Inducible Chimeric Antigen Receptors vector development for use in acute Myeloid Leukaemia

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

The treatment of AML continues to pose significant challenges, and a large number of patients, particularly those over 60 years of age, relapse after initial chemotherapy to induce remission. Majority of patients are ineligible for a bone marrow transplant due to comorbidities. Chimeric Antigen Receptor (CAR) T-cells therapy has produced promising results in the treatment of some blood cancers such as B-cell Acute Lymphoblastic Leukaemia (ALL) and B-cells Non-Hodgkin Lymphoma. Several myeloid leukaemia associated antigens have been recently discovered as possible targets for an AML CAR-based therapy and amongst these, CD123 and CD33 are under investigation by our group either as a bridge to transplant o as long-term remission or consolidation therapy. CAR T-cell therapy can cause some life-threating side effects such as Cytokines Release Syndrome (CRS), Neurotoxicity and several On-target off-tumour effects. At the present moment, CAR expression on T-cells is an all or nothing process, resulting in a full T-cell activation which can be clinically challenging to moderate.

Here in this PhD, I have developed a proof-of-concept CAR construct whose expression can be regulated by an approved clinical drug: Rapamycin. It has been previously shown that a mutant form of FK506 Binding Protein 12KDa (FKBP12) aggregates in the ER and that this aggregation is disturbed by Rapamycin and allows the protein to leave the ER. I constructed a CD123 scFv CAR in-frame with 3 repeats of FKBP12 separated by a Furin cleavage site called “RapaCAR”. Rapamycin administration in micromolar quantities allows CAR expression modulation without recurring to an “on/off mechanism”. The receptor shows turn over of less than 24 hours and the modulation persist in transduced cells for more than 100 days. The Multiplicity of Infection (MOI) used for cell transduction is crucial to reduce the leakage in the system, hence the regulatability of the CAR expression. The system has been also tested with a Rapalog that is also commonly used in the clinic, but at higher concentration than Rapamycin and with faster pharmacokinetics. RapaCAR has been tested in K562 and Jurkat cells, showing that the regulation of the CAR expression is achievable in T-cells. RapaCAR would allow to interrupt and/or modulate the treatment as needed in a clinical setting. Furthermore, I have generated new CAR vectors for CD123 with CD137 as co-activation domain instead of CD28 already available in the Department. In previous studies this co-activator has shown to activate the T-cell in a similar way to CD28, but with a prolonged T-cells survival. These constructs offer exciting opportunities for the future to development of Regulable CAR for long-term treatments. Finally, I have generated a clean cellular system for either CD123 or CD33 or both antigens using Ramos cell line. These cells are ideal targets for future cytotoxicity assays with high expression of myeloid antigens (CD123, CD33), thereby permitting testing of CAR construct potency in standardised conditions.
Date of Award1 Dec 2022
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorGhulam Mufti (Supervisor) & Joop Gaken (Supervisor)

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