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Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain cancer, for which effective therapies are urgently needed. Chimeric antigen receptor (CAR)-based immunotherapy represents a promising therapeutic approach, but it is often impeded by highly immunosuppressive tumor microenvironments (TME). Here, in an immunocompetent, orthotopic GBM mouse model, we show that CAR-T cells targeting tumor-specific epidermal growth factor receptor variant III (EGFRvIII) alone fail to control fully established tumors but, when combined with a single, locally delivered dose of IL-12, achieve durable anti-tumor responses. IL-12 not only boosts cytotoxicity of CAR-T cells, but also reshapes the TME, driving increased infiltration of proinflammatory CD4+ T cells, decreased numbers of regulatory T cells (Treg), and activation of the myeloid compartment. Importantly, the immunotherapy-enabling benefits of IL-12 are achieved with minimal systemic effects. Our findings thus show that local delivery of IL-12 may be an effective adjuvant for CAR-T cell therapy for GBM., Glioblastoma multiform (GBM) is a common and aggressive type of primary brain cancer that currently has no effective therapy. Here, the authors show, using a mouse GBM model and EGFRvIII-targeting chimeric antigen receptor (CAR)-T cells, that Intratumoral injection of interleukin-12 helps condition the microenvironment and promote anti-tumor immunity.

Significance: Green-fluorescent protein (GFP)-like fluorescent proteins are used extensively as genetic reporters in fluorescence imaging due to their distinctive ability to form chromophores independent of external enzymes or cofactors. However, their use for photoacoustic (PA) imaging has not been demonstrated in mammalian tissues because they possess low PA signal generation efficiency in their native state. By engineering them to become nonfluorescent (NF), their PA generation efficiency was increased. This enabled the generation of in vivo contrast in mice, making it possible for GFP-like proteins to be used as PA genetic reporters in mammalian tissues. Aim: The aim was to develop a darkened GFP-like protein reporter by modifying E2 crimson fluorescent protein (FP) in order to generate NF mutant proteins with high PA signal generation efficiency for in vivo imaging. Approach: The absorbance, fluorescence, and PA amplitude spectra of purified protein solutions of the FP and engineered NF mutants were measured in order to identify the mutant with the highest PA signal generation efficiency. This mutant, referred to as NFA, and the native FP were then stably expressed in LS174T human colorectal tumor cells using a retroviral vector and tested for photostability under continuous pulsed illumination. To demonstrate the improvement in PA signal generation in vivo, cells expressing the FP and NFA mutant were injected subcutaneously in mice and imaged using a Fabry–Perot based PA scanner. Results: The NF mutants of E2 crimson exhibited fluorescence that was 2 orders of magnitude lower than the FP and a higher PA signal generation efficiency; the NFA-generated PA signal was approximately three times higher than the FP. Tumor cells expressing the NFA mutant provided sufficient image contrast to be visualized in vivo against a background of strong vascular contrast, whereas the FP-expressing cells did not generate visible contrast. Conclusion: A GFP-like protein has been demonstrated as a genetic reporter for PA imaging in mammalian tissue for the first time. This was achieved by a mutation, which darkened the FP and increased the PA signal generation efficiency. The approach taken suggests that GFP-like proteins could be a promising addition to the current cohort of genetic reporters available for in vivo PA imaging.

Treatment with T-cells redirected to tumour specificity with a chimeric antigen receptor (CAR) may be well suited to treat intracranial tumours due to the ability of T-cells to access the central nervous system and migrate to infiltrative sites of disease. In adult glioblastoma, a case report of local and distant eradication of intracranial and spinal tumour deposits following intraventricular infusion of IL13Ra2-CAR T-cells indicates the potential of this approach. However, in contrast to the sustained complete remissions observed in haematological malignancies, in the majority of patients with glioblastoma CAR T-cell therapy has not resulted in clinical benefit. Tumour heterogeneity and the highly immune inhibitory tumour microenvironment (TME) are likely key barriers to achieving durable anti-tumour immunity. Here use intra-tumoural administration of IL-12 to enable CAR T-cell immunity. We employed CAR-T cells targeting the tumour-specific epidermal growth factor variant III (EGFRvIII). In an immunocompetent orthotopic mouse model of high-grade glioma, we show that CAR-T cells alone failed to control fully established tumour, but when combined with a single, locally delivered dose of IL-12, durable antitumor responses were achieved. IL-12 not only boosted cytotoxicity of CAR T-cells, but also reshaped the TME driving increased infiltration of proinflammatory CD4+ T-cells, decreased numbers of regulatory T-cells (Tregs) and activation of the myeloid compartment. Critically, immunotherapy enabling benefits of IL-12 were achieved with minimal systemic effects. Our findings show that local delivery of IL-12 is an effective adjuvant for CAR-T cell therapy for high-grade glioma. Assessment of application in high-risk childhood brain tumours is ongoing.