Goodwin, Sydney2026-07-172026-07-172026https://hdl.handle.net/1828/24105Adoptive cell therapies (ACTs) have transformed cancer treatment, yet their efficacy is often limited by poor T cell persistence and sustained functionality. Effective anti-tumour immunity requires durable co-stimulatory signalling, particularly through pathways such as CD28, while inhibitory checkpoint pathways including PD-1 and CTLA-4 suppress T cell activity within the tumour microenvironment. Although immune checkpoint inhibitors (ICIs) can relieve inhibitory signalling, they remain dependent on pre-existing anti-tumour immunity. One potential approach involves the use of agonistic antibodies to stimulate co-stimulatory receptors on T cells; however, systemic administration of co-stimulatory agonists is associated with significant toxicity due to indiscriminate activation of endogenous immune populations. Another attractive approach is the use of switch receptors that combine the extracellular domain of a checkpoint protein with the intracellular domain of a co-stimulatory protein, such that endogenous ligand binding delivers a targeted stimulatory signal to adoptively transferred T cells. However, switch receptors are limited by endogenous ligand expression that varies between patients and cancer types, reducing their versatility and predicted efficacy. To address these limitations, we hypothesized that clinically approved ICIs could be repurposed as selective activators of chimeric co-stimulatory receptors (CCRs), enabling antibody-mediated enhancement of adoptively transferred T cells while avoiding broad systemic immune activation. To test this hypothesis, we engineered multiple CCR constructs fusing the extracellular domains of immune checkpoint proteins (PD-1, PD-L1, and CTLA-4) with the intracellular domain of CD28. Human T cells expressing these CCRs were evaluated for functional activation following stimulation by clinically relevant ICIs. In vitro, CCR-expressing T cells demonstrated robust activation in response to checkpoint antibodies, including enhanced IL-2 secretion, proliferation, and cytotoxicity that exceeded responses induced by conventional CD28 agonist antibodies. These findings established that ICIs can be functionally repurposed to deliver potent co-stimulatory signals through our engineered receptors. However, despite strong in vitro activity, in vivo adoptive transfer studies in NSG mouse models revealed a profound and unexpected loss of CCR-expressing T cells following systemic ICI administration. This depletion occurred across multiple CCR constructs and antibody subclasses, suggesting that antibody engagement may trigger deleterious immune-mediated mechanisms that override the intended co-stimulatory effects. Mechanistic studies investigating antibody Fc interactions demonstrated that Fc engineering strategies altered these effects in vitro but did not restore CCR+ T cell persistence in vivo. These findings implicate complex contributions from Fcγ receptor interactions, complement activation, and/or other immune processes. Collectively, this work establishes checkpoint-converting CCRs as a novel and conceptually compelling platform to improve cellular immunotherapies, while also identifying a major translational barrier to their implementation. More broadly, this thesis highlights the importance of Fc biology, host immune interactions, and preclinical model selection in the development of antibody and cell-based combination strategies. Although substantial mechanistic challenges remain, resolving the pathways responsible for CCR+ T cell depletion may enable the future development of next-generation ACT platforms that leverage existing clinically approved immunotherapies to enhance anti-tumour responses.enAvailable to the World Wide Webcancerimmunotherapyadoptive cell therapychimeric antigen receptor therapyswitch receptorsimmune checkpoint inhibitorsanimal modelsovarian cancerThesis