采用装载ICP34.5的G47Δ溶瘤单纯疱疹病毒治疗PAX3-FOXO1腺泡状横纹肌肉瘤，与抗PD1抗体联合给药时可带来生存获益

Background. Patients diagnosed with PAX3-FOXO1 (P3F) positive alveolar rhabdomyosarcomas (aRMS) have much worse prognosis than those with fusion-negative aRMS or embryonal rhabdomyosarcomas. The low immunogenicity of P3F+ aRMS tumors has impaired efforts to treat patients with immune checkpoint blockade, and the lack of an accessible PAX3-FOXO1 expressing murine tumor model has limited efforts to address this unmet therapeutic need. Here we generate a novel oncolytic herpes virus (oHSV) and show it improves mouse survival in a PAX3-FOXO1 expressing M3-9-M murine tumor model, when combined with anti-PD1 treatment. Methods. We designed a P3F-responsive promoter by combining a PRS1 element with a minimal Myogenin promoter. This synthetic promoter was inserted into the oHSV G47∆, to drive the cytotoxic ICP34.5 gene. We tested rG47-PRS1 cytotoxicity and growth in human and murine RMS cell lines, as well as safety in normal human muscle. To determine efficacy of oncolytic herpes viruses in RMS tumor bearing C57/BL6 mice, we implanted M3-9-M:PAX3-FOXO1 tumors into the gastrocnemius muscle, followed by intratumoral injection of 1E8 PFU of virus with or without IP injection of anti-PD1 antibody. Tumor infiltrating lymphocytes were observed via flow cytometry and IHC upon harvest. Additionally, we explored the gene expression response of human and murine RMS cell lines to infection via RNA sequencing, RTqPCR, and western blot. Results. In vitro assays showed the PRS1 promoter resulted in expression of GFP or luciferase transgene in the presence of PAX3-FOXO1, but not its absence. Infection of RMS cells with rG47-PRS1 resulted in ICP34.5 protein expression. Recombinant rG47-PRS1 kills RMS lines faster than its parental virus while displaying the same safety profile as parental G47∆ in normal human muscle cells in vitro and is safe for intra-muscular injections in mice up to 1E9 PFU. Mice harboring M-3-9M RMS tumors expressing PAX3-FOXO1 had no response to anti-PD1 treatment alone but responded to combination therapy with either G47∆ or rG47-PRS1 virus (mean tumor volume p<0.001; survival G47∆+PD1 vs PD1 alone p<0.0012). Tumors from combination treated mice had higher CD45+ lymphocyte infiltration of their tumors and significantly reduced PD1 positive CD4 and CD8 T cells as compared to mock or single treatment. RNA sequencing data revealed increased NF-kB pathway activation in aRMS tumor lines after infection, which we observed via p65, p100, cFOS, JNK1, and PI3K western blot and RTqPCR for AP-1 transcription factor elements. Conclusions. We observe oHSV sensitizes PAX3-FOXO1 aRMS to anti-PD1 therapy in mice. Infection of aRMS leads to upregulation of the NF-kB pathway, which may have a role in T cell engagement or tumor biology for this pediatric malignancy.