Abstract 601: Oncolytic and immune effects of RAPTOR: a novel oncolytic Herpes Simplex Virus 1 expressing PTEN-L for brain tumor therapy
Authors: Russell Luke , Sprague Alexander , Jaime-Ramirez Alena Cristina , Bolyard Chelsea , Banasavadi-Siddegowda Yeshavanth , Yoo JiYoung , Wang Pin Yi , Cripe Timothy , Kaur Balveen 
Published: July 2017

Abstract
Glioblastoma (GBM) is a World Health Organization grade IV astrocytoma with poor patient survival, characterized by extreme heterogeneity and invasiveness, and by resistance to radiation and chemotherapeutic drugs. Brain metastases are 2-3x more frequent than GBM, with breast cancer among the top 3 brain metastasizing tumor types. The tumor suppressor phosphatase and tensin homolog on chromosome ten (PTEN) is mutated or deleted in ~50% of all high grade gliomas and is lost in 33-49% of all breast cancers. We propose to target the dysregulated PTEN pathway in brain tumors to enhance patient survival.
Oncolytic viruses are engineered to destroy tumor cells while sparing non-neoplastic cells, and can be designed to incorporate payloads that enhance their oncolytic effects. PTEN-L is a recently discovered, longer form of PTEN which is secreted and membrane permeable, and retains its phosphatase activity after secretion and re-entry into cells. We have generated a novel oncolytic herpesvirus (RAPTOR) that secretes PTEN-L from infected cells for the treatment of GBM and breast cancer brain metastases (BCBM). In vitro assays to evaluate virally mediated tumor cell killing, virus replication, transgene expression, and flow cytometry to evaluate intracellular pAkt and cell surface programmed death ligand 1 (PD-L1) were performed. In vivo studies using mice bearing orthotopic human GBM (nude mice) or murine BCBM (nude and FVB/N) were used to evaluate brain immune infiltrates and survival.
Western blot analyses of infected cell lysates and conditioned media show that RAPTOR produces functional secreted PTEN-L, as confirmed by intracellular pAkt FACS. Tumor cell killing and replication were equivalent between RAPTOR and control HSVQ. In vivo, treatment of mice bearing established orthotopic BCBM revealed improved anti-tumor efficacy of RAPTOR. Significantly, 9/10 RAPTOR treated mice were long term survivors in immunocompetent FVB/N mice vs. 4/10 HSVQ treated mice (p=0.044). Treatment of nude mice bearing the same tumors did not produce long term survivors, implicating the development of antitumor immunity in RAPTOR treated animals. Flow cytometry revealed significant down regulation of the T-cell repressor PD-L1 on RAPTOR vs. HSVQ treated cells. Consistent with this, FACS of virus treated tumor bearing brain hemispheres revealed RAPTOR induced a significant influx in antigen presenting, NK, and CD8 T cells 7 days post treatment.
Our findings demonstrate that RAPTOR enhances survival of brain tumor bearing mice using a two-pronged approach: lytic tumor cell death and immune cell education and activation against the brain tumor. RAPTOR inhibits virally induced PD-L1 expression on tumor cells, and could pave the way for a paradigm shift in immune therapy where immune checkpoint inhibition occurs locally within the tumor rather than globally, overriding toxicity of neutralizing antibodies.
Citation Format: Luke Russell, Alexander Sprague, Alena Cristina Jaime-Ramirez, Chelsea Bolyard, Yeshavanth Banasavadi-Siddegowda, JiYoung Yoo, Pin Yi Wang, Timothy Cripe, Balveen Kaur. Oncolytic and immune effects of RAPTOR: a novel oncolytic Herpes Simplex Virus 1 expressing PTEN-L for brain tumor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 601. doi:10.1158/1538-7445.AM2017-601