Background Glioblastoma is a highly aggressive cancer type and despite aggressive therapy, patients’ survival remains poor. Immunotherapy of brain cancer is particularly difficult because of its location behind the blood-brain-barrier and the immunosuppressive tumour microenvironment. In order to (re-)activate the immune system, and reverse the local immunosuppression, we employ the pro-inflammatory cytokine interleukin 12 (IL-12). This highly potent immune-stimulatory agent is known for its anti-cancer effect. Unfortunately, IL-12 was found to induce severe toxicity when applied intravenously, impeding its way into clinics. Thus, currently the only valid therapeutic option is local application into the tumour site.
Materials and Methods Engineered proteins were expressed in HEK293T cells and purified by affinity chromatography. In vivo experiments were performed in glioma-bearing mice using intracranial injection of bioluminescent GL-261 cell line. Treatments were performed on day 21 and 28 post tumour injection through intracranial injection using a step-catheter modelling convection enhanced delivery in mice. Blood or tissue was analysed using immunohistochemistry, flow cytometry and ELISA.
Results Based on an IL-12-IgG fusion protein, we engineered a molecule for exclusively local therapy of brain cancer. We showed anti-cancer efficacy and increased tissue retention of the fusion molecule in glioma in mice. However, molecular analysis of treated tissue confirmed an upregulation of the immunosuppressive molecule PD-L1 in the tumour microenvironment. This means that, despite its efficacy, IL-12 induces an adaptive resistance mechanism, counteracting the therapeutic effect. We thus hypothesised that local IL-12 therapy combined with local blockade of the PD-1/PD-L1-axis would further improve therapeutic efficacy, while exclusively local administration would avoid increased side effects, which usually accompany combination immunotherapy. We showed significantly enhanced long-term survival of glioma-bearing mice treated with IL-12 therapy in combination with PD-L1 blockade compared to single or control treatments. In a next step, we engineered a novel, bifunctional molecule. Optimized for local application and minimized leakage into the systemic circulation, it combines immune-stimulation and checkpoint blockade in one entity. We showed anti-cancer efficacy and increased tissue retention in glioma in mice.
Conclusions The potent anti-cancer effect of the cytokine IL-12 can be used in therapy when applied locally into the brain tumour. Besides fusion to IgG, we introduced several specific modifications on the molecule, which are crucial to prevent systemic exposure and associated toxic side effects. To overcome the dampening of the immune reaction through induced PD-L1 expression, we introduced a combination therapy of IL-12 with a PD-L1-blocking antibody in a single molecule. We showed this combination superior to single treatments in the context of exclusively local brain tumour therapy.
Disclosure Information L. Schellhammer: None. M. Beffinger: None. S. Pantelyushin: None. T. Buch: None. J. vom Berg: None.