Optimising antigen targeting in next generation regulatory T cell therapy

Thesis

Conventional immunosuppression causes adverse effects, such as infection and malignancy, in transplant recipients, and is unable to eliminate the risk of rejection. Early-phase clinical trials demonstrated that autologous polyclonal regulatory T cell (Treg) therapy can enable safe reduction of conventional immunosuppression. However, there are concerns of limited potency and a theoretical risk of non-specific immunosuppression. Antigen-specific Treg strategies such as donor antigenreactive Tregs are limited by difficulties in cell manufacturing, and chimeric antigen receptor (CAR) Tregs are limited by their single-antigen specificity.

To overcome these challenges, Tregs with a SNAPtag modular receptor which can be programmed against different antigens by covalently binding to antibody adaptor molecules through its cognate ligand (benzylguanine) were developed with the goal of broadening the applicability of antigen-specific Treg therapy. 

To achieve this, three specific aims were investigated: 

1. To design, generate and evaluate Tregs transduced to express SNAPtag receptors via lentiviral vectors for antigen-specific activation and suppression in vitro (Chapter 3) 

2. To design, generate and evaluate Tregs transfected to express SNAPtag receptors via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas gene editing for antigen-specific activation and suppression in vitro (Chapter 4) 

3. To establish in vivo models for the evaluation of human Tregs with programmable antigen specificity (Chapter 5) 

This work demonstrates that Tregs transduced with a SNAPtag modular CAR and preloaded with anti-HLA and other antibodies could be activated specifically. However, these effects were inconsistent and modest compared to conventional CAR Tregs, and SNAP-CAR Tregs were not consistently more suppressive compared to polyclonal Tregs. Novel SNAPtag constructs designed to increase functional surface SNAPtag receptor expression over time and potentially improve Treg suppressive function, using both lentiviral and CRISPR-Cas12a gene-editing techniques, did not improve Treg function.

Through this work, a Treg enrichment and sorting process utilising vortex-actuated cell sorting was optimised and was shown to improve early Treg viability and expansion. Preliminary work to establish a humanised mouse skin transplantation model using HLA-A2 transgenic donor mice and cell tracking using bioluminescence was performed.  

These findings indicate that while SNAPtag modular receptors remain a promising strategy for broadening antigen-specific Treg therapy, further refinements are required to improve potency and consistency, and their efficacy remains to be established in vivo.

Authors: Ho, QY

• DOI: 10.5287/ora-2rg8qkvg0
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: cell separation; HLA antigens; fluorescence-activated cell sorting; magnetic-activated cell sorting; regulatory; cell- and tissue-based therapy; chimeric antigen; CRISPR-CAS systems; vortex-actuated cell sorting; t-lymphocytes; autoimmune disease; gene editing; immune tolerance; cell survival; transplantation; receptors; lentivirus
• Subjects: Immunology; T cells; Suppressor cells; Cell receptors