Marta Giménez-Alejandre

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.

VCN-01 is an oncolytic adenovirus designed to replicate selectively in tumor cells with high abundance of free E2F-1, a consequence of a dysfunctional RB1 pathway. Thus, we reasoned that VCN-01 could provide targeted therapeutic activity against even chemoresistant retinoblastoma. In vitro, VCN-01 effectively killed patient-derived retinoblastoma models. In mice, intravitreous administration of VCN-01 in retinoblastoma xenografts induced tumor necrosis, improved ocular survival compared with standard-of-care chemotherapy, and prevented micrometastatic dissemination into the brain. In juvenile immunocompetent rabbits, VCN-01 did not replicate in retinas, induced minor local side effects, and only leaked slightly and for a short time into the blood. Initial phase 1 data in patients showed the feasibility of the administration of intravitreous VCN-01 and resulted in antitumor activity in retinoblastoma vitreous seeds and evidence of viral replication markers in tumor cells. The treatment caused local vitreous inflammation but no systemic complications. Thus, oncolytic adenoviruses targeting RB1 might provide a tumor-selective and chemotherapy-independent treatment option for retinoblastoma.

Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense desmoplastic stroma that limits the delivery of anticancer agents. VCN-01 is an oncolytic adenovirus designed to replicate in cancer cells with a dysfunctional RB1 pathway and express hyaluronidase. Here, we evaluated the mechanism of action of VCN-01 in preclinical models and in patients with pancreatic cancer. Methods VCN-01 replication and antitumor efficacy were evaluated alone and in combination with standard chemotherapy in immunodeficient and immunocompetent preclinical models using intravenous or intratumoral administration. Hyaluronidase activity was evaluated by histochemical staining and by measuring drug delivery into tumors. In a proof-of-concept clinical trial, VCN-01 was administered intratumorally to patients with PDAC at doses up to 1×10 11 viral particles in combination with chemotherapy. Hyaluronidase expression was measured in serum by an ELISA and its activity within tumors by endoscopic ultrasound elastography. Results VCN-01 replicated in PDAC models and exerted antitumor effects which were improved when combined with chemotherapy. Hyaluronidase expression by VCN-01 degraded tumor stroma and facilitated delivery of a variety of therapeutic agents such as chemotherapy and therapeutic antibodies. Clinically, treatment was generally well-tolerated and resulted in disease stabilization of injected lesions. VCN-01 was detected in blood as secondary peaks and in post-treatment tumor biopsies, indicating virus replication. Patients had increasing levels of hyaluronidase in sera over time and decreased tumor stiffness, suggesting stromal disruption. Conclusions VCN-01 is an oncolytic adenovirus with direct antitumor effects and stromal disruption capabilities, representing a new therapeutic agent for cancers with dense stroma. Trial registration number EudraCT number: 2012-005556-42 and NCT02045589 .

Chimeric antigen receptor (CAR)-T cell therapy for solid tumors faces significant hurdles, including T-cell inhibition mediated by the PD-1/PD-L1 axis. The effects of disrupting this pathway on T-cells are being actively explored and controversial outcomes have been reported. Here, we hypothesize that CAR-antigen affinity may be a key factor modulating T-cell susceptibility towards the PD-1/PD-L1 axis. We systematically interrogate CAR-T cells targeting HER2 with either low (LA) or high affinity (HA) in various preclinical models. Our results reveal an increased sensitivity of LA CAR-T cells to PD-L1-mediated inhibition when compared to their HA counterparts by using in vitro models of tumor cell lines and supported lipid bilayers modified to display varying PD-L1 densities. CRISPR/Cas9-mediated knockout (KO) of PD-1 enhances LA CAR-T cell cytokine secretion and polyfunctionality in vitro and antitumor effect in vivo and results in the downregulation of gene signatures related to T-cell exhaustion. By contrast, HA CAR-T cell features remain unaffected following PD-1 KO. This behavior holds true for CD28 and ICOS but not 4-1BB co-stimulated CAR-T cells, which are less sensitive to PD-L1 inhibition albeit targeting the antigen with LA. Our findings may inform CAR-T therapies involving disruption of PD-1/PD-L1 pathway tailored in particular for effective treatment of solid tumors.

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.