Development and characterisation of a novel chimeric antigen receptor against LGR5

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.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Mueller, Nico

Advisor dc:contributor.advisor

• de la Roche, Maike

• CAR T cell
• Immunotherapy
• LGR5

Licence

• https://www.repository.cam.ac.uk/bitstreams/27486f07-59a3-481d-90a0-9b895b2eccd9/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.123492

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/392980