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Sarcomas are a relatively rare cancer, but often incurable at the late metastatic stage. Oncolytic immunotherapy has gained attention over the past years, and a wide range of oncolytic viruses have been delivered via intratumoral injection with positive safety and promising efficacy data. Here, we report preclinical and clinical results from treatment of sarcoma with oncolytic adenovirus Ad5/3-D24-GMCSF (CGTG-102). Ad5/3-D24-GMCSF is a serotype chimeric oncolytic adenovirus coding for human granulocyte-macrophage colony-stimulating factor (GM-CSF). The efficacy of Ad5/3-D24-GMCSF was evaluated on a panel of soft-tissue sarcoma (STS) cell lines and in two animal models. Sarcoma specific human data were also collected from the Advanced Therapy Access Program (ATAP), in preparation for further clinical development. Efficacy was seen in both in vitro and in vivo STS models. Fifteen patients with treatment-refractory STS (13/15) or primary bone sarcoma (2/15) were treated in ATAP, and treatments appeared safe and well-tolerated. A total of 12 radiological RECIST response evaluations were performed, and two cases of minor response, six cases of stable disease and four cases of progressive disease were detected in patients progressing prior to virus treatment. Overall, the median survival time post treatment was 170 days. One patient is still alive at 1,459 days post virus treatment. In summary, Ad5/3-D24-GMCSF appears promising for the treatment of advanced STS; a clinical trial for treatment of refractory injectable solid tumors including STS is ongoing., (© 2013 UICC.)

Background: Oncolytic viruses have shown potential as cancer therapeutics, but not all patients seem to benefit from therapy. Polymorphisms in Fc gamma receptors (FcgRs) lead to altered binding affinity of IgG between the receptor allotypes and therefore contribute to differences in immune defense mechanisms. Associations have been identified between FcgR polymorphisms and responsiveness to different immunotherapies. Taken together with the increasing understanding that immunological factors might determine the efficacy of oncolytic virotherapy we studied whether FcgR polymorphisms would have prognostic and/or predictive significance in the context of oncolytic adenovirus treatments., Methods: 235 patients with advanced solid tumors were genotyped for two FcgR polymorphisms, FcgRIIa-H131R (rs1801274) and FcgRIIIa-V158F (rs396991), using TaqMan based qPCR. The genotypes were correlated with patient survival and tumor imaging data., Results: In patients treated with oncolytic adenoviruses, overall survival was significantly shorter if the patient had an FcgRIIIa-VV/ FcgRIIa-HR (VVHR) genotype combination (P = 0,032). In contrast, patients with FFHR and FFRR genotypes had significantly longer overall survival (P = 0,004 and P = 0,006, respectively) if they were treated with GM-CSF-armed adenovirus in comparison to other viruses. Treatment of these patients with unarmed virus correlated with shorter survival (P < 0,0005 and P = 0,016, respectively). Treating FFHH individuals with CD40L-armed virus resulted in longer survival than treatment with other viruses (P = 0,047)., Conclusions: Our data are compatible with the hypothesis that individual differences in effector cell functions, such as NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and tumor antigen presentation by APCs caused by polymorphisms in FcgRs could play role in the effectiveness of oncolytic virotherapies. If confirmed in larger populations, FcgR polymorphisms could have potential as prognostic and predictive biomarkers for oncolytic adenovirus therapies to enable better selection of patients for clinical trials. Also, putative associations between genotypes, different viruses and survival implicate potentially important mechanistic issues.

Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cancer cell death. We hypothesized that the combination of oncolytic adenovirus with low-dose temozolomide (TMZ) is safe, effective, and capable of inducing antitumor immune responses. Metronomic low-dose cyclophosphamide (CP) was added to selectively reduce regulatory T-cells. Preclinically, combination therapy inhibited tumor growth, increased autophagy, and triggered immunogenic cell death as indicated by elevated calreticulin, adenosine triphosphate (ATP) release, and nuclear protein high-mobility group box-1 (HMGB1) secretion. A total of 41 combination treatments given to 17 chemotherapy-refractory cancer patients were well tolerated. We observed anti- and proinflammatory cytokine release, evidence of virus replication, and induction of neutralizing antibodies. Tumor cells showed increased autophagy post-treatment. Release of HMGB1 into serum--a possible indicator of immune response--increased in 60% of treatments, and seemed to correlate with tumor-specific T-cell responses, observed in 10/15 cases overall (P = 0.0833). Evidence of antitumor efficacy was seen in 67% of evaluable treatments with a trend for increased survival over matched controls treated with virus only. In summary, the combination of oncolytic adenovirus with low-dose TMZ and metronomic CP increased tumor cell autophagy, elicited antitumor immune responses, and showed promising safety and efficacy.

Twenty-five patients with chemotherapy refractory cancer were treated with a fully serotype 3-based oncolytic adenovirus Ad3-hTERT-E1A. In mice, Ad3 induced higher amounts of cytokines but less liver damage than Ad5 or Ad5/3. In humans, the only grade 3 adverse reactions were self-limiting cytopenias and generally the safety profile resembled Ad5-based oncolytic viruses. Patients that had been previously treated with Ad5 viruses presented longer lasting lymphocytopenia but no median increase in Ad3-specific T-cells in blood, suggesting immunological activity against antigens other than Ad3 hexon. Frequent alterations in antitumor T-cells in blood were seen regardless of previous virus exposure. Neutralizing antibodies against Ad3 increased in all patients, whereas Ad5 neutralizing antibodies remained stable. Treatment with Ad3-hTERT-E1A resulted in re-emergence of Ad5 viruses from previous treatments into blood and vice versa. Signs of possible efficacy were seen in 11/15 (73%) patients evaluable for tumor markers, four of which were treated only intravenously. Particularly promising results were seen in breast cancer patients and especially those receiving concomitant trastuzumab. Taken together, Ad3-hTERT-E1A seems safe for further clinical testing or development of armed versions. It offers an immunologically attractive alternative, with possible pharmacodynamic differences and a different receptor compared to Ad5.

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., (©2012 AACR.)

Augmenting antitumor immunity is a promising way to enhance the potency of oncolytic adenoviral therapy. Granulocyte-macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific CD8(+) cytotoxic T-lymphocytes. Serotype 5 adenoviruses (Ad5) are commonly used in cancer gene therapy. However, expression of the coxsackie-adenovirus receptor is variable in many advanced tumors and preclinical data have demonstrated an advantage for replacing the Ad5 knob with the Ad3 knob. Here, a 5/3 capsid chimeric and p16-Rb pathway selective oncolytic adenovirus coding for GMCSF was engineered and tested preclinically. A total of 21 patients with advanced solid tumors refractory to standard therapies were then treated intratumorally and intravenously with Ad5/3-D24-GMCSF, which was combined with low-dose metronomic cyclophosphamide to reduce regulatory T cells. No severe adverse events occurred. Analysis of pretreatment samples of malignant pleural effusion and ascites confirmed the efficacy of Ad5/3-D24-GMCSF in transduction and cell killing. Evidence of biological activity of the virus was seen in 13/21 patients and 8/12 showed objective clinical benefit as evaluated by radiology with Response Evaluation Criteria In Solid Tumors (RECIST) criteria. Antiadenoviral and antitumoral immune responses were elicited after treatment. Thus, Ad5/3-D24-GMCSF seems safe in treating cancer patients and promising signs of efficacy were seen.