Jha, Ram;
(2022)
Development of a protein-protein displacement platform using an anti-minocycline single domain antibody and a dedicated displaceable peptide for the generation of an OFF-switch CAR T cell.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in the clinic; however, the treatment carries potential risks of significant toxicities. A versatile small-molecule control system is desirable to increase control and mitigate toxicity in clinical settings. I set about creating a two-component small-molecule control system based on disruption of protein-protein interactions. Given its wide availability, excellent bio-distribution, and lack of toxicity, this system was developed around minocycline. The protein-protein displacement system was composed of an anti-minocycline single-domain antibody (sdAb) and a minocycline displaceable sdAb-specific cyclic peptide. Minocycline-specific sdAbs were generated by immunising an alpaca with KLH-conjugated minocycline. The highest affinity sdAb-minocycline binding was measured at 31 nM. Displaceable cyclic peptides were isolated by panning a cysteine-constrained heptapeptide phage library against the sdAb and eluted using minocycline. Peptide sequence, ACPGWARAFC, presented the highest affinity sdAb binding at 111 nM. Utilising the displacement system, an OFF-switch split CAR was developed, made up of a physically separated antigen-binding chain bearing a tumour-specific sdAb and the anti-minocycline sdAb which interacts with a cyclic peptide bearing signaling chain forming a functional CAR heterodimer (MinoCAR). MinoCAR showed cytotoxicity and cytokine release against tumour cells comparable to a conventional CAR. Transient suppression of MinoCAR was achieved via the administration of minocycline in a dose-dependent and reversible manner. To apply the system to control secretion of cellular payloads, the KDEL retention motif was fused to the sdAb thus anchoring it to the ER/Golgi. The pro-inflammatory cytokine IL-12 was fused to ACPGWARAFC thus causing its cellular retention. Upon the addition of minocycline, the sdAb-peptide complex was displaced, enabling the secretion of IL-12. This work allows remote-controlled regulation of a novel minimally immunogenic OFF-switch CAR using an FDA approved drug which may permit clinicians to precisely control the timing, intensity and safety of the tumour targeted response.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Development of a protein-protein displacement platform using an anti-minocycline single domain antibody and a dedicated displaceable peptide for the generation of an OFF-switch CAR T cell |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. 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 > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10156551 |
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