Background: Lisavanbulin (BAL101553, prodrug of BAL27862) is a novel tumor checkpoint controller that promotes tumor cell death by modulating the spindle assembly checkpoint. BAL27862 is a lipophilic, small molecule (MW 387) shown in rodents to penetrate the brain (1:1 plasma ratio) with promising antitumor activity in orthotopic models of glioblastoma (GB) as monotherapy or in combination with radiotherapy (RT) ± chemotherapy. In a completed phase 1 study (Lopez et al. ESMO 2020, NCT02490800) with daily oral lisavanbulin in patients with recurrent GB or high-grade glioma, the RP2D was determined at 25 mg/day. In this phase 1 study, two patients (out of 20 patients) with GB show a long-lasting (> 2 years) clinical benefit with improvement in clinical symptoms and in target and/or non-target GB lesions as per RANO criteria. Both patients show strong end-binding protein 1 (EB1) expression in their GB tissues as assessed by immunohistochemistry staining. EB1, a protein located on the plus-ends of microtubules, is involved in microtubule (MT) function and has been associated with stemness of glioma cells and a more aggressive disease. Data from GB mouse models suggest that EB1 is a predictive marker for response to lisavanbulin. The prevalence of EB1-positivity in GB is estimated at ̃5%. This ongoing phase 2 study is an extension of the completed Phase 1 study and is conducted to confirm prospectively whether EB1 is a response-predictive biomarker for lisavanbulin in GB. Methods: This is an ongoing multicenter, open-label, phase 2 study using a Simon Two-Stage design to assess the efficacy of lisavanbulin in patients with recurrent GB. The study is being performed in the UK, Switzerland and Germany. Patients with histologically-confirmed GB and recurrent disease after prior RT with alkylating chemotherapy (de-novo/primary GB) or after prior chemotherapy or RT (secondary GB), are eligible for enrollment if their GB archival tumor tissue is EB1-positive. EB1-positivity is defined as moderate to strong EB1-staining in at least 70% of GB tumor cells using a CE-marked immunohistochemistry Clinical Trial Assay (Targos Molecular Pathology GmbH). The primary study objective is the overall response rate by RANO, with MRI scans being performed every 8 weeks. Secondary endpoints include progression-free survival and overall survival. Adverse events are assessed using CTCAEv5. To develop a potential RNA-based response signature, molecular profiling of tumor tissue is performed using whole transcriptome sequencing (RNAseq) in each patient enrolled in the study to define the genomic expression profiles in patients with EB1-positive GB. Nine evaluable patients are to be enrolled in Stage 1, and an additional 10 patients will be enrolled in stage 2 if at least 2 objective responses per RANO criteria are observed in stage 1. Clinical trial information: 02490800.