Extended Data Fig. 1: (related to Sub-heading, Safety of CAN-3110 in patients with rHGG/rGBM) Clinical trial design and treatment strategy.

(a) Dose-escalation schema- Subjects with a previous diagnosis of rHGG (Glioblastoma, Grade IV or III astrocytoma or anaplastic astrocytoma, grade III anaplastic oligodendroglioma, including molecular grading with or without a mutation in IDH and with or without hypermethylation of the MGMT promoter) were eligible for the trial. At the time of stereotactic biopsy, the neuropathologist had to confirm that there was histologic evidence consistent with glioma to exclude inclusion of subjects with radiation necrosis and/or infection. The first 9 cohorts of subjects underwent one stereotactic inoculation of CAN-3110 at a tumour site selected to be different from the antecedent biopsy site (to avoid blood contamination of the injectate) in a 3 + 3 dose-escalation design, starting from 10⁶ pfus up to 10¹⁰ pfus in half-log increments. The biopsy and injections for each subject were carried out in an intraoperative MRI to visualize injections in gadolinium-enhancing tumour. The volume of injectate was 1 ml delivered over 5 min using the SmartFlow cannula (ClearPoint Neuro, Inc.) that minimizes reflux. When all 30 subjects in the first 9 cohorts were treated (September 2017- February 2020) without a dose limiting toxicity, the protocol was amended to include a tenth cohort of 12 subjects, where up to 5 regions of tumour were injected with a dose of 10⁹ pfus divided into 1 to 5 mls based on tumour diameter (e.g., for each mm of tumour diameter, 1 ml of CAN-3110 was injected). No DLTs were encountered. Subjects in cohort 10 were accrued from June, 2020 until January, 2021. (b-c) Representative intraoperative MRIs during (b) and after (c) CAN-3110 injection. b: Subject 021 was positioned prone in the intraoperative MRI. The SmartFlow cannula is shown in the occipital area penetrating the skull through a drilled burr hole (white arrow). The T2 dark area shows the needle trajectory through the occipital and temporal lobe to reach the area of rGBM where the tip of the needle (yellow arrowhead) is placed for injection. The gadolinium-enhanced tumour was manually overlaid with purple colour. c: Representative intraoperative MRI (subject 002) showing the view from the intraoperative console after injection of CAN-3110 (10⁶ pfus/1 ml). The T1 dark injectate is indicated by the blue and yellow cross-hatch, with the red dot showing where the tip of injection needle was after injection and needle removal, showing persistence of the injectate at site of injection with minimal reflux. The rGBM consisted of a bifrontal mass and the needle was inserted from the frontal vertex to reach an area of gadolinium-enhancement located in the inferior frontal lobe. The 3 images shown in the console are from the same brain section in coronal, sagittal and axial planes.