Background Glioblastoma (GBM) is largely refractory to immune checkpoint blockers due to the heterogenous nature of this tumor type within individuals as well as a highly immunosuppressive tumor microenvironment (TME). Novel therapeutic avenues are challenged by the necessity of a drug to cross the blood-brain barrier. BRG399 is a brain permeant, non-Pgp substrate binding to tubulin eliciting anti-cancer activity. BRG399 was assessed for efficacy in a syngeneic orthotopic glioma rat model with a treatment design allowing to demonstrate an immune memory response exhibiting antitumor activity.

Methods Cell potency (IC₅₀) of BRG399 in in vitro GBM models were assessed in 3 glioma cell lines. In vivo, Sprague Dawley rats were implanted with C6 rat glioma cells into the right entorhinal cortex/subiculum region and tumor growth was assessed by magnetic resonance imaging (MRI). Rats were dosed for 16 days BID p.o with BRG399 (5, 10, 20 mg/kg). Survivors were given a 5-day drug holiday followed by 3 days BID p.o BRG399 treatment (treatment cycle repeated twice). 55 days after the last treatment, survivors were re-challenged by left entorhinal cortex/subiculum implantation. BRG399 biodistribution in plasma and brain was assessed using LC MS/MS and TIMSTOF FLEX spatial OMICS analysis.

Results BRG399 administration resulted in improvement in survival (7.7 ± 2.6 days to 25.5 ± 15.6 days, log-rank test, p:0.007) compared to controls. MRI assessment showed tumors still being present in survivors (two rats from each BRG399 treatment group). However, after animals were left untreated for 5 days, followed by two more treatments spaced apart by 5 days, a significant reduction in tumor size, tumor fragmentation, or complete disappearance of tumor was observed. After 97 days post tumor implantation, 4/6 animals were still alive with no reappearance of tumor. Moreover, no tumor growth was observed in long-term survivors after re-challenge by implantation of tumor cells into the left-brain hemisphere.

Conclusions These results demonstrate that BRG399 sufficiently distributes in the brain to have a therapeutic effect on orthotopic glioma rat model. The constrain of tumor growth and regression of tumor after the introduction of a drug holiday suggests an involvement of immune components in the TME, possibly due to induction of immunogenic cell death and/or regulation of the immunosuppressive TME.