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Precision Cytokine Therapy: Engineering and Delivery of CD8- and CAR-Tropic Interleukin-2 Variants https://doi.org/10.17863/CAM.131432 Ghodsinia, Arman 000900040642926X Liston, Adrian Dooley, James Glioblastoma is the most aggressive form of brain cancer, with median survival rates of less than 15 months. As the current standard of care is not curative, there is an urgent need to develop novel therapeutic approaches. Immunotherapy represents one potential strategy, although the unique immune-privileged status of the brain poses a major challenge to its success. Immune privilege refers to the limited ability of the central nervous system (CNS) to generate strong immune responses against foreign antigens. This results from the specialised CNS microenvironment and, in particular, the markedly low numbers of cytotoxic CD8+ T cells present in the brain. This scarcity of CD8+ T cells contributes to uncontrolled tumour progression once glioblastoma arises. To address this problem, my PhD project focused on increasing CD8+ T cell numbers in the brain by expressing modified versions of interleukin 2 (IL2), a key T cell growth factor, using an adeno-associated viral (AAV) gene delivery system. Previous work by Yshii et al. (2022) demonstrated that AAV-mediated expression of wild type IL2 in the brain expanded immunosuppressive CD4+ regulatory T cells (Tregs) and protected mice from pathological neuroinflammation. To avoid preferential Treg expansion, I developed IL2 variants with two features: (i) reduced binding to CD25, the IL2 receptor subunit highly expressed on Tregs but very lowly on naïve CD8+ T cells; and (ii) enhanced specificity for CD8+ T cells via fusion to a CD8-targeting single-chain variable fragment (scFv). This construct, termed scFv-IL2, displayed over one million-fold greater selectivity for CD8+ T cells compared to wild type IL2. AAV mediated expression of scFv-IL2 in the brain increased CD8+ T cell numbers by more than 2,000-fold, from ~500 cells at baseline to ~1,000,000 cells, without causing toxicity or weight loss. Indeed, testing of scFv-IL2 in syngeneic orthotopic glioblastoma models transiently controlled tumour growth and extended overall survival. Furthermore, experiments of purified proteins against an MC38 subcutaneous tumour model in vivo demonstrated the superior anti-tumour properties of scFv-IL2 relative to wild type IL2. In addition, IL2 variants designed to selectively support chimeric antigen receptor (CAR) T cells were developed. The most promising constructs employed the SpyTag/SpyCatcher protein coupling system. In vitro experiments showed that SpyTagged-IL2 significantly enhanced phospho-STAT5 levels in SpyCatcher-expressing T cells, providing proof-of-concept that IL2 can be precisely targeted to engineered T cell populations. Future studies will test these IL2 variants in humanized mouse models of glioblastoma. Collectively, this PhD presents novel tools to expand CD8+ T cells and CAR T cells, with potential implications for advancing glioblastoma immunotherapy. University of Cambridge 2025-12-22 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/405096 embargo 2027-06-23 https://www.repository.cam.ac.uk/bitstreams/99d5afbd-ed41-4b01-9929-811f49582c74/download f3fa8b082bc5af51467d61eb94a4db54 https://www.repository.cam.ac.uk/bitstreams/8abc737c-26a4-421d-af30-d4357d2d640e/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved Anti-GD2 CAR T Glioblastoma Immunotherapy Interleukin-2 orthogonal IL2 SpyCatcher SpyTag