Cancer cells take up arginine from the extracellular space to drive cell proliferation and viability. Similar mechanisms are applied by immune cells, resulting in the competition between conventional T cells, or indeed chimeric antigen receptor (CAR) T cells and tumour cells, for the limited availability of arginine within the environment. T cells are susceptible to the low-arginine microenvironment because of the low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC). We demonstrate that T cells can be reengineered to express functional ASS or OTC enzymes to  increase CAR T cell proliferation and function.

Metabolic adaptation enables T cells to fuel their extraordinary functions. The co-stimulatory domains in chimeric antigen receptor (CAR)-constructs influence the T cell’s metabolic profile. We find that this results in a differential ability of CAR T cells to function in nutrient-restricted environments in vitro and in patients in vivo, ultimately impacting treatment outcome.

The general inaccessibility of the tumour microenvironment hampers effectiveness of CAR T cells for application in solid tumours. Direct targeting of the tumour endothelium is a highly effective way of inhibiting tumour growth, in part through alleviating immune suppression. Our strategy is to employ CAR T cells specifically targeting antigens ubiquitously overexpressed by tumour endothelial cells in multiple solid tumour types as a way to overcome these hurdles.

The tumour microenvironment (TME) presents physical, chemical, and cellular barriers hindering CAR T cell activity. Addressing collagen-rich matrix, acidic tumour interstitial fluid, and immunosuppressive cells is crucial to enhance CAR T cell efficacy. In this presentation, we will show that targeting specific antigens in the tumour matrix, such as the extra domain A from fibronectin (EDA), and equipping CAR T cells with transporters to counteract the acidic tumour pH or compete for nutrients in the nutrient-deprived TME can overcome TME challenges and enhance the anti-tumour activity of CAR T cells.

Targeting solid cancers with CAR T cells is limited by the lack of tumour-specific antigens and the immunosuppressive, desmoplastic tumour microenvironment. We hypothesized that targeting endosialin (CD248), expressed by tumour-associated pericytes, would circumvent these challenges. Endosialin-directed CAR T cells demonstrated specific activity in vivo, depleting target stromal cells, resulting in reduced tumour growth and substantial impairment of metastatic outgrowth, highlighting endosialin as an exciting antigen for CAR T cell therapy.

Despite the clinical success of therapy with chimeric antigen receptor–engineered T cells (CAR T) in hematology, a significant percentage of patients eventually relapses, and several challenges still hinder the application in solid tumours. Designing chimeric receptor systems using rationale combinations of targets, costimulatory signals, and logic-gating expression circuits can lead to the next-generation of CAR T cell therapy with broader applicability and improved efficacy and safety profile.

Our group developed both ON- and OFF-switch CARs allowing the remote control of engineered T cells upon application of a clinically-approved small molecule. In addition, we optimised a lentiviral vector enabling constitutive expression of a CAR and independent activation-inducible production of immunomodulatory gene-cargo like cytokines or mIR-based shRNAs to knock-down inhibitory genes. Our engineering tools can be used to improve both the safety and function of CAR T cell therapy.

The therapeutic use of chimeric antigen receptor T cells has achieved significant success in the treatment of B cells malignancies. Despite promising results in mouse tumour models, a similar outcome hasn’t yet been observed in solid tumours. Specifically, in glioblastoma (GBM), several clinical trials only showed a modest efficacy, partly due to the high tumour heterogeneity and immunosuppressive microenvironment. In this setting, we aim to develop an "à-la-carte" CAR T cell strategy that targets a panel of GBM antigens. We use transiently expressed RNA CAR T cells, allowing to achieve the dual goal of reducing potential side effects while delivering multiple CAR T cell infusions. Through a phage display screening, we generated various novel binders against GBM-associated cell surface or secreted targets. That allowed us to build a library of CAR constructs that we tested in vitro and in vivo in immunocompetent or compromised glioma mouse models in our laboratory. Our results point to validating the use of RNA CAR Ts as an attractive new manufacturing platform in the GBM setting. My group has also collaborated in various approaches to enhance anti tumor activity of our CAR T cell products with other immune cells, among these approaches the combination with engineered dendritic cell progenitors show impressive tumour response rates in hard to treat glioma in vivo models.

To address the formidable challenges of antigen loss and tumour heterogeneity, we coupled the cytotoxicity of chimeric antigen receptor (CAR) T cells with the antigen-independent specificity of tumour-colonizing bacteria to create a platform of probiotic-guided CAR T cells (ProCARs). By engineering bacteria to release synthetic CAR targets in situ, we show effective and antigen-agnostic use of the ProCAR platform across multiple models of xenograft and syngeneic cancers.

Our immune system interacts with many diseases in a multidimensional manner involving substantial biological, chemical, and physical exchanges. Manipulating the disease-immunity interactions may afford novel immunotherapies to better treat diseases such as cancer. My lab aims to develop novel strategies to engineer the multidimensional immunity-disease interactions (or termed ‘immunoengineering’) to create safe and effective therapies against cancer. We leverage the power of metabolic and cellular bioengineering, synthetic chemistry and material engineering, and mechanical engineering to achieve controllable modulation of immune responses. In this talk, I will share our recent discovery of IL-10 as a metabolic reprogramming agent that reinvigorates the terminally exhausted CD8+ tumor infiltrating lymphocytes. This strategy has been extended to develop metabolically armoured CAR T cells with IL-10 secretion to counter exhaustion-associated dysfunction in the tumour microenvironment for enhanced anticancer immunity. This new CAR T cell therapy, i.e. IL-10-secreting CAR T, has shown promise in several on-going IIT clinical trials (ClinicalTrials.gov ID: NCT05715606, NCT05747157, NCT06120166) in the treatment of refractory/relapsed CD19+ B cell leukemia and lymphoma.