Utilising anhydride reactivity to target endogenous human proteins

Covalent conjugation between proteins has been used to develop novel drug delivery methods, enhance therapeutic efficacy, and improve diagnostic sensitivity. Proximity-directed ligation methods enable coupling to unmodified endogenous proteins. A diverse range of proteins contain autoproteolytic domains that cleave at an Asp-Pro bond via an aspartic anhydride intermediate. In this thesis, the anhydride is harnessed as an electrophilic warhead for targeted conjugation. I developed NeissLock, a technology that uses the self-processing module (SPM) of the FrpC protein from Neisseria meningitidis. SPM is fused to the C-terminus of the binder protein so that the anhydride generated upon calcium-induced activation reacts with proximal nucleophiles on the target. NeissLock, using the standard genetic code, enables rapid and specific coupling under mild conditions. I discovered that the FrpA SPM variant has an enhanced activation rate compared to the first-generation NeissLock. I developed a bacterial surface display system for directed evolution of SPM, towards improving the cleavage yield and stability of the domain. Finally, I established the split SPM system by splitting the domain into 2 fragments to provide another level of control over SPM activation. The fusion of SpyTag003/SpyCatcher003 to the split SPM fragments increased the coupling efficiency. Split SPM facilitates the mammalian expression of binder-SPM fusions, broadening the applicability of NeissLock. I fused various human red blood cell-binding peptides to SPM to test for coupling to human red blood cells. Covalent conjugation of therapeutic proteins to red blood cells using NeissLock would increase the stability of coupling compared to non-covalent methods, potentially enhancing drug delivery using red blood cell carriers.