Adam Curnock, Emma Henderson, Rita Figueiredo, Katherine Wiseman, Ronan O’Dwyer, David Overton, Veronica Gonzalez, Nicola Smit, Lorraine Whaley, Hemza Ghadbane, Shephen Hearty, Tara Mahon, Peter Weber.
The success of immunosuppressive therapies to preserve pancreatic islets and treat Type I Diabetes (T1D) has, so far, been limited either due to safety concerns or lack of persistent efficacy. To overcome these issues, we designed a novel targeted immune-suppressive approach, called ImmTAAI, that binds specifically β-cell in pancreatic islets, engages autoreactive T cells and suppresses their effector functions only when bound to target cells. By restricting T cell inhibition to the pancreas, we aim to achieve potent localized immune suppression while avoiding systemic immunosuppression.
To create a β-cell specific PD-1 agonist ImmTAAI, an affinity-enhanced T cell receptor (TCR) specific for the peptide-HLA-A*02 complex of pre-pro-insulin PPI15-24, a well-characterized autoantigen found on pancreatic β-cells, was fused to an agonistic PD-1 antibody to mimic the activity of PD-L1, the endogenous ligand of the PD-1. To verify that the TCR targeting domain is able to bind its cellular target, β-cell lines and disaggregated β-islets derived from healthy donors were stained with biotinylated PPI15-24 TCR. Furthermore, the subcellular localisation of PPI PD-1 agonist ImmTAAI at the β-cell – T cell interface was studied by confocal microscopy. To evaluate the impact of the β-cell bound PD-1 agonist bispecific on TCR signalling, a β-cell line – Jurkat NFAT cellular reporter assay was used. Finally, inhibition of T cell functions by cell-bound ImmTAAI was measured in cytotoxicity co-culture imaging experiments using CD8+ T cell clones that recognize the PPI6-14 peptide-HLA presented by EndoH-BH2 target cells.
Summary of Results
Microscopy studies showed that the PPI TCR bound specifically to immortalized β-cell lines and primary β-cells from disaggregated pancreatic islets. In co-culture localization experiments we observed that, once bound to β-cells, the PPI PD-1 agonist ImmTAAI engaged the PD-1 receptor on attacking T cells, accumulated at the T cell synapse and inhibited TCR signaling. The engineered bispecific did not compete with PD-L1 binding to PD-1 and was additive with PD-L1 in inhibiting TCR signaling. At picomolar concentrations, the PPI PD-1 agonist ImmTAAI suppressed secretion of inflammatory cytokines and inhibited β-cell killing by autoreactive CD8+ T cell clones. Notably, this PD-1 agonist was unable to inhibit T cells when free in solution and not targeted to β-cells.
We have generated a TCR bispecific inhibitory ImmTAAI molecule that is targeted to β-cells to suppress autoreactive T cells and protect pancreatic islets. These data support the concept that this bispecific inhibitor with the potential to bind β-cells with high specificity, enhance natural immune suppression delivered by PD-L1 and inhibit attacking T cells. Importantly, the molecule is inactive when free in solution and therefore has the potential to deliver localized immune suppression in islets while avoiding systemic immunosuppression. These features make PPI PD-1 agonist ImmTAAI molecules an attractive and novel approach to potentially treat T1D.