Parekh, Farhaan;
(2020)
Novel CD123 Binders for CAR-T Cell Therapy.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
CD123- the a-chain of the IL-3 cytokine receptor (IL3RA)- is a promising new immunotherapeutic target due to its surface expression on a multitude of leukemic disorders and possible link to proliferative advantage. In particular, CD123 has potential in the treatment of acute myeloid leukaemia (AML). Refractory AML is challenging to treat using conventional methods of treatment due to its cellular heterogeneity, with relapse normally occurring due to the survival of AML stem cells post induction therapy leading to re-initiation of disease. CD123 is highly expressed on both AML stem cells hereby termed LSCs (leukemic stem cells) and bulk disease. It is thus proposed that targeting CD123 in AML, using potent therapies such as engineered CAR-T cells has the potential to ablate both bulk disease and LSCs, reducing the probability of relapse in refractory AML patients. The work in this thesis investigates the development and application of novel anti-CD123 binders for use in CAR-T cells. CD123 binders were generated by different approaches. First, a naïve synthetic library based on the single domain shark derived vNAR was constructed and selected against CD123 using the PURE ribosome display system. The challenges associated with this approach led to the adoption of an immune library strategy wherein a rat based immune scFv (variable heavy and light immunoglobulin chains fused by flexible linker) phage library of 109 was constructed, and used to isolate six novel anti-CD123 binders through manual picking post enrichment. A Next Generation Sequencing (NGS) based selection strategy was applied to the final round of selection and facilitated the identification of a further 23 novel anti-CD123 heavy chains in unique 42 variable-heavy/variablelight (VH-VL) combinations. Six lead clones were biophysically characterised to ascertain their stability and binding kinetics prior to incorporation into 2nd generation CAR format and integration to T-cells for the evaluation of their function in cytotoxic activity and proliferative capacity. All the new antiCD123 CAR-T cells showed potent cytotoxicity toward physiologically relevant AML cell lines in a dose dependant manner whist showing minimal activity against CD123 negative control cells. The results obtained from the work in this thesis demonstrate the feasibility of generating potent novel engineered CAR-T cells with specificity for CD123 positive AML cell lines. These new agents will now be further explored as potential new cellular therapies for treatment of patients with refractory AML.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Novel CD123 Binders for CAR-T Cell Therapy |
| Event: | UCL (University College London) |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2020. 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 Medical Sciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10091010 |
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