{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/392980"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/392980","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Development and characterisation of a novel chimeric antigen receptor against LGR5","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Mueller, Nico"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["de la Roche, Maike"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-08-02","date_published":"2025-08-02","updated_at":"2026-07-24T01:33:21Z","subjects":["CAR T cell","Immunotherapy","LGR5"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/27486f07-59a3-481d-90a0-9b895b2eccd9/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.123492","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["de la Roche, Maike"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Cancer Research UK"]},{"key":"dc:creator","label":"Author","values":["Mueller, Nico"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2025-08-02"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/392980"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["CAR T cell","Immunotherapy","LGR5"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/27486f07-59a3-481d-90a0-9b895b2eccd9/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-12-01"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.123492"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/63460af8-d9db-4200-ad04-b3c3cb32421e/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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). 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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. 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