Retinoblastoma is a pediatric solid tumor of the retina activated upon homozygous inactivation of the tumor suppressor RB1. 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.

Cancer immunotherapy targeting immune checkpoint inhibitors shows efficacy in several human cancers, but “cold tumors” that lack immune cells are typically unresponsive. Among the potential therapeutic approaches that could “heat” or promote lymphocyte infiltration of cold tumors, oncolytic viruses have attracted interest for their lytic and immunogenic mechanisms of action. In this article, we review the use of oncolytic adenoviruses in cancer immunotherapy, with a particular focus on preclinical and clinical data of oncolytic adenovirus-triggered immune responses against tumor antigens. We also discuss parameters to consider in clinical trial design and the combination of oncolytic adenoviruses with conventional treatments or other immunotherapies.

Several studies have evaluated the efficacy of using human oncolytic adenovirus (OAdv)–loaded mesenchymal stem cells (MSC) for cancer treatment. For example, we have described the antitumor efficacy of CELYVIR, autologous bone marrow–mesenchymal stem cells infected with the OAdv ICOVIR-5, for treatment of patients with neuroblastoma. Results from this clinical trial point out the role of the immune system in the clinical outcome. In this context, a better understanding of the immunophenotypic changes of human MSCs upon adenoviral infection and how these changes affect human autologous or allogeneic peripheral blood mononuclear cells (PBMC) could guide strategies to improve the antitumor efficacy of infected MSCs. In this work, we show how infection by an OAdv induces toll-like receptor 9 overexpression and activation of the NFĸB pathway in menstrual blood–derived MSCs, leading to a specific cytokine secretion profile. Moreover, a proinflammatory environment, mainly mediated by monocyte activation that leads to the activation of both T cells and natural killer cells (NK cell), is generated when OAdv-loaded MSCs are cocultured with allogeneic PBMCs. This combination of allogeneic PBMCs and OAdv-loaded MSCs enhances antitumor efficacy both in vitro and in vivo, an effect partially mediated by monocytes and NK cells. Altogether our results demonstrate not only the importance of the immune system for the OAdv-loaded MSCs antitumor efficacy, but in particular the benefits of using allogeneic MSCs for this therapy.

Oncolytic viruses (OVs) preferentially infect and selectively replicate in cancer cells. OVs have been tested in clinical trials as monotherapy or in combination with chemotherapy, radiotherapy, and immunotherapy. However, the dense extracellular matrix hampers the intratumoral spreading and efficacy of OVs. Previously we described VCN-01, an oncolytic adenovirus expressing a soluble version of human sperm hyaluronidase (hyal) PH20, which exhibited enhanced intratumoral distribution and antitumor activity in different models. Here, we present two oncolytic adenoviruses designed to increase the secretion of PH20 compared to VCN-01. ICO15K-40SAPH20, encoding PH20 under an Ad40 splice acceptor, and ICO15K-E1aPH20 expressing PH20 fused to the E1A gene by P2A peptide. We demonstrate that increased hyal activity improves antitumor efficacy in both a sensitive immunodeficient model and an immunocompetent model. Moreover, we show that hyal activity impacts T cell accumulation in tumors, highlighting the value of a hyaluronidase-expressing virus for combinations with other immunotherapies in cancers involving dense stroma.

Regulatory T cells (T _reg cells) hold promise for sustainable therapy of immune disorders. Recent advancements in chimeric antigen receptor development and genome editing aim to enhance the specificity and function of T _reg cells. However, impurities and functional instability pose challenges for the development of safe gene-edited T _reg cell products. Here, we examined different T _reg cell subsets regarding their fate, epigenomic stability, transcriptomes, T cell receptor repertoires, and function ex vivo and after manufacturing. Each T _reg cell subset displayed distinct features, including lineage stability, epigenomics, surface markers, T cell receptor diversity, and transcriptomics. Earlier-differentiated memory T _reg cell populations, including a hitherto unidentified naïve-like memory T _reg cell subset, outperformed late-differentiated effector memory–like T _reg cells in regulatory function, proliferative capacity, and epigenomic stability. High yields of stable, functional T _reg cell products could be achieved by depleting the small effector memory–like T _reg cell subset before manufacturing. Considering T _reg cell subset composition appears critical to maintain lineage stability in the final cell product.

Clinical results with oncolytic adenoviruses (OAds) used as antitumor monotherapies show limited efficacy. To increase OAd potency, transgenes have been inserted into their genome, a strategy known as “arming OAds”. Here, we review different parameters that affect the outcome of armed OAds. Recombinant adenovirus used in gene therapy and vaccination have been the basis for the design of armed OAds. Hence, early region 1 (E1) and early region 3 (E3) have been the most commonly used transgene insertion sites, along with partially or complete E3 deletions. Besides transgene location and orientation, transcriptional control elements, transgene function, either virocentric or immunocentric, and even the codons encoding it, greatly impact on transgene levels and virus fitness.