Oncolytic HSV Immunovirotherapy for brain tumors

Glioblastoma (GBM) is a fatal cancer of the brain showing little change in survival over decades of therapeutic trials. Development of oncolytic viruses (OV) is iterative and extends from our first description of a genetically- engineered OV to our G-series of oncolytic herpes simplex viruses (oHSVs), ie., G47, which has been shown

to be both efficacious for GBM and safe for use in the human brain. Our overarching hypothesis is that increased efficacy will be produced by simultaneously stimulating intratumoral immune responses with an IL- 12-expressing oHSV and diminishing the immune suppression of adenosine (ADO) in the tumor

microenvironment (TME) of GBM. The extracellular ADO (eADO) pathway is important to both tumor cells and immune cells. Indeed, eADO in the TME has been considered an immune checkpoint that can regulate both innate and adaptive immunity. Much of eADO is produced by plasma membrane ectonucleotidases CD39 and

CD73, and metabolized by ADO degrading enzymes (ADE). Elevated ADO levels are strongly immunosuppressive in tumors; inhibiting cytotoxic T-cells (CTL), tumor-associated macrophages (TAM), and NK cells by binding to the ADO receptor A2aR and also augmenting the immunosuppressive activity of Tregs. The TME of tumors can have ADO levels many logs higher than normal tissues and high eADO can induce

aggressive traits in GBM. Moreover, hypoxia is common in GBM and hypoxia induces release of ATP or NAD+, both possible sources of ADO. ADO also stimulates angiogenesis, a common feature of GBM. In light of this, it should be noted that our oHSV G47-IL12, has potent anti-angiogenic activity, is not inhibited by

hypoxia, and is immunotherapeutic. The possibility of improving tumor therapy by blocking ADO has been demonstrated in mice but thus far has had only limited success in the clinic for cancer in general and has only minimally been explored for GBM. Nonetheless, multiple potential targets exist. Among the targets shown to

influence the immunosuppressive activities of ADO are CD39, CD73, nucleoside transporters such as ENT-1, ADO receptors such as A2aR, and the ADEs, adenosine deaminase (ADA) and adenosine kinase (ADK). Therefore, we plan to determine if agents inhibiting CD39, CD73, or A2aR or a combination of these can act

synergistically in vitro to improve GBM immunovirotherapy. For this, we have a panel of 5 non-immunogenic mouse glioblastoma stem-like cell (GSC) lines with variable expression of the ADO pathway. These and human GSCs and immune cells will be used to interrogate ADO signaling, as well as the effects of CD39,

CD73, and A2aR inhibitors in vitro. Next, we plan to make two novel oHSVs expressing ADA or ADK and test their effects both in vitro and in vivo in immunocompetent mice. Finally, we hypothesize that the combination of intratumoral G47mIL12 or G47mIL12ADE plus systemic inhibitors of the ADO pathway can synergistically

improve efficacy in vivo. These studies should identify an oHSV-ADO pathway inhibitor combination that warrants translation to the clinic for GBM patients.