Successful virotherapy requires efficient virus spread within tumors. We tested whether the expression of hyaluronidase, an enzyme which dissociates the extracellular matrix (ECM), could enhance the intratumoral distribution of an oncolytic adenovirus and improve its therapeutic activity. As a proof of concept, we demonstrated that intratumoral coadministration of hyaluronidase in mice-bearing tumor xenografts improves the antitumor activity of an oncolytic adenovirus. Next, we constructed a replication-competent adenovirus expressing a soluble form of the human sperm hyaluronidase (PH20) under the control of the major late promoter (MLP) (AdwtRGD-PH20). Intratumoral treatment of human melanoma xenografts with AdwtRGD-PH20 resulted in degradation of hyaluronan (HA), enhanced viral distribution, and induced tumor regression in all treated tumors. Finally, the PH20 cDNA was inserted in an oncolytic adenovirus that selectively kills pRb pathway-defective tumor cells. The antitumoral activity of the novel oncolytic adenovirus expressing PH20 (ICOVIR17) was compared to that of the parental virus ICOVIR15. ICOVIR17 showed more antitumor efficacy following intratumoral and systemic administration in mice with prestablished tumors, along with an improved spread of the virus within the tumor. Importantly, a single intravenous dose of ICOVIR17 induced tumor regression in 60% of treated tumors. These results indicate that ICOVIR17 is a promising candidate for clinical testing.

Purpose: Recent data from randomized clinical trials with oncolytic viral therapies and with cancer immunotherapies have finally recapitulated the promise these platforms demonstrated in preclinical models. Perhaps the greatest advance with oncolytic virotherapy has been the appreciation of the importance of activation of the immune response in therapeutic activity. Meanwhile, the understanding that blockade of immune checkpoints (with antibodies that block the binding of PD1 to PDL1 or CTLA4 to B7-2) is critical for an effective antitumor immune response has revitalized the field of immunotherapy. The combination of immune activation using an oncolytic virus and blockade of immune checkpoints is therefore a logical next step. Experimental Design: Here, we explore such combinations and demonstrate their potential to produce enhanced responses in mouse tumor models. Different combinations and regimens were explored in immunocompetent mouse models of renal and colorectal cancer. Bioluminescence imaging and immune assays were used to determine the mechanisms mediating synergistic or antagonistic combinations. Results: Interaction between immune checkpoint inhibitors and oncolytic virotherapy was found to be complex, with correct selection of viral strain, antibody, and timing of the combination being critical for synergistic effects. Indeed, some combinations produced antagonistic effects and loss of therapeutic activity. A period of oncolytic viral replication and directed targeting of the immune response against the tumor were required for the most beneficial effects, with CD8⁺ and NK, but not CD4⁺ cells mediating the effects. Conclusions: These considerations will be critical in the design of the inevitable clinical translation of these combination approaches. Clin Cancer Res; 21(24); 5543–51. ©2015 AACR. See related commentary by Slaney and Darcy, p. 5417

Purpose: Tumor targeting upon intravenous administration and subsequent intratumoral virus dissemination are key features to improve oncolytic adenovirus therapy. VCN-01 is a novel oncolytic adenovirus that combines selective replication conditional to pRB pathway deregulation, replacement of the heparan sulfate glycosaminoglycan putative-binding site KKTK of the fiber shaft with an integrin-binding motif RGDK for tumor targeting, and expression of hyaluronidase to degrade the extracellular matrix. In this study, we evaluate the safety and efficacy profile of this novel oncolytic adenovirus. Experimental Design: VCN-01 replication and potency were assessed in a panel of tumor cell lines. VCN-01 tumor-selective replication was evaluated in human fibroblasts and pancreatic islets. Preclinical toxicity, biodistribution, and efficacy studies were conducted in mice and Syrian hamsters. Results: Toxicity and biodistribution preclinical studies support the selectivity and safety of VCN-01. Antitumor activity after intravenous or intratumoral administration of the virus was observed in all tumor models tested, including melanoma and pancreatic adenocarcinoma, both in immunodeficient mice and immunocompetent hamsters. Conclusions: Oncolytic adenovirus VCN-01 characterized by the expression of hyaluronidase and the RGD shaft retargeting ligand shows an efficacy–toxicity prolife in mice and hamsters by intravenous and intratumoral administration that warrants clinical testing. Clin Cancer Res; 21(6); 1406–18. ©2014 AACR.

Purpose: Twenty-one patients with cancer were treated with a single round of oncolytic adenovirus ICOVIR-7. Experimental Design: ICOVIR-7 features an RGD-4C modification of the fiber HI-loop of serotype 5 adenovirus for enhanced entry into tumor cells. Tumor selectivity is mediated by an insulator, a modified E2F promoter, and a Rb-binding site deletion of E1A, whereas replication is optimized with E2F binding hairpins and a Kozak sequence. ICOVIR-7 doses ranged from 2 × 10¹⁰ to 1 × 10¹² viral particles. All patients had advanced and metastatic solid tumors refractory to standard therapies. Results: ICOVIR-7 treatment was well tolerated with mild to moderate fever, fatigue, elevated liver transaminases, chills, and hyponatremia. One patient had grade 3 anemia but no other serious side effects were seen. At baseline, 9 of 21 of patients had neutralizing antibody titers against the ICOVIR-7 capsid. Treatment resulted in neutralizing antibody titer induction within 4 weeks in 16 of 18 patients. No elevations of serum proinflammatory cytokine levels were detected. Viral genomes were detected in the circulation in 18 of 21 of patients after injection and 7 of 15 of the samples were positive 2 to 4 weeks later suggesting viral replication. Conclusions: Overall, objective evidence of antitumor activity was seen in 9 of 17 evaluable patients. In radiological analyses, 5 of 12 evaluable patients had stabilization or reduction in tumor size. These consisted of one partial response, two minor responses and two cases of stable disease, all occurring in patients who had progressive disease before treatment. In summary, ICOVIR-7 treatment is apparently safe, resulting in anticancer activity, and is therefore promising for further clinical testing. Clin Cancer Res; 16(11); 3035–43. ©2010 AACR.

E2F acts as a transcriptional repressor when bound to unphosphorylated RB during the G₁ or G₀ phase. Upon phosphorylation, E2F is released from the E2F–RB complexes to activate transcription. Tumor cells are characterized by an increase in the level of "free" E2F as a consequence of the absence or hyperphosphorylation of RB. The E2F-1 promoter is a well-characterized E2F-responsive promoter, and it can be used to control adenovirus E1a gene expression as a strategy to achieve tumor-selective expression and replication of an adenovirus. ICOVIR-5 (Ad-DM-E2F-K-24RGD) is an optimized oncolytic adenovirus that combines E1a transcriptional control by an insulated form of the E2F promoter with the 24 mutation of E1a to improve the therapeutic index of Ad24RGD. ICOVIR-5 also contains the Kozak sequence at the E1a start codon, which is important to restore E1a expression and viral replication to AdwtRGD levels in tumor cells. The unique combination of genetic elements in ICOVIR-5 allows the selectivity for cells with a deregulated E2F–RB pathway to be increased and potent anti-tumoral activity to be maintained. Dose–response toxicological and efficacy studies after a single systemic administration in pre-clinical models in mice are presented to demonstrate that this virus holds promise for treatment of disseminated cancer.