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PDF file - 48K, Description of disease at the time of first treatment.

PDF file - 1MB, PBMCs of a healthy donor was stimulated with supernatant collected from virus (CGTG-401 or control virus not coding CD40L) infected A549 lung cancer cells and IL-8 production was assessed 12, 24, 48, and 72 h later with FACSArray. The increase demonstrated at later time points indicates biological activity of the virus produced CD40L.

PDF file - 1.1MB, (A) Patient serum samples were analyzed for Th1 induced cytokines interferon-� (IFN-�), tumor necrosis factor-� (TNF-�) and interleukin-2 (IL-2) and Th2 cytokines interleukin-4 (IL-4), interleukin-5 (IL-5) and Interleukin 10 (IL-10). The ratio between Th1 and Th2 type cytokines was assessed. Cytokines levels are reported relative to their baseline level, which was set as 1 and a ratio between Th1/Th2 was calculated for each time point. (B) Malignant ascites of patient R8 and pleural effusion of patient T181 were analyzed for Th1 induced cytokines IFN-� and IL-2, and for Th2 induced IL-10 before and after treatment.

PDF file - 1.2MB, Blood samples for all 9 patients were collected before (baseline) and at several time points after the treatment. Soluble CD40L (sCD40L) and RANTES concentrations in the serum were assessed. Data are presented as median � SD.

PDF file - 64K, Adverse reactions. 9 patients total, 6 received concomitant low-lose cyclophosphamide 50 mg/d, 3 did not. Numbers of patients experiencing the reaction at any time during their treatment (including 28d follow-up) is shown. The data is shown for the two subgroups separately in parentheses (cyclophosphamide/none).

PDF file - 41K, Response evaluation of injected and non-injected lesions.

PDF file - 58K, Prior therapies.

PDF file - 2MB, Serum levels for (A) IL-6, (B) IL-8, (C) IL-10, (D) IL-12, (E) TNF-alpha, and (F) IFN-gamma was assessed before and after the treatment. Data are presented separately for patients receiving virus treatment with or without concomitant low dose cyclophosphamide (50 mg/d) and shown as median � SD. NA, sample not available.

PDF file - 69K, Description of historical controls (Pesonen et al. Gene Ther 2010;17:892-904) treated with unarmed oncolytic adenovirus Ad5/3-cox2L-D24.

The immunosuppressive environment of advanced tumors is a primary obstacle to the efficacy of immunostimulatory and vaccine approaches. Here, we report an approach to arm an oncolytic virus with CD40 ligand (CD40L) to stimulate beneficial immunologic responses in patients. A double-targeted chimeric adenovirus controlled by the hTERT promoter and expressing CD40L (CGTG-401) was constructed and nine patients with progressing advanced solid tumors refractory to standard therapies were treated intratumorally. No serious adverse events resulting in patient hospitalization occurred. Moderate or no increases in neutralizing antibodies were seen, suggesting effective Th1 immunologic effects. An assessment of the blood levels of virus indicated 17.5% of the samples (n = 40) were positive at a low level early after treatment, but not thereafter. In contrast, high levels of virus, CD40L, and RANTES were documented locally at the tumor. Peripheral blood mononuclear cells were analyzed by IFN-γ ELISPOT analysis and induction of both survivin-specific and adenovirus-specific T cells was seen. Antitumor T-cell responses were even more pronounced when assessed by intracellular cytokine staining after stimulation with tumor type–specific peptide pools. Of the evaluable patients, 83% displayed disease control at 3 months and in both cases in which treatment was continued the effect was sustained for at least 8 months. Injected and noninjected lesions responded identically. Together, these findings support further clinical evaluation of CGTG-401. Cancer Res; 72(7); 1621–31. ©2012 AACR.

The safety of oncolytic viruses for treatment of cancer has been shown in clinical trials while antitumor efficacy has often remained modest. As expression of the coxsackie-adenovirus receptor may be variable in advanced tumors, we developed Ad5-D24-RGD, a p16/Rb pathway selective oncolytic adenovirus featuring RGD-4C modification of the fiber. This allows viral entry through alpha-v-beta integrins frequently highly expressed in advanced tumors. Advanced tumors are often immunosuppressive which results in lack of tumor eradication despite abnormal epitopes being present. Granulocyte-macrophage colony stimulating factor (GMCSF) is a potent activator of immune system with established antitumor properties. To stimulate antitumor immunity and break tumor associated immunotolerance, we constructed Ad5-RGD-D24-GMCSF, featuring GMCSF controlled by the adenoviral E3 promoter. Preliminary safety of Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of human cancer was established. Treatments with Ad5-D24-RGD (N = 9) and Ad5-RGD-D24-GMCSF (N = 7) were well tolerated. Typical side effects were grade 1-2 fatigue, fever and injection site pain. 77% (10/13) of evaluable patients showed virus in circulation for at least 2 weeks. In 3 out of 6 evaluable patients, disease previously progressing stabilized after a single treatment with Ad5-RGD-D24-GMCSF. In addition, 2/3 patients had stabilization or reduction in tumor marker levels. All patients treated with Ad5-D24-RGD showed disease progression in radiological analysis, although 3/6 had temporary reduction or stabilization of marker levels. Induction of tumor and adenovirus specific immunity was demonstrated with ELISPOT in Ad5-RGD-D24-GMCSF treated patients. RGD modified oncolytic adenoviruses with or without GMCSF seem safe for further clinical development.

Patients with advanced solid tumors refractory to and progressing after conventional therapies were treated with three different regimens of low-dose cyclophosphamide (CP) in combination with oncolytic adenovirus. CP was given with oral metronomic dosing (50 mg/day, N = 21), intravenously (single 1,000 mg dose, N = 7) or both (N = 7). Virus was injected intratumorally. Controls (N = 8) received virus without CP. Treatments were well tolerated and safe regardless of schedule. Antibody formation and virus replication were not affected by CP. Metronomic CP (oral and oral + intravenous schedules) decreased regulatory T cells (Tregs) without compromising induction of antitumor or antiviral T-cell responses. Oncolytic adenovirus given together with metronomic CP increased cytotoxic T cells and induced Th1 type immunity on a systemic level in most patients. All CP regimens resulted in higher rates of disease control than virus only (all P < 0.0001) and the best progression-free (PFS) and overall survival (OS) was seen in the oral + intravenous group. One year PFS and OS were 53 and 42% (P = 0.0016 and P < 0.02 versus virus only), respectively, both which are unusually high for chemotherapy refractory patients. We conclude that low-dose CP results in immunological effects appealing for oncolytic virotherapy. While these first-in-human data suggest good safety, intriguing efficacy and extended survival, the results should be confirmed in a randomized trial.

Successful tumor targeting of systemically administered oncolytic adenoviruses may be hindered by interactions with blood components.Blood distribution of oncolytic adenoviruses featuring type 5 adenovirus fiber, 5/3 capsid chimerism, or RGD-4C in the fiber knob was investigated in vitro and in patients with refractory solid tumors.Virus titers and prevalence in serum of patients increased over the first post-treatment week, suggesting replication. Detection of low virus loads was more sensitive in blood clots than in serum, although viral levels500 viral particles/mL did not differ significantly between both sample types. While adenovirus bound to erythrocytes, platelets, granulocytes, and peripheral blood mononuclear cells in vitro, the virus was mainly detectable in erythrocytes and granulocytes in cancer patients. Taken together with a temporary post-treatment decrease in thrombocyte counts, platelet activation by adenovirus and subsequent clearance seem likely to occur in humans. Fiber modifications had limited observed effect on virus distribution in blood cell compartments. Neutrophils, monocytes and cytotoxic T lymphocytes were the major leukocyte subpopulations interacting with adenoviruses.Serum and blood clots are relevant to estimate oncolytic adenovirus replication. Insight into viral interactions with blood cells may contribute to the development of new strategies for tumor delivery.