UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Tunable CAR T Cell Responses Achieved by the Disruption of Protein-Protein Interactions via the Administration of a Small Molecule

Costa Guerra, José António; (2021) Tunable CAR T Cell Responses Achieved by the Disruption of Protein-Protein Interactions via the Administration of a Small Molecule. Doctoral thesis (Ph.D), UCL (University College London).

[thumbnail of Costa Guerra_10120857_thesis_redacted.pdf] Text
Costa Guerra_10120857_thesis_redacted.pdf
Access restricted to UCL open access staff until 1 March 2022.

Download (10MB)

Abstract

Introduction: The adoptive transfer of T-cells engineered to express chimeric antigen receptors (CARs) has significantly widened the applicability of passive immunotherapy in cancer. Up until now, several studies of CAR therapy have emerged as a promising treatment for cancer, although there are serious concerns regarding potential risk of acute and chronic toxicity. Given the ability of CAR T-cells to engraft and proliferate in the patient, toxicity from excessive activity can be progressive and fulminant. This highlights the need for technologies to remotely control and tune the potency of the CAR T-cells after administration. Suicide gene approaches allow a level of control of CAR T-cell therapy. However, as suicide switches result in permanent loss of all engineered T-cells, a new strategy which can temporarily reduce or stop CAR T-cell activity without triggering CAR T-cell apoptosis is desirable. One way to achieve this is to use small molecules that present desired pharmacologic properties, such as tetracycline and analogues doxycycline and minocycline. Results: Therefore, we developed a Tet-Off signalling switch that enables tetracycline-dependent, titratable, and reversible control over CAR T-cells. The CAR was split into two: an antigen recognition component fused with TetR (Tet repressor) and a signalling component fused with TiP (TetR interacting peptide). Both TiP and tetracycline are known to bind to TetR with a high affinity. We predicted that although TiP bound to the same part of TetR as tetracycline, Tetracycline would bind with a much higher affinity than TiP and hence rapidly displace TiP. First with eGFP, we showed that in the absence of tetracycline such a system results in membrane localization of a TiP fusion protein, with the eGFP being displaced to the cytoplasm in the presence of tetracycline. Next as a CAR, we showed that in the absence of tetracycline, the two CAR components associate and the CAR signals in the presence of cognate antigens. On the addition of tetracycline, TiP was displaced and the receptor stopped signalling. Depending on the concentration of tetracycline present, tetCAR T-cells showed titratable activity, from as strong as that of conventional CAR T-cells to undetectable. Similar results were obtained when using the analogues doxycycline and minocycline. We tested the effectiveness of this tetCAR in murine and human T-cells. These results included IL-2 and IFN-γ production, the tetCAR T-cell ability to control target cell killing and also the killing of multiple types of target cells by the tetCAR T-cells. Regarding reversibility, we showed that the inhibition process is reversible. Once we wash off the tetracycline from the cultures, tetCAR T-cells regain activity. Conclusion: We successfully generated and controlled the activity of tetCAR T-cells by tetracycline administration.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Tunable CAR T Cell Responses Achieved by the Disruption of Protein-Protein Interactions via the Administration of a Small Molecule
Event: UCL (University College London)
Language: English
Additional information: Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/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.ucl.ac.uk/id/eprint/10120857
Downloads since deposit
3Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item