Background: The prognosis for patients (pts) with recurrent (r) glioblastoma (GBM) is poor, with no highly effective approved therapies. Treatment failure may result from poor penetration of drugs through the blood-brain barrier and the immunosuppressive nature of the tumor microenvironment (TME). PVSRIPO, a recombinant poliovirus (PV):rhinovirus chimera, is a novel, non-neurovirulent, intratumoral immunotherapy. Trial results in rGBM pts show greater long-term survival with PVSRIPO monotherapy (21%, 36-60 months [mos]) vs criteria-matched external controls (4%, 36 mos; 2%, 60 mos; Desjardins 2018 NEJM). PVSRIPO targets CD155 (PV receptor), expressed on solid tumors and on APC. PVSRIPO infection results in inflammatory-mediated destruction of tumor cells but non-lethal lingering infection in TME APC. This leads to type I/III interferon-dominant inflammation and, ultimately, tumor antigen-specific T cell activation and recruitment (Brown 2017 Sci Transl Med), which is potentiated by immunologic recall to intratumoral replicating virus via prior vaccination. Induction of type 1 IFN dominant inflammation and compensatory activation of the PD-1:PD-L1 immune checkpoint (IC) pathway support investigation of PVSRIPO in combination with PD-1/L1 IC inhibitors. Immunologically cold mouse glioma models show PVSRIPO+anti-PD-1 therapy resulted in greater anti-tumor response than either agent. Methods: LUMINOS-101 is a phase 2, multicenter, open-label, single-arm study of intratumoral infusion of PVSRIPO (Day 1: 5x10⁷ TCID₅₀) followed by the anti-PD-1 monoclonal antibody pembrolizumab (200mg IV q3w) in adult pts with rGBM. The trial objective is to evaluate anti-tumor activity and safety and tolerability of the combination. Eligibility criteria include pts ≥18 years who had prior PV and boost IPOL^® immunizations, histologically confirmed supratentorial rGBM, infusible 1 to ≤5.5cm enhancing disease, confirmed disease progression following prior therapies, and KPS ≥70. Key exclusion criteria include multifocal disease; discontinuation of prior anti-PD-1/L1 agent for toxicity; prior intratumoral therapy, immunotherapy, or radiotherapy within 12 weeks; high-dose systemic corticosteroids; chemotherapy, anti-VEGF, or TTF therapy ≤1-6 weeks depending on the therapy; serious cerebral herniation syndrome; extensive leptomeningeal, subependymal, or ≥1cm enhancing disease crossing the midline; and severe active comorbidities. Primary endpoints are objective response rate, duration of response, and safety. Secondary endpoints include overall and progression-free survival and disease control rate and duration. Exploratory endpoints include assessment of tumor and blood for biomarkers of response. The initially planned safety lead-in period is now fully enrolled. Recruitment is ongoing in the US, and results will inform the design of a randomized phase 3 trial. Clinical trial information: NCT04479241