Targeted cancer immunotherapy with oncolytic adenovirus coding for a fully human monoclonal antibody specific for CTLA-4.

Promising clinical results have been achieved with monoclonal antibodies (mAbs) such as ipilimumab and tremelimumab that block cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CD152). However, systemic administration of these agents also has the potential for severe immune-related adverse events. Thus, local production might allow higher concentrations at the target while reducing systemic side effects. We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Δ24aCTLA4 expressing complete human mAb specific for CTLA-4 and tested it in vitro, in vivo and in peripheral blood mononuclear cells (PBMCs) of normal donors and patients with advanced solid tumors. mAb expression was confirmed by western blotting and immunohistochemistry. Biological functionality was determined in a T-cell line and in PBMCs from cancer patients. T cells of patients, but not those of healthy donors, were activated by an anti-CTLA4mAb produced by Ad5/3-Δ24aCTLA4. In addition to immunological effects, a direct anti-CTLA-4-mediated pro-apoptotic effect was observed in vitro and in vivo. Local production resulted in 43-fold higher (P<0.05) tumor versus plasma anti-CTLA4mAb concentration. Plasma levels in mice remained below what has been reported safe in humans. Replication-competent Ad5/3-Δ24aCTLA4 resulted in 81-fold higher (P<0.05) tumor mAb levels as compared with a replication-deficient control. This is the first report of an oncolytic adenovirus producing a full-length human mAb. High mAb concentrations were seen at tumors with lower systemic levels. Stimulation of T cells of cancer patients by Ad5/3-Δ24aCTLA4 suggests feasibility of testing the approach in clinical trials.

Oncolytic adenovirus based on serotype 3.

Oncolytic adenoviruses have been safe in clinical trials but the efficacy has been mostly limited. All published trials have been performed with serotype 5 based viruses. The expression level of the Ad5 receptor CAR may be variable in advanced tumors. In contrast, the Ad3 receptor remains unclear, but is known to be abundantly expressed in most tumors. Therefore, we hypothesized that a fully serotype 3 oncolytic adenovirus might be useful for treating cancer. Patients exposed to adenoviruses develop high titers of serotype-specific neutralizing antibodies, which might compromise re-administration. Thus, having different serotype oncolytic viruses available might facilitate repeated dosing in humans. Ad3-hTERT-E1A is a fully serotype 3 oncolytic adenovirus controlled by the promoter of the catalytic domain of human telomerase. It was effective in vitro on cell lines representing seven major cancer types, although low toxicity was seen in non-malignant cells. In vivo, the virus had anti-tumor efficacy in three different animal models. Although in vitro oncolysis mediated by Ad3-hTERT-E1A and wild-type Ad3 occurred more slowly than with Ad5 or Ad5/3 (Ad3 fiber knob in Ad5) based viruses, in vivo the virus was at least as potent as controls. Anti-tumor efficacy was retained in presence of neutralizing anti-Ad5 antibodies whereas Ad5 based controls were blocked. In summary, we report generation of a non-Ad5 based oncolytic adenovirus, which might be useful for testing in cancer patients, especially in the context of high anti-Ad5 neutralizing antibodies.

Prolonged systemic circulation of chimeric oncolytic adenovirus Ad5/3-Cox2L-D24 in patients with metastatic and refractory solid tumors.

Eighteen patients with refractory and progressive solid tumors were treated with a single round of triple modified oncolytic adenovirus (Ad5/3-Cox2L-D24). Ad5/3-Cox2L-D24 is the first non-Coxsackie-adenovirus receptor-binding oncolytic adenovirus used in humans. Grades 1-2 flu-like symptoms, fever, and fatigue were seen in most patients, whereas transaminitis or thrombocytopenia were seen in some. Non-hematological grades 3-5 side effects were seen in one patient with grade 3 ileus. Treatment resulted in high neutralizing antibody titers within 3 weeks. Virus appeared in serum 2-4 days after treatment in 83% of patients and persisted for up to 5 weeks. One out of five radiologically evaluable patients had partial response (PR), one had minor response (MR), and three had progressive disease (PD). Two patients scored as PD had a decrease in tumor density. Tumor reductions not measurable with Response Evaluation Criteria In Solid Tumors (RECIST) were seen in a further four patients. PR, MR, stable disease, and PD were seen in 12, 23.5, 35, and 29.5% of tumor markers analyzed, respectively (N=17). Ad5/3-Cox2L-D24 appears safe for treatment of cancer in humans and extended virus circulation results from a single treatment. Objective evidence of anti-tumor activity was seen in 11/18 (61%) of patients. Clinical trials are needed to extend these findings.

Cancer gene therapy with oncolytic adenoviruses.

Metastatic cancer remains difficult to treat effectively and treatments are in most cases not curative despite significant side effects. Novel, targeted approaches such as gene therapy hold promise for the treatment of various tumor types. Among the most promising cancer gene therapy approaches are oncolytic adenoviruses, which are able to infect, replicate in and lyse tumor cells. Recent data from clinical trials with these vectors have shown that they are safe. However, antitumor efficacy needs to be improved to make oncolytic adenoviruses a viable treatment alternative for cancer patients. This review focuses on targeting strategies to improve tumor cell transduction and cancer cell selective replication. Strategies to improve antitumor efficacy by arming the virus with therapeutic transgenes are also discussed. Furthermore, an overview of the most important clinical approaches with oncolytic adenoviruses is given.

Ad5/3-9HIF-Delta24-VEGFR-1-Ig, an infectivity enhanced, dual-targeted and antiangiogenic oncolytic adenovirus for kidney cancer treatment.

Despite good safety data in clinical trials, oncolytic adenoviruses have not been efficient enough to make them a viable treatment alternative for cancers. As more potent viruses are being made, transcriptional and transductional targeting to tumor tissues becomes increasingly appealing. To improve antitumor efficacy, oncolytic adenoviruses can be armed with therapeutic transgenes, such as the antiangiogenic soluble vascular endothelial growth factor receptor 1-Ig fusion protein. We hypothesized that an infectivity enhanced, targeted, vascular endothelial growth factor receptor 1-Ig armed oncolytic adenovirus would exhibit improved specificity and antitumor effect in murine kidney cancer models. Two hypoxia inducible factor-sensitive promoters were evaluated for renal cancer specificity using a novel in vivo dual luciferase-imaging system. Earlier data had shown usefulness of the 5/3-serotype chimera capsid modification for kidney cancer. Therefore, we constructed Ad5/3-9HIF-Delta24-VEGFR-1-Ig, which showed good specificity and oncolytic effect on renal cancer cells in vitro and resulted in antitumor efficacy in a subcutaneous in vivo model, in which vascular endothelial growth factor receptor 1-Ig expression and a concurrent antiangiogenic effect were confirmed. In an intraperitoneally disseminated kidney cancer model, significantly enhanced survival was observed when compared with control viruses. These results suggest that a targeted, antiangiogenic, oncolytic adenovirus might be a valuable agent for testing in kidney cancer patients.

Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo.

Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.