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Title: The molecular and cellular function of gene-modified T-cells
Author: Bridgeman, John Stephen
ISNI:       0000 0001 3479 5663
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2008
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Chimeric Immune Receptors (CIR) consisting of a carcinoembryonic antigen (CEA)specific single chain antibody fragment (scFv), fused to the CD3s signalling chain from the T-cell receptor (TCR), induce T-cell activation on CEA ligation. The study sought to investigate some of the structural aspects of CIRs with the objective of potentially optimising CIR function. Analysis of several model systems identified Jurkat T-cells as a useful model for assessing CIR-mediated responses to antigen. CIR-expressing Jurkat T-cells demonstrated a dose-dependent increase in CD69 expression in response to CEA. Jurkat T-cells expressing a CIR with the wild-type CD3s transmembrane domain exhibited a significantly higher surface expression of the TCR associated CD3e than cells expressing receptors with any other transmembrane domain. This upregulation of TCR-associated but not TCR-non associated receptors was abrogated by a transmembrane C2G mutation, which effectively prevented dimerisation, or by replacing the transmembrane domain with a heterologous non-dimerising domain. The concentration of CEA required for 50% maximal CD69 expression (ECso) was also significantly increased by both of these modifications. Moving the disulphide bridge to the extracellular domain, but not any other position in the transmembrane domain, permitted redimerisation and partially restored optimal receptor function. This difference in function may be attributed to TCR interactions as wild-type dimeric but not monomeric receptors, as well as inducing an upregulation in TCR complexes in Jurkat T-cells, could also reconstitute TCR expression in CD3s deficient MA5.8 cells. Potential TCR-CIR interactions were further investigated by mutational analysis to the charged transmembrane D6 residue responsible for wild-type CD3s-TCR interactions. Conservative D6N/E/Q mutations permitted dimerisation but significantly impacted on receptor efficiency; however, non-conservative D6K mutations affected receptor dimerisation and had a more severe impact on receptor function. This knowledge obtained from this study was applied to clone a novel CD28-fusion receptor containing the CD3s transmembrane domain with analysis in Jurkat T-cells demonstrating this CIR had a significantly lower ECso than existing CD28-fusion receptors containing the CD28 transmembrane domain. The second aim of this study was to investigate the role of CD2 as a novel costimulatory module. scFv.CD2.CD3s and scFv.CD28.CD2.CD3s activated Jurkat T-cells more efficiently than scFv.CD28.CD3s. However, in primary human T-cells only scFv.CD2.CD3s expressed at levels to observe functional responses. In these cells CD2 costimulation induced higher IFNy secretion than CD28 costimulation. In summary the report highlights the importance of the CD3s transmembrane domain in optimal CIR function and demonstrates the potential for CD2-based CIRs for future clinical trials.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available