Development of a class B evasin-derived peptide for inhibiting islet inflammation

Thesis

Type 1 diabetes (T1D) is a disease characterised by the migration of CD8⁺ and CD4⁺ T-cells to pancreatic islets, where they cause inflammation (“insulitis”) and destroy insulin-producing beta cells. T-cell targeting drugs have been approved for treating T1D, but are associated with significant immunosuppressive side effects. Targeting T-cell trafficking to islets is an alternative strategy that has the potential to minimise such side effects. Chemokines direct T-cell migration to islets, but the redundancy of the chemokine network has hindered therapeutic development. Tick evasins neutralise the redundant chemokine network, with class A and B evasin proteins inhibiting CC and CXC-chemokines, respectively. Peptides derived from class A evasins are potent inhibitors of chemotaxis. This work hypothesises that potent chemokine-inhibiting peptides can be identified from class B evasins. Phage display library screening of evasin B-derived peptides against biotinylated chemokine targets identified a novel peptide that can bind and inhibit multiple CC and CXC class chemokines, as determined by in vitro chemotaxis assays. Combinatorial saturation mutagenesis and phage display selection were used to enhance its affinity for multiple chemokine targets. RNA-sequencing data from cytokine-stimulated human islets were used to model the variety of chemokines in insulitis, providing an approximation of their abundance. The mutated peptide, CM1629, inhibited the migration of CD8⁺ and CD4⁺ T-cells to this insulitis chemokine pool. CM1629 did not inhibit monocyte migration, indicating its specificity for T-cells. Mutagenesis resulted in a 19-fold increase in potency for CM1629 compared to the parental peptide, with CM1629 exhibiting nanomolar potency against the insulitis chemokine pool and CD8⁺ T-cells. AlphaFold3 modelling reveals that tryptophan substitutions enhanced hydrophobic inter-chain interactions, suggesting a mechanism for enhanced potency. CM1629 may be an effective starting point for developing therapeutics that target T-cell trafficking in T1D.

Authors: Payne, ME

• DOI: 10.5287/ora-00gde6x6m
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English