Halliwell, Emma;
(2021)
Optimisation of a chimeric antigen receptor targeting anaplastic lymphoma kinase for immunotherapy for childhood solid cancers.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Cancer immunotherapy with chimeric antigen receptor (CAR) engineered T cells has shown great promise in the treatment of haematological malignancies. Paediatric neuroblastoma is one of the most common solid extracranial tumours found in children and currently accounts for ~15% of deaths from childhood cancers. Despite multimodal chemotherapy and immunotherapeutic strategies, high-risk patients suffer relapse and treatment related morbidities. This project aims to optimise a novel CAR, targeting the neuroblastoma associated antigen, anaplastic lymphoma kinase (ALK) and we hypothesised that ALK will represent a suitable target this therapy. A panel of second generation anti-ALK CAR T cells was investigated containing multiple anti-ALK single chain variable fragments (scFvs) and stalk regions combined with CD28 and CD3ζ signalling endodomains. In vitro investigations demonstrated effective functional activity at high target antigen densities, which significantly diminishes at lower antigen expression levels. ALK8-CD8-28ζ demonstrated antigen dependent and ALK restricted cytotoxic activity, cytokine production and proliferation and was therefore identified as the ‘lead’ CAR. Interestingly, when the same anti-ALK binder was combined with an Fc spacer of differing origin and length, there was an indication of antigen independent proliferation, indicative of tonic signalling. Despite this differing activity, there were minimal differences found between the ALK-CAR T cells’ activation, exhaustion and memory immunophenotypes. ALK8-CD8-28ζ was compared to another panel of ALK-CAR T cells combining a previously published anti-ALK scFv with various stalk and transmembrane regions, however ALK8-CD8-28ζ demonstrated superior activity in cytokine and proliferation assays. Finally, ALK was combined into a dual-targeting CAR T cell system with another neuroblastoma associated antigen, GD2, in an attempt to increase in vitro activity at low target antigen densities. Despite evidence of background single endodomain CAR activity, the engagement of the double targeting CAR T cell demonstrated increased levels of antigen specific proliferation, proinflammatory cytokine release, activation and degranulation.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Optimisation of a chimeric antigen receptor targeting anaplastic lymphoma kinase for immunotherapy for childhood solid cancers |
| Event: | UCL |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10131856 |
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