Abstract
Solid organ transplantation is the standard treatment for end-stage organ disease. However, its impact is short-lived due to allograft recognition. Adoptive transfer of regulatory T-cells (Tregs) protects from allograft rejection, and the safety has been demonstrated in clinical trials. Although antigen-specific Tregs are superior to polyclonal Tregs, they are still MHC-restricted. As well as antigen specificity, genetic engineering offers a solution to the unmet need of improving Treg function for clinical therapy as CARs are customisable building blocks. Tregs expressing chimeric antigen receptors (CAR) confer antigen-specificity in a non-MHC restricted manner. Thus, two major approaches have been explored in this thesis.The first approach was applying fourth-generation CAR technology, i.e. those that release an additional cytokine into the respective microenvironment. Therefore, this thesis focuses on design, construction, and investigation of an IL-10-releasing HLA-A2- targeting CAR Treg in protecting an allograft. Additionally, the ability to track Tregs in vivo such as inclusion of the sodium-iodide symporter (NIS) within the constructs, would inform us of Treg biodistribution and longevity, aiding therapy development and personalised monitoring.
Herein, the IL-10 HLA-A2 NIS TagRFP CAR was designed and constructed. Tregs were transduced and their potency was compared to control Tregs in vitro. The IL-10 A2 CAR Treg was shown to be superior to the HLA-A2 CAR Treg. A skin transplant model using skin from an HLA-A2 expressing transgenic mouse was performed. The IL-10 CAR Treg functions potently in vitro, however the skin transplantation and imaging model need further optimisation to reliably assess CAR Treg efficacy and detection.
The second approach was to comprehensively analyse a panel of different costimulatory molecules in second-generation CARs to identify one that may be optimal for Treg function. Herein, I performed a pilot study comparing five endodomains to the CD3ζ-CD28 molecule – CD27, OX40, 4-1BB, ICOS, DR3. There are no significant differences in function or phenotype, although incorporating a DR3 endodomain preliminarily seems to suggest a weaker suppressive capacity.
To conclude, this is the first attempt in creating a fourth-generation CAR Treg, and an independent validation of CAR Tregs with different signalling domains. This study introduces a broad range of possibilities in manipulating Tregs, with clinical implications for transplantation and autoimmunity.
| Date of Award | 1 Apr 2021 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Giovanna Lombardi (Supervisor) & Gilbert Fruhwirth (Supervisor) |