PROJECT SUMMARY – PROJECT 4 The ultimate goal of this proposal is to develop a novel, effective, and safe oncolytic herpes simplex viral (oHSV)- based immunotherapy for the treatment of glioblastoma (GBM), a highly fatal and the most common malignant brain tumor. oHSV treatment of GBM relies on cancer-specific replication of the virus leading to tumor destruction with minimal toxicity to adjacent non-neoplastic tissue. Its safety in patients has been proven, yet efficacy remains to be improved. Project 4 will focus on enhancing anti-tumor immune responses by arming oHSVs with powerful immune-modulation payloads by testing two newly generated novel oHSVs, termed OV-Cmab-CCL5 and OV- αCD47-IgG1, designed to induce immune infiltration and block a checkpoint, respectively, as monotherapies in addition to their combination. The two oHSVs are expected to synergize with each other to maximize immune responses in the GBM microenvironment by targeting both immune stimulation and immune suppression to create an overall pro-inflammatory tumor microenvironment, which has been demonstrated to positively correlate with patient survival in the rQNestin34.5 clinical trial of Project 2. We generated OV-Cmab-CCL5, an oHSV expressing a secretable single-chain variable fragment of the epidermal growth factor receptor (anti-EGFR) IgG1 antibody cetuximab linked to CCL5 by an Fc knob-into-hole system that produces heterodimers and prevents homodimers. To target a checkpoint on innate immune cells, we also engineered oHSV to express a full-length, soluble anti-CD47 antibody with a human IgG1 scaffold (so-called OV-αCD47-G1), for locoregional control of GBM. The antibody αCD47-IgG1 secreted by virus-infected GBM cells blocks the CD47 “don't eat me” signal and exerts additional functions of Fc receptor-mediated antibody-dependent cellular phagocytosis by macrophages and antibody-dependent cellular cytotoxicity by NK cells. Our data show that in GBM mouse models, both oHSVs reduced tumor size and prolonged survival, owing to enhanced anti-tumor immune responses. We hypothesize that OV-Cmab-CCL5 is a safe and effective agent that can improve GBM therapy with multiple mechanisms of action, and the combination therapy of OV-Cmab-CCL5 and OV-αCD47-IgG1 will have better efficacy than the respective monotherapies. We have manufactured Good Manufacturing Practice (GMP)-grade viruses at our in-house GMP facility and now propose to conduct Investigational New Drug (IND) enabling studies so that our innovative oHSVs will be ready for testing in future clinical trials. Project 4 has three Specific Aims: (1) dissect systemic and regional immune responses, identify a marker(s) in the peripheral blood of mouse models or clinical samples from Project 2 that correlates with anti-tumor activity, and improve the efficacy of rQnestin34.5.v2 in GBM mouse models after OV-Cmab-CCL5 treatment; (2) perform IND-enabling in vivo efficacy and toxicity studies using GMP-grade OV-Cmab-CCL5; and (3) de...