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Development and characterisation of a novel chimeric antigen receptor against LGR5 https://doi.org/10.17863/CAM.123492 Mueller, Nico 0009000883586428 de la Roche, Maike Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a target gene of the Wnt signalling pathway, is lowly expressed in some stem cell niches and overexpressed in multiple cancers, including pre-B acute lymphoblastic leukaemia (pre-B ALL), hepatocellular carcinoma (HCC) and colorectal cancer (CRC). LGR5 has thus emerged as a promising therapeutic target and has spurred the development of a range of LGR5-targeting immunotherapeutics. In this thesis, I report the generation, characterisation and preclinical validation of novel 2nd generation LGR5scFv-Chimeric Antigen Receptor (CAR) NK and CD8 T cells using flow cytometry, functional in vitro assays and in vivo tumour xenograft models. While LGR5scFv-CAR NK92 cells exhibited high specificity, they had poor efficacy in vitro and failed to survive in vivo. In contrast, LGR5scFv-CAR T cells showed specific and potent killing of LGR5high tumour cells in vitro, and significant anti-tumour activity across pre-B ALL, HCC and CRC tumour xenograft models in vivo. Despite significant reduction in tumour growth, complete eradication was not achieved, likely due to limited CAR T cell persistence. Generally, poor persistence remains a major challenge for CAR T cell therapy and often contributes to tumour relapse. While other morphogen pathways such as Notch and Wnt have been shown to have important roles in T cell differentiation and memory development – and our group has uncovered roles for Hh signalling in CD8 T cell cytotoxicity, migration and CD4 T cell polarisation – a role for Hh signalling in T cell memory formation had not been explored. I used knockout mouse models of the Hh ligand Ihh and Hh transcription factor Gli1 in a series of in vitro cytokine polarisation assays, in vivo homeostatic proliferation models and antigen-driven memory formation and rechallenge influenza A virus infection models. Across all systems tested, loss of Ihh or Gli1 in T cells had no effect on memory formation. Taken together, I have developed a novel CAR T cell ready for clinical development and found that Hh components Ihh and Gli1 are dispensable for T cell memory formation. University of Cambridge 2025-08-02 Thesis Doctoral Doctor of Philosophy (PhD) eng Cancer Research UK https://www.repository.cam.ac.uk/handle/1810/392980 embargo 2026-12-01 https://www.repository.cam.ac.uk/bitstreams/63460af8-d9db-4200-ad04-b3c3cb32421e/download 5e7c5910c2e29f2a784519bc7aa725d3 https://www.repository.cam.ac.uk/bitstreams/27486f07-59a3-481d-90a0-9b895b2eccd9/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved CAR T cell Immunotherapy LGR5