Chief Investigator: Dr Timothy Sadlon

Funding Amount: $74,915

Recipient: Women’s and Children’s Health Network

Overview:

Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing pancreatic beta-cells. There is no cure for T1D, and patients require lifelong daily insulin injections. In T1D, Regulatory T cells (Tregs) fail and can no longer prevent the autoimmune response. Giving Tregs to T1D children is a potential new therapy but has been hampered by an inability to make large numbers of the ‘right’ Treg that will work in the pancreas. We propose to overcome this by reprogramming Treg with autoantigen targeting chimeric antigen receptors to test their suitability as a new T1D therapy.

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Research Outcomes:

Researchers: Dr Tim Sadlon, Professor Simon Barry

Research Completed: 2020

Research Findings: Regulatory T cells (Tregs) are specialised immune cells that control inappropriate immune reactions. If Tregs fail in their function autoimmune diseases like T1D result. Injection of Tregs into animals can delay, prevent or reverse T1D in animals and is the foundation for numerous T1D clinical trials. These trials currently use a mix of different Treg cells, only a tiny fraction of which are the correct type that will work in the pancreas. This means large numbers of Tregs need to be isolated and grown up before being transferred into patients to treat the disease. Animal models indicate that purifying the correct type of Tregs (antigen-specific Tregs) are more potent and require many fewer cells to be effective. However, isolation and growing up rare antigen-specific Tregs remains extremely challenging. Our novel approach to overcome this limitation is to reprogram all isolated Treg by adding a chimeric autoantigen receptor to them which should turn all the cells into T1D fighting Treg (CaAR-Tregs) . A second aim is to strengthen the CaAR-Treg cell so that are better able to survive and function in the inflamed pancreas of a T1D patient.

Key Outcomes: Although new data in the field has forced us to change approach (see below) delaying completion of the project we have recently recruited a master of philosophy student Ms Jacqui Scaffidi to continue this work to ensure successful completion. In addition, a collaboration with Professor Toby Coates (Co-supervior of Ms Scaffidi) has been established to extend this project to include testing of CaAR-Treg in animal models.

The original chimeric autoantigen receptors (CaAR) fused the variable regions of the heavy and light chains of the GAD65 antibody b96.11 monoclonal antibody into a single chain variable fragment (scFv). This scFV was then fused to an extracellular hinge region (three lengths-see below), a transmembrane domain and a second-generation intracellular signalling domain. Based on our work on anti-cancer CARs (expressed in effector T cells ie non-Treg cells) the intracellular signalling domain of the GAD65 CaAR consisted of the CD3zeta chain and 4-1BB co-receptor. However, a publication this year (Boroughs, A .C., et al JCI insight, 2019) in which the 4-1BB and an alternative CD28 intracellular signalling domain were compared in human Treg cells showed that the inclusion of 4-1BB signalling domain strongly interfered with the suppressive activity of Treg cells. This strongly indicating that our original CaAR would not function and could in fact be detrimental. Based upon this new information, we needed to replace the 4-1BB domain with the intracellular CD28 domain in our GAD65 recognising CaAR constructs. This has caused a delay in the completion of the project as we remake the GAD65 recognising CaAR constructs. Three different vectors are being remade that contain various lengths of a modified lgG4 Hinge domain that separates the scFv domain from the CD8 transmembrane and intracellular signalling domains. As the length of the linker can be a critical determinate of activity, through providing the correct special distance for a functional immune synapse to form three vectors with varying length hinge regions will be tested. Following vector construction and virus production these will be used to create CaAR-Treg for testing.

Lessons from the cancer immunotherapy field on the use of chimeric antigen CAR-T cells has emphasised the importance of the correct cellular phenotype of the cells transferred into patients for effective and long-lived responses. A critical component of this is to maintain a high proportion of cells with a stem/memory cell-like phenotype and limit proliferation exhaustion during the process of expanding the cells in the laboratory. This balance of growing up sufficient cells and maintaining the correct phenotype is dependent upon both the correct combination of growth factors (cytokines) added to the cultures as well as the intracellular signalling domain of the Chimeric antigen receptor itself. We have trialled a number of different cytokine cocktails to promote both good proliferation and maintenance of a high proportion of cells with a memory/stem- like phenotype.

We found that a combination of low IL2 together with IL7 and ILlS yielded the best results both in terms of phenotype and numbers of cells.

We have also optimisation the timing and lentiviral vector required for efficient transduction of human Tregs. Although, lentiviral vector are able to transduce non-dividing cells, the rate of pro-virus integration into the genome is much higher in activated cells. We therefore assessed the optimal time post activation for Treg transduction. Treg cells were activated by culturing cells with a 3:1 ratio of anti-CD3/anti-CD28 beads that cross-link the TCR receptor on the cell surface and at day 1,2 or 3, known amounts of infectious units of lentivirus particles (MDI; multiplicity of infection) were added to the cells. The Lentivirus used co-expresses the green fluorescent protein GFP allowing the transduction rate to be calculated. We determined that the addition of a MOI of 20 and virus exposure on day 2 post-transduction gave the highest transduction rate.

Research Papers: Nil to date.

Related Publications: We have not yet reached a point in the research that would allow publication. The in vitro creation and testing of the suppressive function of the CaAR-Treg cells, the next step in the research proposal, would generate suitable data for a publication. This project is now being continued as a masters of philosophy project by Ms Jacqui Scaffidi as a collaboration between Professor Simon Barry’s and Professor Toby Coates’ (Medicine, University of Adelaide) research groups. This will ensure the completion of the project as well as testing of the cells within a TlD animal model and high impact publications. The support of CRF will be appropriately acknowledged with sufficient prominence in any publications, papers, reports or articles resulting from research projects funded by CRF.

Future Outcomes: We have yet to generate any new intellectual property however if we can demonstrate antigen-specific suppressive function of CaAR-Treg in vitro it would be of potential commercial interest and we seek to protect this intellectual property