Manel Cascallo

Oncolytic Adenovirus ICOVIR-7 in Patients with Advanced and Refractory Solid Tumors

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.

Hyaluronidase Expression by an Oncolytic Adenovirus Enhances Its Intratumoral Spread and Suppresses Tumor Growth

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.

ICOVIR-5 shows E2F1 addiction and potent antiglioma effect in vivo

During 2007, approximately 200,000 people in the United States will be diagnosed with brain tumors. Gliomas account for 77% of primary malignant brain tumors, and the prognosis has hardly changed in the past 20 years, with only 30% of patients with malignant glioma surviving 5 years after diagnosis. Oncolytic adenoviruses are promising therapies for the treatment of gliomas. Here, report the antiglioma activity of the tumor-selective ICOVIR-5 adenovirus, which encompasses an early 1A adenoviral (E1A) deletion in the retinoblastoma (Rb) protein-binding region, substitution of the E1A promoter for E2F-responsive elements, and an RGD-4C peptide motif inserted into the adenoviral fiber to enhance adenoviral tropism. Mechanistic studies showed a dramatic addiction of ICOVIR-5 to the E2F1 oncogene in vitro and in vivo. This addiction was mediated by the occupancy of the ectopic adenoviral E2F1-responsive elements by the endogenous E2F1 protein resulting in high level of E1A expression in cancer cells and potent antiglioma effect. Importantly, we showed for the first time the ability of oncolytic adenoviruses to enhance E2F transcriptional activity in vivo, and we provided direct evidence of the interaction of the E2F1 protein with native and ectopic adenovirus promoters. Restoration of Rb function led to the association of Rb/E2F1 repressor complexes with ICOVIR-5 ectopic E2F1 promoter and subsequent down-modulation of E1A, dramatically impairing adenoviral replication. In xenografted mice, intratumoral injection of ICOVIR-5 resulted in a significant improvement of the median survival (P < 0.0001), and furthermore, led to 37% of long-term survivors free of disease. The antitumor activity of ICOVIR-5 suggests that it has the potential to be an effective agent in the treatment of gliomas.

Bioselection of a Gain of Function Mutation that Enhances Adenovirus 5 Release and Improves Its Antitumoral Potency

Genetic bioselection of a mutagenized Ad5wt stock in human tumor xenografts led us to isolate AdT1, a mutant displaying a large-plaque phenotype in vitro and an enhanced systemic antitumor activity in vivo. AdT1 phenotype correlates with an increased progeny release without affecting total viral yield in different human tumors and cancer-associated fibroblasts. An approach combining hybrid Ad5/AdT1 recombinants and sequencing identified a truncating insertion in the endoplasmic reticulum retention domain of the E3/19K protein (445A mutation) which relocates the protein to the plasma membrane and is responsible for AdT1s enhanced release. E3/19K-445A phenotype does not correlate with the proteins ability to interact with MHC-I or induce apoptosis. Intracellular calcium measurement revealed that the 445A mutation induces extracellular Ca(2+) influx, deregulating intracellular Ca(2+) homeostasis and inducing membrane permeabilization, a viroporin-like function. E3/19K-445A mutants also display enhanced antitumoral activity when injected both intratumorally and systemically in different models in vivo. Our results indicate that the inclusion of mutation 445A in tumor-selective adenoviruses would be a very powerful tool to enhance their antitumor efficacy.

Oncolytic viruses from the perspective of the immune system

Cancer treatment with oncolytic viruses is at a crucial intersection from where two very different routes can be taken. The key role of the immune system needs to be addressed proactively to succeed. An immunocentric point of view posits that the intense immunosuppression induced by tumors can be outbalanced by the natural immunogenicity of viruses. To their advantage, viruses can be safely armed to be even more immunostimulatory. The microbe-associated inflammatory response is optimal for antigen presentation and helps to reveal the hidden tumor antigens. The induced immune effector cells patrol the organs to destroy disseminated tumor cells out of the reach of the oncolytic virus. However, as tumor immunosuppression is localized, this concept needs to be revisited because every tumor focus will have to be reached by the oncolytic virus. By contrast, virocentrics see the immune system as an obstacle to virotherapy. A virus is so immunogenic that it dominates all the elicited immunity to the detriment of a response towards tumor antigens. For them immunosuppression is the way to go, and the intense immunosuppression in and around the tumor is now an advantage, offering a privileged site for virus replication. A better oncolytic virus evades the immune system, but such a virus should be very tumor-selective to be safe. Although the trend favors immunocentrics, clinical results have been more often documented in immunocompromised patients. Trials of comparative interventions on the immune system will validate immunocentrism or virocentrism. What seems clear is that at this intersection one should take one route or the other to overcome the current limitations of virotherapy.

Minimal RB-responsive E1A Promoter Modification to Attain Potency, Selectivity, and Transgene-arming Capacity in Oncolytic Adenoviruses

Oncolytic adenoviruses are promising anticancer agents due to their ability to self-amplify at the tumor mass. However, tumor stroma imposes barriers difficult to overcome by these agents. Transgene expression is a valuable strategy to counteract these limitations and to enhance antitumor activity. For this purpose, the genetic backbone in which the transgene is inserted should be optimized to render transgene expression compatible with the adenovirus replication cycle and to keep genome size within the encapsidation size limit. In order to design a potent and selective oncolytic adenovirus that keeps intact all the viral functions with minimal increase in genome size, we inserted palindromic E2F-binding sites into the endogenous E1A promoter. The insertion of these sites controlling E1A-Δ24 results in a low systemic toxicity profile in mice. Importantly, the E2F-binding sites also increased the cytotoxicity and the systemic antitumor activity relative to wild-type adenovirus in all cancer models tested. The low toxicity and the increased potency results in improved antitumor efficacy after systemic injection and increased survival of mice carrying tumors. Furthermore, the constrained genome size of this backbone allows an efficient and potent expression of transgenes, indicating that this virus holds promise for overcoming the limitations of oncolytic adenoviral therapy.

Replacement of Adenovirus Type 5 Fiber Shaft Heparan Sulfate Proteoglycan-Binding Domain with RGD for Improved Tumor Infectivity and Targeting

Tumor targeting on systemic adenovirus administration is key to improve the prospects of adenovirus-mediated gene therapy and virus therapy of cancer. Despite many genetic and ligand conjugation approaches this objective remains elusive. Ablation of human adenovirus type 5 (Ad5) binding to its natural receptors in airway epithelial cells, that is, the coxsackievirus and adenovirus receptor (CAR) and integrins, does not impact on the preeminent liver tropism of adenovirus in the bloodstream. This is explained by a distinct entry pathway mediated by blood factors and heparan sulfates. Mutation of the KKTK heparin sulfate-binding domain of the fiber shaft to GATK results in liver transduction detargeting, but it is not compatible with otherwise useful HI-loop tumor-targeting ligand insertions such as the insertion of Arg-Gly-Asp (RGD). To circumvent this problem we have mutated the KKTK domain to RGDK, and analyzed the liver-detargeting and tumor-targeting transduction properties of this replacement mutant. Similar to RGD at the HI-loop, RGD at this new shaft location efficiently enhances the infectivity of adenovirus and improves the tumor-to-liver transduction ratio in vivo.

Adenovirus-mediated wt-p16 reintroduction induces cell cycle arrest or apoptosis in pancreatic cancer.

Pancreatic cancer has long carried poor prognosis. The development of new therapeutic approaches is particularly urgent. Inactivation of the tumor-suppressor gene p16 INK4a/CDKN2 , a specific inhibitor of the cyclin-dependent kinases CDK4 and CDK6, is the most common genetic alteration in human pancreatic cancer, making it an ideal target for gene replacement. Here we transfected tumor cells using a recombinant adenovirus containing the wt- p16 cDNA (Ad5RSV- p16 ). The overexpression of p16 decreased cell proliferation in all four human pancreatic tumor cell lines (NP-9, NP-18, NP-29, and NP-31). However, G1 arrest and senescence were observed in only three. In contrast, the fourth (NP-18) showed a significant increase in apoptosis. This differential behavior may be related to the differences found in the expression level of E2F-1. Experiments on subcutaneous pancreatic xenografts demonstrated the effectiveness of p16 in the inhibition of pancreatic tumor growth in vivo. Taken together, our results indicate that approaches involving p16 replacement are promising in pancreatic cancer treatment. Cancer Gene Therapy (2001) 8, 740–750

Modulation of drug cytotoxicity by reintroduction of wild-type p53 gene (Ad5CMV-p53) in human pancreatic cancer.

Chemotherapy does not significantly improve prognosis in pancreatic cancer. New therapeutical approaches involving p53 gene replacement appear to be very encouraging due to the key role of p53 in the cell response to DNA damage. Here, we have evaluated the effectiveness of combining wild-type p53 (wt-p53) gene reintroduction (Ad5CMV-p53) and exposure to two genotoxic drugs, gemcitabine and cisplatin, in several human pancreatic cell lines. The efficiency of the combinations was clearly dependent upon timing, as assessed by cell survival determinations. Although wt-p53 transduction before drug treatment induced chemoresistance, p53 transduction in cells treated previously with gemcitabine increased cytotoxicity. Cell cycle profiles showed significant decreases in the percentage of cells in the S phase as a consequence of arrests provoked by the expression of exogenous p53, reducing the number of cells susceptible to the drug. The sensitivity of cells to cisplatin, which has a lower degree of S-phase specificity, was not modified as much by p53 gene replacement. In contrast, the recognition of the previous drug-induced DNA damage by the newly expressed wt-p53 elicited increases in sub-G1 populations, consistent with the annexin determinations and bax/bcl-2 ratios observed. Experiments on subcutaneous pancreatic xenografts corroborated the effectiveness of this approach in vivo. Thus, the combination of p53 transduction and chemotherapy, under a correct schedule of administration, appears to be a very promising therapy for human pancreatic cancer.

Safety and Efficacy of VCN-01, an Oncolytic Adenovirus Combining Fiber HSG-Binding Domain Replacement with RGD and Hyaluronidase Expression

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.