggest that Ad5-Δ24RGD has potential for use in OS treatment.

Tissue Inhibitor of Metalloproteinase-3 Expression from an Oncolytic Adenovirus Inhibits Matrix Metalloproteinase Activity In vivo without Affecting Antitumor Efficacy in Malignant Glioma

Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, AdDelta24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus AdDelta24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by AdDelta24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2-activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the AdDelta24TIMP-3-treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87deltaEGFR) tumors with AdDelta24TIMP-3 and AdDelta24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of AdDelta24 did not significantly affect the antitumor efficacy of this oncolytic agent.

Differential Effects of Combined Ad5-Δ24RGD and Radiation Therapy in In vitro versus In vivo Models of Malignant Glioma

Purpose: The integrin-targeted conditionally replicating adenovirus Ad5-Δ24RGD has been shown to possess strong oncolytic activity in experimental tumors and is currently being developed toward phase I clinical evaluation for ovarian cancer and malignant glioma. Previously, we reported that combination therapy of Ad5-Δ24RGD with irradiation led to synergistic antitumor activity in s.c. glioma xenografts. In the current study, the underlying mechanism of action to this synergy was studied and the effects of combined therapy were assessed in an orthotopic glioma model. Experimental Design and Results: Sequencing studies in U-87 monolayers showed that delivery of irradiation before Ad5-Δ24RGD infection led to a greater oncolytic effect than simultaneous delivery or infection before irradiation. This effect was not due to enhanced virus production or release. Experiments using a luciferase-encoding vector revealed a small increase in transgene expression in irradiated cells. In tumor spheroids, combination therapy was more effective than Ad5-Δ24RGD or irradiation alone. Staining of spheroid sections showed improved penetration of virus to the core of irradiated spheroids. Mice bearing intracranial tumors received a combination of Ad5-Δ24RGD with 1 × 5 Gy total body irradiation or with 2 × 6 Gy whole brain irradiation. In contrast to the in vitro data and reported results in s.c. tumors, addition of radiotherapy did not significantly enhance the antitumor effect of Ad5-Δ24RGD. Conclusions: Combined treatment with Ad5-Δ24RGD and irradiation shows enhanced antitumor activity in vitro and in s.c. tumors, but not in an orthotopic glioma model. These differential results underscore the significance of the selected tumor model in assessing the effects of combination therapies with oncolytic adenoviruses.

Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome‐wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta‐analysis of findings from genome‐wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.—Meliopoulos, V. A., Andersen, L. E., Birrer, K. F., Simpson, K. J., Lowenthal, J. W., Bean, A. G. D., Stambas, J., Stewart, C. R., Tompkins, S. M., van Beusechem, V. W., Fraser, I., Mhlanga, M., Barichievy, S., Smith, Q., Leake, D., Karpilow, J., Buck, A., Jona, G., Tripp, R. A. Host gene targets for novel influenza therapies elucidated by high‐throughput RNA interference screens. FASEB J. 26, 1372‐1386 (2012). www.fasebj.org